Medicinal Chemistry Approaches to Heart Regeneration - American

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Perspective

Medicinal Chemistry Approaches to Heart Regeneration Dennis Schade, and Alleyn T. Plowright J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Aug 2015 Downloaded from http://pubs.acs.org on August 19, 2015

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Journal of Medicinal Chemistry

Medicinal Chemistry Approaches to Heart Regeneration Dennis Schade1*, Alleyn T. Plowright2 1

TU Dortmund University, Department of Chemistry & Chemical Biology,

Otto-Hahn-Str. 6, 44227 Dortmund, Germany 2

AstraZeneca, Department of Medicinal Chemistry, Cardiovascular and

Metabolic Diseases Innovative Medicines, Pepparedsleden 1, Mölndal, 43183, Sweden * Corresponding author: [email protected], +49-231-7557083

KEYWORDS cardiomyocytes, heart muscle, cardiac regeneration, stem cells, drug discovery, small molecules

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ABSTRACT

Due to the minimal and clearly insufficient ability of the adult heart to regenerate after ischemic injury, there is a great opportunity to identify biological mechanisms, substances and factors that enhance this process. Hence, innovative therapeutic management of heart failure following infarction requires a paradigm shift in pharmacotherapy. Spurred by tremendous progress in the field of stem cell and cardiac biology, several attractive approaches for regeneration of lost cardiomyocytes and supporting vasculature have emerged. Research in this area focuses on restoring the hearts’ original function via proliferation and differentiation

of

cardiac

progenitor

cells,

proliferation

of

pre-existing

cardiomyocytes and reprogramming of cardiac fibroblasts. In this Perspective, we outline these principal strategies, putative biological targets or signaling pathways and chemical agents, with a focus on small molecules, to achieve therapeutic heart regeneration. We also point out the many remaining questions and challenges, particularly for translating in vitro discoveries to in vivo application.

ACS Paragon Plus Environment

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1. INTRODUCTION According to the Federal Statistical Office (2013) cardiovascular diseases (CVDs) account for 39.7% mortality in the German population, and therefore, represent the major cause of death.1 Among these CVDs, chronic ischemic heart disease (8.2%), myocardial infarction (MI) (6.1%) and heart failure (5.1%) belong to the top three causes of death in Germany. These numbers translate approximately to the world population and it has been estimated that along with the demographic change CVD-related mortality will continuously increase, whereby ischemic heart disease will remain the leading cause of death.2 From a pathophysiological and etiological perspective, such CVDs are closely allied and mostly mutually dependent. Particularly after MI, heart failure is an inevitable consequence when the size of the ischemic site reaches a critical limit. Therapeutic approaches are largely aimed at reducing cardiovascular risk factors, lowering high blood pressure and “economizing” cardiac performance. Unfortunately, the available pharmacological regimen is stretched to its limits as it often deals mainly with the symptoms. Real progress can thus only be made via a better understanding of heart muscle regeneration and remodeling post-MI. During MI, the affected heart tissue undergoes necrotic processes leading to a loss of ca. one billion cardiomyocytes (CMs).3 As a consequence of these massive acute and chronic inflammatory responses, lost heart muscle is eventually replaced


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by collagen-rich fibrotic tissue that severely impairs the biomechanical and electrophysiological function of the heart, resulting in heart failure.3 Unfortunately, the innate regenerative capacity of the adult mammalian heart is very limited as opposed to that of lower vertebrates. However, a large body of evidence suggests that the renewal of CMs continues even after the neonatal period.4 Although the numbers strongly depend on the techniques used, the majority of studies suggest an annual CM turnover rate of 1-2% at the age of 25 that gradually decreases with ageing.5-8 Moreover, the regenerative potential after injury or in diseased hearts has neither been conclusively quantified nor well-understood. From a therapeutic point of view, transplantation of the entire heart, which represents a last option for the patient in severe cases of heart failure, is restricted due to limited numbers of matching donor organs as well as life-long immunosuppression. For these reasons, it is highly attractive to identify and develop methods that would restore the hearts’ original function through replacing and/or regenerating lost CMs. In principal, two main strategies are being pursued in the field: Firstly, regeneration via transplantation of pluripotent stem cell (PSC)-derived cardiac progenitor cells (CPCs) or adult stem cells, CMs or engineered cardiac tissue.9 Secondly, the stimulation of in vivo regenerative (endogenous) processes which are mainly attributed to pre-existing CMs (proliferation) and multipotent progenitor cells (see Fig. 1).10, 11 However, in vivo heart regeneration also includes approaches


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beyond the actual de novo generation of CMs such as improving vascularization and minimizing cardiac fibrosis and remodeling.11-14 Undoubtedly, the field of “regenerative cardiology” has benefitted tremendously from milestone discoveries in stem cell research. The nobel prize-awarded finding by Yamanaka that somatic cells can be reprogrammed to an induced pluripotent stem cell-like state (iPSCs) as well as the ability to generate heart muscle cells from these iPSCs using directed differentiation methods have provided a fertile ground for many applications.15 Such applications are not only of therapeutic nature (i.e., regenerative medicine) but also include technologies for the drug discovery process (i.e., safety pharmacology and toxicology) and disease modeling approaches for basic and applied research in cardiac biology.16, 17


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Figure 1. Possible cell sources and strategies for endogenous therapeutic heart regeneration in vivo.

The development of regenerative therapies in this field not only involves cellbased methods but increasingly appreciates the use of chemical modalities, i.e. chemical agents or chemically-defined methods.18-20 Such modalities potentially provide several advantages over (stem) cell-based approaches as their use may avoid or minimize issues of ethical controversies, technical obstacles (e.g., efficient cell application, retention, survival etc.), mutations and immunogenicity. A variety of such chemical modalities have been developed for regenerative applications, ranging from RNA therapeutics (e.g., miRNAs, anti-miRNAs),


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peptides and proteins to small molecular agents.20, 21 While each modality displays its own strengths and weaknesses, all of them are of tremendous value for the many applications in (cardiac) regenerative medicine. Small molecules are of particular interest to the medicinal chemist and in general have already found entry and widespread acceptance in the field of stem cell and regenerative biology.18, 19, 22, 23

Small molecules add another layer of potential advantages over other chemical

modalities since they are (physico)chemically well-defined, cell-permeable and amenable

to

medicinal

chemistry-driven

pharmacodynamic

(PD)

and

pharmacokinetic (PK) optimization.22 In addition, small molecules have the potential to be precisely delivered to the heart and, with good understanding of PK–PD relationships, the doses can be titrated according to the response, offering an advantage in a dynamic clinical setting. This review covers mainly small molecule-based approaches to heart regeneration with a particular focus on cardiomyogenic agents that have been developed to stimulate CM formation from several types of cell sources (see Fig. 1). Some challenges associated with the respective compounds and the underlying therapeutic concepts will be discussed, along with several strategies that may be of therapeutic value but for which no small molecules (early tools or drugs) are, to date, available.


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2. CHEMICAL CONTROL OF CARDIAC DIFFERENTIATION 2.1. Introduction to cardiomyocyte differentiation from stem cells Until 5-6 years ago, only a handful of small molecular agents had been reported to affect cardiac differentiation from pluripotent stem cells (PSCs) with many exhibiting rather unspecific modes of action.24 Among the earliest examples (2003) was ascorbic acid (52, Fig. 9) which was discovered from a focused, small-scale screen using a mouse embryonic stem cells (mESC)-Myh6-GFP reporter cell line.25 Similarly, in 2004, the diaminopyrimidine compound class of cardiogenols (18, Fig. 4) was reported to stimulate cardiac differentiation from mESCs, although the primary large-scale screen was performed in mouse embryonic carcinoma cells (P19).26 At the same time, isoxazolyl-serine-based agonists of the peroxisome proliferator-activated receptor (PPAR) were disclosed with cardiogenic activity on mESCs.27 Ever since, tremendous progress has been made to efficiently generate CMs from stem cells, especially from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hIPSCs), which are increasingly used in cardiovascular research.16,

17, 28

In this regard, robust conditions for directed CM differentiation

have been developed and key steps for efficient, high-yielding protocols were identified along with appropriate modulators.29-32 These protocols were not only aimed at delivering high yields of CMs but also at making use of chemically-


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defined conditions devoid of animal-derived additives (serum, growth factors, cytokines etc.). In fact, chemically-defined conditions are key requirements for scalable, reproducible and inexpensive procedures for hPSC-based CM differentiation. In general, cardiopoietic differentiation can be simplified into four key steps: 1) mesoderm induction, 2) mesoderm structuring, 3) formation of cardiac mesoderm (cardiac progenitor cells) and 4) CM induction followed by processes of maturation. As indicated above, many cardiopoietic (natural) factors involved in these processes are known, and for further details, the reader is referred to more specialized reviews.33, 34 Here, we primarily focus on human PSC differentiation followed by complementary examples from other organisms and assay systems. From an “induced signaling perspective”, directed differentiation protocols can be divided into two principal categories or strategies, either driving bone morphogenetic protein (BMP) signaling- or Wnt/β-catenin signaling as emphasized by Zhang et al..35 The role of Wnt signaling is biphasic as the pathway needs to be stimulated for mesoderm induction but subsequently inhibited for cardiac specification. Utilizing the knowledge of such key signaling pathways, protocols for (largely) chemically-defined conditions were systematically developed using the known small molecule modulators (summarized in Fig. 2). Early reports primarily disclosed the use of Wnt/β-catenin stimulators and


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inhibitors for directed cardiac differentiation.31,

36-38

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Gonzalez et al. additionally

made use of the Nodal/Activin/TGFβ inhibitor SB-431542 (16, Fig. 3) and the Hedgehog agonist purmorphamine (20, Fig. 4) in a unique chemically-defined system.39 Most recently, Burridge et al. reported on a systematic protocol optimization in which a larger set of small molecule pathway modulators was evaluated. These efforts led to a powerful cardiac differentiation platform from hPSCs, ultimately using mainly small molecule-mediated Wnt stimulation and inhibition.32 Similarly, the Greber group published an exquisite protocol that allows universal cardiac induction from hPSCs in 2D and 3D formats.35 Notably, they used a defined combination of bFGF, BMP4 and Wnt stimulation that strictly cooperates in a dose- and time-dependent fashion, yielding highly efficient mesoderm induction while using minimal amounts of the respective growth factors. For instance, BMP-only approaches otherwise typically require several day treatment with BMP4 at 5- to 25-fold higher doses.30, 40, 41 Balancing BMP versus Wnt signaling in early cardiac development was also shown to be critical for lineage commitment towards epicardial cells and CMs from hPSCs.42 This study underlines once more the tremendous potential in using directed differentiation procedures from pluripotent stem cells for various applications within basic and applied research.


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In the following sections, small molecule modulators of these key cardiogenic pathways or mechanisms are summarized and discussed.


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Figure 2. Schematic illustration of cardiac differentiation from human pluripotent stem cells (hPSCs), key cardiopoietic factors and pathways together with corresponding small molecule modulators for directed differentiation protocols. Image modified according to Burridge and colleagues.34 Green arrows = pathway stimulation, red arrows = pathway inhibition.


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2.2. Chemical perturbation of cardiopoietic signaling pathways 2.2.1. Targeting Wnt/β β-catenin signaling Wnt pathway stimulators. As outlined above, canonical Wnt signaling needs to be stimulated for efficient mesoderm induction and this is widely used for directed differentiation protocols. Inhibition of glycogen synthase kinase 3-β (GSK3β) represents the most well-established means to activate the Wnt/β-catenin pathway (Fig. 2). Although various small molecule GSK3β inhibitors are known to date, either BIO (13) or CHIR99021 (14) are typically used in the field for directed differentiation (Fig. 3b). To the best of our knowledge, thus far, only Burridge et al. systematically explored different chemotypes of GSK3β inhibitors and tested which was best suited in their differentiation protocol.32 In addition to 13 and 14, TWS119, 1-azakenpaullone, TDZD-8, ARA014418 and 3F8 were evaluated. Interestingly, only 13 and 14 induced cardiac differentiation, whereas the other compounds exhibited pronounced toxic effects. Aside from targeting GSK3β, few alternative options (based on chemical modalities) exist to stimulate Wnt, such as the Wntepanes or QS11 that only work in synergy with Wnt3a stimulation.43, 44


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Wnt pathway inhibitors. In contrast to Wnt pathway stimulation, more cellular targets and chemical modalities exist to inhibit Wnt signaling and many have been explored for cardiac differentiation. In principal, the pathway can be inhibited at different levels within the cascade: at the cell surface/receptor level, signaling can be inhibited via porcupine inhibitors (e.g., 1 (IWP-2), 2 (IWP-3)) which prevent Wnt ligand palmitoylation, an essential step for the excretion of Wnt proteins (Fig. 3a).32, 45, 46 Activation of the β-catenin destruction complex leads to a reduction of β-catenin protein levels. Small molecule inhibition of axin-destabilizing tankyrases (TNKSs) (e.g., 3 (IWR-1), 4 (XAV939)

45, 47

or activation of CK1α (8,

pyrvinium)48 mediate this effect (Fig. 3a). It should be noted that a direct effect of pyrvinium on CK1 isoforms has been questioned in a recent study by Venerando and colleagues.49 The authors argued that pyrvinium interferes with the Wnt pathway via promotion of Akt/PKB phosphorylation and GSK3β activation. Moreover, several compounds have been reported to inhibit the pathway downstream of β-catenin. Some of these molecules inhibit specific protein-protein interactions (PPIs) of β-catenin with transcription factors or cofactors for the transcriptional machinery, respectively. For further details on such modulators, the reader is referred to more specialized reviews.50 However, in the context of cardiac differentiation, a small molecule modulator of the CREB-binding protein (CBP)/βcatenin interaction (i.e., 6, ICG-001) has been reported to be beneficial.51 In


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addition, the functional counterpart, i.e. IQ-1 (7), an inhibitor of p300/β-catenin is also depicted (Fig. 3a). To date, primarily TNKS and porcupine inhibitors are being used by investigators in the field for small molecule-mediated cardiac differentiation, although a large panel of Wnt inhibitors has not been extensively studied thus far. An early example was given by Lanier et al. who compared several distinct inhibitor classes within an approach to systematically optimize the IWR-class of Wnt inhibitors for cardiogenesis from hPSCs (see below for more details).52 More recently, Burridge et al. compared six different Wnt inhibitors, i.e. 1, 3, 4, 6, IWP4 and Wnt-C59 (38, Fig. 6). Similar to Lanier et al., the authors found that the IWR-class of compounds exhibited a superior performance over a large dose range (1-10 µM), implicating lowest toxicity, counterproductive side effects or off-target activity. From the tested porcupine inhibitors, 38 turned out to be the most effective at low doses with first signs of toxicity >5 µM. Additional cardiomyogenic small molecule Wnt inhibitors are KY02111 (11) and cardionogen-1 (12) for which the cellular target(s) have not yet been identified (Fig. 3a). Triazolothiadiazole 12 was discovered in an elegant in vivo small molecule screen (4000 compounds) in zebrafish and characterized as an inhibitor of β-catenin-dependent Wnt signaling.53 Interestingly, this activity appeared to be reserved for certain cell types and tissues (e.g., heart) since cardionogens were


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inactive in classic HEK cell-based Wnt assays. The benzothiazole 11 was developed based on hits from a small molecule screen of 9600 compounds in a monkey ESC-based cardiac assay and further established for directed CM differentiation protocols in different hES and hIPS cell lines. 11 was characterized as a Wnt inhibitor that appears to act downstream of the β-catenin destruction complex. It exhibits an interesting activity profile as it has been described to supersede any other cardiac factor during directed differentiation from hPSCs, although it worked best in combination with other Wnt inhibitors such as 1 or 4 suggesting a cooperative effect.37 It is thus tempting to speculate that, even though being well-characterized as a Wnt/β-catenin inhibitor, a specific multi-target profile is responsible for 11’s extraordinary cardiogenic activity.


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Figure 3. Cardiomyogenic small molecules targeting Wnt/β-Catenin and TGFβ superfamily ligands. a) Wnt/βcatenin inhibitors, b) Wnt/β-catenin activators, c) TGFβ superfamily inhibitors.


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2.2.2. Targeting the TGFβ β superfamily of ligands Bone Morphogenetic Protein (BMP). The role of BMP for cardiac myogenesis is well-established,54 and it has already been discussed above in the context of mesoderm induction (Fig. 2). A finely regulated interplay between BMP, Nodal/Activin A and Wnt is of critical importance for effective mesoderm induction.34 In this regard, BMP stimulation is required and typically achieved by using recombinant BMP4 and Activin A without a small molecule agonist being available. Notably, the Greber group reported that Activin is dispensable for effective mesoderm induction in hESCs and hIPSCs.35 However, an early report by Hao et al. indicated that BMP inhibition by dorsomorphin (15, Fig. 3) in very early stages of mESC differentiation promotes cardiac development.55 15 was originally discovered as a BMP inhibitor in a small molecule high-content imaging screen in zebrafish, monitoring the development of the embryonic dorso-ventral axis.56 The approach to use 15 for cardiomyogenesis was based on the idea of replacing the endogenous BMP inhibitor (i.e., Noggin) (Fig. 2).57 Along with a systematic medicinal chemistry-driven optimization campaign of 15, the authors later described the utility of a highly potent and selective analog (i.e., dorsomorphin homologue 1, DMH-1, 45, see Fig. 7) in later stages of cardiac differentiation, allowing for the enrichment of pro-cardiac progenitors that respond to small molecule Wnt inhibition.58 The original


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compound 15 simply was not selective enough for Smad-dependent BMP signaling but also affected AMP kinase, VEGF and PDGF signaling which may have contributed to its distinct effects on Brachuyry T+ mesoderm and Mesp1+ early cardiovascular progenitor cells in their first study. It is now widely accepted that in later stages of differentiation, after mesoderm has been formed, BMP and Nodal/Activin/TGFβ signaling is important for structuring towards cardiac mesoderm and cardiac progenitor cells.30, 40, 41 In these stages of cardiac mesoderm, small molecule-mediated inhibition of BMP and Nodal/Activin/TGFβ promote the formation of cardiac progenitors. Kattman et al. demonstrated the combination of 15 with 16, a pan-TGFβ inhibitor lacking selectivity for Nodal/Activin A and TGFβ, as an effective means to promote formation of cardiac progenitors and CMs from PSCs.41 However, it is to be noted that their studies underline the critical role of endogenously produced signaling agonists (cytokines) which depend on the cell lines used. The authors concluded that the optimization of endogenous (natural factors) versus exogenous (recombinant cytokines, small molecules) signaling determines the efficiency of cardiac differentiation. Transforming Growth Factor β (TGFβ β). Yet another level of complexity to the role of TGFβ/Activin/Nodal signaling during cardiac differentiation was added by studies from the Mercola group who discovered a novel class of TGFβ inhibitors in


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the context of cardiac differentiation from a high-content screen of 17.000 compounds in mESCs.59 A specific subclass of b-annelated 1,4-dihydropyridines (e.g., ITD-1, 17, Fig. 3) stimulated cardiogenesis from murine and human ESCs at stages where uncommitted mesoderm specifies towards a cardiac fate. These compounds exert their pathway inhibitory activity in a novel fashion, namely through inducing the clearance of the TGFβ type II receptor from the cell surface, followed by its proteasomal degradation.59 They were subsequently named ITDs, “inducers of TGFβ receptor degradation”. Using the ITDs, the authors revealed a biphasic role of TGFβ signaling during cardiogenesis in mESCs: ITDs inhibited mesoderm induction when given early during differentiation, and promoted differentiation when given in stages of mesoderm structuring towards the cardiac fate. Together, modulation of the four key signaling pathways of Wnt, BMP, TGFβ/Nodal/Activin and FGF currently represent the main strategies for effective directed CM differentiation from PSCs. All four of these pathways are important in early phases of differentiation (i.e., mesoderm induction), and mainly Wnt/βcatenin pathway stimulators such as 13 and 14 are used for a chemically-defined procedure, along with the still required cytokines BMP4 and bFGF. In addition, in later stages, perturbation of Wnt/β-catenin signaling via the use of small molecule inhibitors represents the most powerful strategy to ensure a chemically-defined


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protocol. However, along with Wnt inhibition, modulation of several members of the TGFβ superfamily of ligands is described by many investigators and may have advantages for a fine control of cardiac specification and late CM differentiation.


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2.2.3. Miscellaneous targets beyond Wnt, FGF and the TGFβ β superfamily pathways Besides the above described compounds that target distinct cardiogenic pathways, several additional small molecules have been reported that stimulate cardiac differentiation from PSCs. Many of them are, to date, not wellcharacterized regarding their modes of action on a cellular or molecular level and it is likely that some, at least in part, are cardiomyogenic by also interfering with some of the above described pathways. Among the few remaining examples of small molecules with a defined annotated activity is the p38MAPK inhibitor 9 (Fig. 3) which was reported in 2008 to promote cardiogenesis from hESCs by 2.5-fold when used at specific concentrations.60 In fact, this was one of the first protocols describing a chemically-defined, serum-free procedure, although factors from END2 cells (conditioned media) were required. Later the same groups provided mechanistic explanations as they observed that insulin/IGF-1 inhibitory effects on cardiogenesis were antagonized by secreted factors from END2 cells.61 This activity could be mimicked by prostacyclin (i.e., prostaglandin I2, PGI2). The combination of PGI2 and 9, therefore, furnished a very first serum- and xeno-free differentiation medium. However, a very interesting follow-up on this story was recently reported by Laco et al. who suggested that the main cardiogenic effects of


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9 likely did not solely originate from its p38MAPK inhibitory activity. Through synthesizing a series of 2,4,5-trisubstituted azole analogs of 9 (e.g., TA-02, 10, Fig. 3), kinase profiling and testing in hESC-based cardiogenesis the authors identified casein kinase 1 (CK1) as a key off-target.62, 63 Since inhibition of CK1 leads to a destabilization of the β-catenin destruction complex, once again, inhibition of canonical Wnt signaling turned out to be a critical mode of action for cardiogenesis. However, it should be noted that additional kinases may be involved as suggested by results from the authors’ kinome profiling. For instance, 10 or alternative compounds based on 9 may also (at least in part) stimulate cardiogenesis by interference with TGFβ signaling in analogy to the ITDs.59, 64 In addition, the p38MAPK component of the biological activity is still of relevance to cardiogenesis, although probably more due to affecting CM proliferation as has been reported by several groups.65, 66 This example underlines the importance of employing high-quality chemical tools for mechanistic studies. A high degree of target and pathway selectivity is desired in order to draw meaningful conclusions, underling the importance of thorough characterization of small molecules that are discovered in phenotypic screens. For one of the earliest discovered cardiogenic small molecules, the 2,4diaminopyrimidine class of cardiogenols (18, Fig. 4), no conclusive mechanism of


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action or target has been reported.26 However, recent studies demonstrated that 18 and SAR-optimized derivatives (e.g., VUT-MK142, 19, a 4,6-diaminopyrimidine analog, Fig. 4) may exert its activity through targeting cardiovascular progenitor cells, promoting their transition towards the cardiac fate.67, 68 Of note, progenitor cells used for these studies were obtained as a phenotypically stable somatic stem cell line described by Hoebaus et al., who reported their successful maintenance via construction of an articial niche environment.69 From a mechanistic point of view, it would be interesting to characterize such compounds in a human PSCbased cardiac differentiation protocol. The authors’ studies eventually suggested a mechanism that would position these compounds in a similar time frame of differentiation as the above described Wnt, BMP and TGFβ pathway modulators. An impressive consecutive series of reports was reported by the Schneider group, who used a high-throughput screening campaign as a fruitful starting point.70 The authors screened a 147.000-member compound library in P19CL6 cells harboring a Nkx2.5 luciferase reporter construct as a surrogate marker for early cardiac progenitor cells. They found an array of small molecule scaffolds that increased the number of Nkx2.5+ cells, such as the sulfonylhydrazones and isoxazoles, with the latter being substantially followed-up as described below (see chapter 4).71, 72 The sulfonylhydrazones (e.g., Shz-1, 21, Fig. 4) appeared to act early in the time frame of cardiac differentiation since it activated the mesodermal marker


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Brachuyry T as well as early cardiogenic program genes (e.g., Nkx2.5, myocardin).70 Interestingly, mechanistic studies by Sadek et al. suggested that 21 unveils pro-cardiac activity independent from the key known cardiopoietic pathways (such as Wnt, BMP and FGF) but might be mediated by MAPK/ERKdependent signaling. Moreover, the authors showed that 21 was capable of enhancing the cardioregenerative potential of granule colony-stimulating factormobilized peripheral blood mononuclear cells which represent a viable strategy for cellular therapy after MI. Later, Quattrocelli et al. reported on the cardiac activity of 21 in murine iPSCs when given early (i.e., during embryoid body (EB) formation). They suggested that mechanistically 21-induced up-regulation of several miRNAs with miR-1 and miR-133 at early differentiation stages and miR208a at later stages.73 However, little follow-up work has been reported since the first disclosure of 21, probably due to its main activity on very early stages of cardiac differentiation. Another quite comprehensively characterized – yet rather promiscuous – cardiogenic small molecule is a natural product, the prenyl flavonoid icariin (22, Fig. 4), which was first reported in 2005 to stimulate cardiac differentiation from mESCs, although to a rather moderate extent.74 Several studies have been performed since to shed light on the possible mechanisms involved. These included

a

modulation

of

cell

cycling

and

p53-mediated

apoptosis,75


25


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p38MAPK/ERK1,2-mediated signaling and transcription factors NF-kB and AP1,76 involvement of reactive oxygen species77,

78

and effects on the ubiquitine-

proteasome system.79 An interesting chemical class of cardiogenic compounds was introduced by Oh and coworkers.80 Using a mESC cardiac differentiation assay, the authors identified a distinct class of peptidomimetics from a small screen of 200 compounds. They showed a clear dose-dependent effect and some structure activity relationships for specific β-turn mimetics, with CW209E (23, Fig. 4) exhibiting highest efficiency in both mESCs and human PSCs. However, 23 was exposed to differentiating cells over a long period of time, making it difficult to draw mechanistic conclusions. The authors excluded effects on Wnt signaling. However, considering that mimetics of β-turn secondary protein structures can serve as protein-protein interaction (PPI) inhibitors,81 23 may act via disruption of key PPIs relevant for cardiac differentiation from PSCs. Similarly, many other not yet well-characterized cardiogenic small molecules, 23 would have to be studied in greater detail regarding the relevant time-window of action as well as molecular mechanisms including target identification. Calcium signaling in cardiogenesis. The role of calcium signaling during cardiogenesis is also widely appreciated and several small molecule modulators have been described in this regard. It is known that calcium signals can control


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expression/activity of transcription factors in a concentration and spatiotemporaldependent fashion, which may vary during differentiation from PSCs towards CMs.82-85 Studies by Nguemo et al. revealed that the L-type calcium channels are already expressed in undifferentiated mESCs and that the calcium channel blocker nifedipine (24, Fig. 4) inhibits mesoderm formation in mESCs and mIPSCs.86 In contrast, another L-type calcium channel blocker of a different chemotype (i.e., verapamil, 25, Fig. 4) increased the number of contracting EBs and differentiation towards CMs. This finding is in accordance with earlier results from Sachinidis et al. who showed that 25 promotes up-regulation of early markers of the cardiac fate (e.g., Nkx2.5, GATA4) in mESCs.87 These conflicting observations may be explained by the distinct binding sites of verapamil and the DHP-class of calcium channel blockers. These binding sites may vary depending on cell lines and developmental stages, and therefore, may affect ability of being targeted by the respective inhibitors.86 Similarly, elegant studies by Wei et al. underlined a significant role for calcium signaling during cardiac differentiation, demonstrated via inhibitory effects of the CD38/cADPR/calcium axis.88 CD38 is a membrane-bound ADP-ribosyl cyclase that catalyzes the formation of cyclic adenosine diphosphoribose (cADPR) from nicotinamide adenine dinucleotide (NAD) which, in turn, mobilizes calcium from


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intracellular storage sites via ryanodine receptors (RyR). Using CZ-27 (26) and 8Br-cADPR (27) (i.e., distinct CD38 inhibitors, Fig. 4), cardiogenesis from mESCs could be efficiently stimulated, as demonstrated by the number of TnT+ cells (ca. 12-fold over vehicle control, when analyzed on day 9 of differentiation). Moreover, the authors showed that CD38/cADPR-mediated calcium mobilization antagonized cardiomyogenesis via inhibition of FGF-4/MAPK signaling. Finally, cinchona alkaloid-based as well as gefitinib-inspired small molecules were reported with cardiogenic activity in mESCs, although both compound classes displayed moderate efficacies and unknown modes of action.89, 90


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O N

OH

HN MeO

O N

N

18 (cardiogenol C)

S O

H N

N

N

OMe

N

N N H

HN HN

N

N

N H 19 (VUT-MK142)

20 (purmorphamine)

OH

HN

O

OMe

N

O

GluO

N

O

Br

OMan

21 (Shz-1)

OH

N

N N

F

O

CF3

O

O

F

22 (icariin)

F 23 (CW209E)

NO2 COOMe

MeOOC

MeO

CN

N

OMe Br

N H

MeO

OMe 25 (verapamil)

24 (nifedipine) O OH P O O

O

O HO

P O P O O O O

OH

N

O

O

N OH N

NH2 N

OH

HO F

OH

HO 27 (8-Br-cADPR) 26 (CZ-27)

Figure 4. Cardiomyogenic small molecules with miscellaneous and/or not-fully understood targets and mechanisms.


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2.3. Current challenges for chemically-defined production of PSC-derived CMs 2.3.1. Towards mature CMs Although much progress has been made to efficiently generate CMs from hPSCs under xeno-free, chemically-defined conditions, some challenges remain to be tackled. For one, the ability to obtain CMs at a degree of maturity that is comparable to adult CMs. Although it has been described by different investigators that long-term in vitro-culturing of PSC-derived CMs significantly improves their level of maturity, all hallmarks of mature, adult CMs cannot be met, i.e. morphological, calcium-handling, electrophysiological and metabolic properties.9194

To the best of our knowledge, the thyroid hormone triiodo-L-thyronine (T3, 28,

Fig. 5) belongs to the very few small molecule-based or -assisted approaches to improve processes of maturation in vitro.95 Notably, 3D culturing and engineering techniques will likely be key and indispensable to achieve the desired mature CMs, in part considering the necessity of electrical and physical cues only achieved in a highly organized 3D environment. A good example of functionally quite mature 3D CM constructs is the “engineered heart tissue (EHT)” techniques introduced by the Eschenhagen/Zimmermann groups.96-98 Along this line, a recent impressive study by Chong et al. demonstrated the feasibility of generating hESC-derived CMs in large quantities and to a sufficient


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degree of viability, purity and maturity for successful, functional integration into a primate heart after MI.99 However, methods to improve successful engraftment of transplanted cells, which also include small molecule-treatments, are beyond the scope of this review. Briefly, a significant issue is the poor survival of transplanted cells and several strategies have been suggested to overcome this obstacle. The above mentioned authors, for example, developed a “pro-survival cocktail” that consists of the small molecule pinacidil, the cell-permeable peptides cyclosporine A, Z-VAD-FMK and Bcl-XL BH4 in conjunction with the cytokine IGF-1 and Matrigel as a matrix.29,

100

From a chemical modality perspective, several small

molecules have been reported to improve cell survival for transplantation (see Xie et al. for an overview in the cardiac context).101 An interesting recent example (and novel approach) was introduced by the Uesugi group. They designed RGDS peptide-adhesamine hybrid molecules to protect cells from anoikis (i.e., a form of detachment-induced apoptosis) through interaction with specific integrins and syndecans.102 Notably, not only sufficient survival and retention of transplanted cells at the infarct site represent a current major obstacle, but also their fully functional integration into the myocardium. It has been suggested that an inadequate expression of several adherens junction proteins, ion channels and gap junction proteins (e.g., Connexin 43) in PSC-derived (immature) cardiomyocytes may impair functional electrical coupling and possibly lead to arrhythmia.9


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2.3.2. Controlling CM subtype specification On the other hand, current differentiation protocols deliver a mixed population of atrial, ventricular and pacemaker cells. Undefined, heterogeneous CM populations hamper the utility for several applications, such as from safety pharmacology or toxicology as well as for therapeutic transplantation. The identification of cellular and molecular mechanisms that control fate decisions for CM subtype specification, along with the development of respective small molecule tools would thus be a great leap forward in the field. Several hints in the literature suggest, in principle, the feasibility of achieving such chemical or pharmacological control. The first example was described by Kleger et al. who showed that activation of the calcium-activated potassium channel of small and intermediate conductance (SKCas, KCa channels) using 1-EBIO (29, Fig. 5) gave both a tremendous boost in cardiogenesis in mESCs (i.e., 59% versus 11% cTnT+ cells),103 and also significantly altered the quality of generated CMs, from 7% pacemaker-like, 7% atrial-like and 86% ventricular CMs to 58% pacemaker-like, 21% atrial and 21% ventricular CMs. Moreover, the authors suggested that the ion channel SK4 (KCa3.1) was mainly responsible and may mediate effects on differentiation and subtype specification by modulation of ERK signaling.


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The Laflamme lab pursued a similar idea and investigated whether neuregulin (NRG)-1β/ErbB signaling is involved in cardiac subtype specification in hESCs, facilitated by usage of a genetic label for nodal-type hESC-derived CMs.104 They found that the ratio of nodal- to working-type cells is indeed regulated by NRG1β/ErbB. Small molecule perturbation of the pathway, using the ErbB antagonist AG1478 (30, Fig. 5), enriched for pacemaker-like cell populations (from 21% to 52%, measured as action potential phenotype) while not changing overall CM yields. In addition, further work in distinct model systems implicated additional factors for cardiac specification such as retinoic acid (RA, 31, Fig. 5) and endothelin signaling, particularly in the context of nodal-type CM development.105, 106 In one of the first studies of RA signaling in the developing heart (mouse), the Rosenthal group revealed key regulatory functions for RA signaling in chamber development and specification.105 Furthermore, work by Keenan et al. (zebrafish) and Lin et al. (mouse) further underlined a regulatory function of RA signaling on cardiac progenitor cell populations, both indicating that retinoic acid restricts the pool of cardiac progenitors.107,

108

However, in the context of directed cardiac

differentiation of mESCs, Honda et al. made contrary observations that RA receptor agonism (PA024) increases CM yields while using antagonists (PA452) has detrimental effects on cardiogenesis.109 Most impressively, Zhang et al.


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demonstrated that atrial versus ventricular differentiation from hESCs can be significantly controlled by modulation of RA signaling. Adding the RA receptor antagonist BMS-189453 (32, Fig. 5) to differentiating cultures yielded mainly ventricular-like cells (83%, electrophysiological characterization) while employing 31 furnished highly atrial-like enriched CMs (94%).110

HO I

I

HN

COOH

N O

NH2

O

MeO

N H

I

N

MeO

N 30 (AG1478)

29 (1-EBIO)

28 (triiodo-L-thyronine, T3)

COOH

31 (retinoic acid, RA)

Cl

COOH

32 (BMS-189453)

Figure 5. Small molecules affecting cardiomyocyte subtype specification and maturation.

In addition to the above described factors and mechanisms Wnt signaling also appears to play a role in CM subtype specification. For instance, studies regarding the discovery of cardionogen-1 in zebrafish (see above) already suggested a CM subtype-specific role of Wnt/β-catenin since treatment with 3 (Fig. 3) only


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developed ventricular chambers.53 In accordance with these results, Karakikes et al. very recently reported on a chemically-defined protocol for the production of ventricular-like (100%) CMs from hPSCs.111 They claimed that the canonical Wnt inhibitor 3 played a key role for subtype specification when exposed to cells for 4 days, after cardiac mesoderm has already been formed. Similar results were obtained by investigators using 11 (Fig. 3) as a novel Wnt inhibitor under cytokine and xeno-free conditions.37 Under these conditions, ca. 60% ventricular-like cells with a minor atrial-like fraction and a lack of pacemaker-like cells could be generated. However, using the IWP-class of Wnt inhibitors for hPSC differentiation in defined B27-supplemented medium produced mainly atrial-like CMs (indicated as MLC2a+/TnT+ cells) from which a slow transition to MLC2v+ cells was observed during long-term culturing. Still, this protocol also generated CMs with mainly a ventricular-like action potential phenotype (91%) and a small portion of atrial-like phenotype (9%).31 Similar observations during long-time culturing of hPSC-derived CMs were made by others.35 From a mechanistic perspective, recent studies suggested that the timing and duration of BMP signaling during differentiation may affect CM subtype commitment of multipotent precursors.112 Moreover, Dorn et al. recently showed that Nkx2.5-mediated suppression of Islet-1 (Isl-1) is required for the development of ventricular CMs during differentiation from mESCs.113


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Taken together, great progress has been made in understanding CM specification, although a universally applicable, chemically-defined protocol for on-demand generation of all different types of CMs from PSCs is still lacking but would unquestionably be desirable.


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3. TRANSLATION OF IN VITRO TOOLS TO IN VIVO REGENERATION In the previous chapter a wide range of cardiogenic small molecules are described. Since most of these compounds were discovered from in vitro stem cellbased assays that served as surrogate systems to mimic processes of in vivo tissue development and regeneration, they are not naturally suited for in vivo applications. In fact, some examples already stressed the importance of a thorough biological characterization before these tool compounds can be used to prove a specific concept and/or utility in an in vivo setting. A well-characterized mode of action in a given phenotypic context is certainly desirable to allow for meaningful interpretation of in vivo effects. In principle, this is the core element for a good “chemical biology tool”.114 However, translation to in vivo applications also requires classic medicinal chemistry and PK aspects. For instance, the tool compounds need to exhibit sufficient solubility, permeability and metabolic stability beyond their biological (or pharmacodynamic) activity. Early examples for successful translation of an in vitro to an in vivo regenerative small molecule come from the hematopoietic system, with the marketed thrombopoetin mimetic eltrombopag.22 In the following chapter we illustrate the typical hit-to-lead optimization campaigns that have been reported to date for several possible in vivo regenerative candidates in the context of cardiac regeneration. Notably, this indication was


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mostly not the primary aim, but optimization was instead desired in a different disease state. 3.1. Wnt/β β-Catenin pathway inhibitors In directed differentiation protocols, modulation of canonical Wnt signaling currently represents the most powerful hub to improve cardiogenesis from uncommitted progenitor cells. Since these cell populations, or developmentally similar cells, are also present in the adult heart (see CPC chapter), small moleculemediated stimulation is a plausible strategy. Notably, the renewal and differentiation of specific progenitor cells (Isl1+ cells) has been shown to be controlled by Wnt/β-catenin.115 Therefore, Wnt/β-catenin inhibitors are valuable and promising candidates for an in vivo regenerative approach and in vivo-suitable lead candidates that target this signaling pathway are needed to demonstrate proofof-concept. Indeed, initial in vivo studies in MI models have been carried out with 6 and 8 (see Fig. 3).48, 51 Both compounds did not improve scar size but cardiac function was improved after infarction. However, it remains to be shown that a true regenerative response is addressed as the underlying mechanisms were not conclusive and differed for both compounds. Although 6 may bring some specific advantages (see below), there are alternative, more drug-like Wnt inhibitors available than the quite toxic compound 8. Such candidates are summarized in the following paragraphs.


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The first reported, systematic medicinal chemistry-driven optimization of a Wnt inhibitor as a cardiogenic agent is a comprehensive SAR study by Lanier et al. who used 3 as a starting point.52 Comparing a diverse set of Wnt inhibitors with distinct modes of action identified the IWR-class of compounds as the most promising molecules for promoting cardiogenesis from hESCs. The tankyrase inhibitor 4 and β-catenin/TCF4 inhibitor 5 were less potent than 3, and the porcupine inhibitor 2 was comparably potent in a dose-dependent fashion for hESC-cardiogenesis.36 In this setting, 8 turned out to be cytotoxic and could not be used in 293T and hES cells.52 From a SAR perspective, 3 was divided into three regions (A, B, C, Fig. 6a) which were systematically explored for potency in a Wnt reporter gene assay in 293T cells. An important aspect of these studies was also to design molecules that would be stable under physiological conditions. For instance, a possible retroDiels-Alder (chemical) degradation was supposed to be avoided by saturating the A-region of the compounds. The authors eventually improved potency of 3 for Wnt inhibition by up to 13-fold (35, IC50 = 2 nM). Importantly, a direct (i.e., dosedependent)

relationship

between

Wnt

inhibition

(in

293T

cells)

and

cardiomyogenesis in hESCs could be demonstrated. Optimized compounds 33, 34 and 35 turned out to be ca. 1.4-1.7-fold more efficient in hESC-cardiogenesis and required up to 30-fold lower doses which is indicative of their increased potency.


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Pharmaceutical industry is also pursuing hit-to-lead programs for Wnt inhibitors, with tankyrases (TNKS) as attractive drug targets. A Novartis research team reported on an optimization of 4 (Fig. 6b), largely employing structure-based methods.116 This campaign was primarily aimed at providing a more stable, druglike and potent tankyrase inhibitor for in vivo validation studies in a cancer context. Despite the identification of several alternate chemotypes of TNKS inhibitors to date, the authors reasoned that 4 still served as an excellent starting point considering its high cellular potency (72 nM), ligand efficiency (LE = 0.55) and lipophilic efficiency (LipE = 4.2). However, liabilities included a low selectivity over other PARP family members, unfavorable chemical and metabolic stability and low solubility. The dihydrothiopyrane ring in 4 was found as a liability for oxidative metabolism and was subsequently replaced with a dihydropyrane. Further optimization was then devoted to the B-region of the molecule, mainly inspired by structural aspects from distinct screening hits which eventually yielded the preclinical lead candidate NVP-TNKS656 (36) with good biochemical and cellular activity, along with excellent physicochemical and PK properties. In a similar, recently disclosed story, AstraZeneca research teams also reported on an optimization program for 4 (Fig. 6b).117 Inspired by in-house screening hits, and with the help of structure-based methods, they initially focused on the Bregion of the molecule and discovered highly potent biaryl derivatives. Since a


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designated goal was to develop a compound with improved physicochemical and PK properties, a series of “solvent groups” was explored as substituents of the distal pyridine. The cis-2,6-dimethylpiperazine moiety eventually provided a good balance between solubility, low intrinsic clearance, acceptable efflux, and good rat PK properties. Finally, the quinazoline core was replaced by a pyrrolopyrimidine to provide the superior candidate from this series, combining all desired features in 37. Taken together, both examples display attractive candidates suitable for advanced studies to probe TNKS-targeting Wnt inhibitors in vivo. In analogy to the above described inhibitors that perturb Wnt signaling at the axin level, porcupine inhibitors have also been subjected to optimization campaigns for advanced in vivo applications, although, such efforts have not yet been published in detail (Fig. 6c). However, several IWP analogs were reported for distinct applications, and these were designed to overcome distinct downsides that one would associate with the original IWP-like structures. For instance, 1 consists of a 2-mercaptoacetamide-based core unit that bridges two large aromatic bicycles, both of which also contain S-atoms. Since physicochemical and metabolic liabilities are not uncommon with such sulfur-rich structures, novel analogs now lack these structural groups. Both Wnt-C59 (38) and LGK974 (39) contain a “central” acetamide moiety which links two biaryl heterocycles and do not contain any sulfur groups. 38 has been disclosed in a patent (GNF, Genomics Institute of


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the Novartis Research Foundation),118 and reported as a more selective and potent Wnt inhibitor for directed cardiac differentiation from human PSCs (see Fig. 2).32, 118

Moreover, 38 already showed a promising profile in a mouse model for

mammary tumors as it was orally bioavailable, not toxic and effectively inhibited Wnt-driven tumor growth.119 Interestingly, 39 proved safe and effective in several in vivo cancer models and has now entered clinical trials (NCT01351103, Novartis).120


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a A

C

B

O O

O

N

O

HN R

N

O

HN O

R= N

N N

3

O

O

Me 35 33 34 (compd 10, 53AH) (compd 29) (compd 34)

Wnt IC50 = 0.026 µM Cardiogenesis = 100%*

Wnt IC50 = Cardiogenesis =

0.004 µM 136%

0.004 µM 176%

0.002 µM 145%

b O

Novartis A

B O

O S

NH N

N

NH

OMe

N O

N

Wnt IC50 = 0.0035 µM log D = 1.3 LipE = 7.0 (TNKS2) oral F = 32% (mouse)

O CF3

O

36 (NVP-TNKS656) NH

4 N Wnt IC50 = 0.078 µM log D = 4.1 LipE = 4.2

N Wnt IC50 = 0.0050 µM log D = 2.5 LipE = 5.7 (TNKS1) oral F = 12% (mouse) 18% (rat)

AstraZeneca N

N NH

37 (compd 25)

c O

N

N

N GNF / Novartis

N

S N

S

N H

O

S

N H 38 (Wnt-C59)

Ph N

O

N

1

O

N

N N

N H 39 (LGK974)


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Figure 6. Hit-to-lead optimization approaches for selected Wnt/β-Catenin inhibitors. a) The axin destabilizer 3 was optimized for cardiogenesis in hESCs; b) The tankyrase inhibitor 4 was optimized towards two distinct early lead candidates by Novartis and Astrazeneca; c) Development of IWP-type of porcupine inhibitors was not disclosed in detail. Wnt IC50 = cell-based, TCF-luciferase reporter system; Cardiogenesis = stimulation of cardiogenesis in hESCs, normalized to 3 (= 100% efficacy); LipE = log IC50(TNKS enzyme assay) – log D; oral F = bioavailability in % after oral administration. (Compound numbers in parentheses refer to those from their original articles)


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Targeting the Wnt pathway is an attractive strategy for numerous disease states and pursued particularly for the treatment of malignant diseases. However, modulating a key developmental signal transduction pathway that is involved in a wide range of (patho)physiological processes also raises safety concerns. Balancing the benefit-risk profile for a given disease (and patient) will certainly be required, especially for putative regenerative applications. Therefore, it would be desirable to develop means of targeting this pathway in the safest possible fashion. The Kahn group addressed this question by introducing an interesting concept, utilizing small molecule tools. They questioned whether exclusive Wnt antagonism or agonism is really needed and proposed that fixing aberrant Wnt signaling should consider its distinct roles in differentiation versus proliferation.121 In this regard, the histone acetyltransferases (HATs) CREB-binding protein (CBP) and p300 might provide a plausible leverage as distinct transcriptional coactivators of β-catenin. Although they interact with many proteins, and thus, can be seen as master regulators of transcription, they appear to exert defined roles in vitro and in vivo (see reviews by Kahn).121, 122 Kahn et al. found that CBP/β-catenin mediates a transcriptional program that promotes proliferation and the maintenance of pluripotency, whereas p300/β-catenin initiates differentiation.123 Strikingly, they later identified specific small molecule inhibitors for the respective β-catenin PPIs. 6 perturbs the CBP/β-catenin interaction thereby increasing p300/β-catenin-


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dependent transcription which was probed in several in vivo disease models for therapeutic relevance, including MI.51 Moreover, PRI-724 (structure not disclosed) appears to be a second generation CBP/β-catenin inhibitor developed by Prism Pharma and Eisei Pharmaceuticals and showed safety in pre-clinical toxicity studies. In fact, PRI-724 also demonstrated acceptable side-effects and toxicity in an open label phase Ia trial with 18 patients.122 Taken together, safely and effectively targeting Wnt signaling may be a matter of addressing subsets of Wnt target genes in a cell-context specific manner. 3.2. BMP pathway inhibitors Asides from its utility for directed cardiac differentiation, inhibition of BMP signaling is also an attractive strategy for the treatment of certain forms of anemia and hyperossification diseases, such as fibrodyplasia ossificans progressiva (“Münchmeyer syndrome”) which is caused by activating activin receptor-like kinase 2 (ALK2) mutations. As outlined above, the Hong lab showed that 15 represents a potent inhibitor of this BMP type-I receptor kinase (ALK2, BMPR-I, IC50 = 0.068 µM)124 but also had significant off-target activity on other kinases, including AMPK (IC50 = 0.23 µM), TGFβR-I (ALK5, IC50 = 10 µM) and VEGFR2 (KDR, IC50 = 0.22 µM).125 Moreover, 15 exhibited poor PK properties with a short plasma half-life after bolus i.p. administration (t1/2 = 10 min) which hampers utility for in vivo pharmacological applications. Initial SAR studies were mainly


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devoted to improve potency and plasma half-life.126 Cuny et al. examined the heterocyclic core and found that the N1 atom was essential for BMP inhibition whereas N4 could also be replaced by a carbon (Fig. 7). Importantly, they significantly improved potency in a cellular BMP assay (IC50 = 0.0045 µM) by replacing the distal piperidinylethoxy substituent with piperazine, yielding LDN193189 (40). This lead-like compound also exhibited a better plasma half-life (t1/2 = 1.6 h). However, a major issue for 40 remained the off-target activity which was even more pronounced for this compound against TGFβ signaling (ALK5, IC50 = 0.5 µM).


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Figure 7. SAR-guided optimization of the BMP inhibitor 15. BMP IC50 = cellbased Id1-promoter or BMP-responsive element luciferase reporter system; activin-like receptor kinase 2 and 3 (ALK2, ALK3) enzymatic inhibition assays (data from reference

124

); dorsalization EC100 = measure of BMP-mediated effects

on embryonic dorso-ventral axis formation in zebrafish, BMP inhibition induces a dorsal fate;125 plasma t1/2 = after i.p. injection in mice. In this regard, the development of 41 represented a big leap forward. Replacement of the piperazine by an iso-propyloxy substituent furnished the most


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selective small molecule BMP inhibitor. Although with losses in potency in the cellular (IC50 = ca. 0.1 µM) and kinase assay (ALK2, IC50 = 0.1 µM), the lack of inhibitory activity against AMPK, TGFβR-I and VEGFR-2 makes 41 a superior pharmacological tool compound. In the context of cardiac differentiation, 41 revealed that the non-BMP activity of 15 was likely responsible for a different cardiogenic profile in PSCs.58 Notably, recent endeavors in the Hong group led to the discovery of an even more selective derivative of 41, i.e. the ALK2 versus ALK3 selective inhibitor ML347 (42, >300-fold selectivity) which may serve as a useful compound in the future.124 However, for both 41 and 42, low solubilities and metabolic liabilities may be issues that need to be tackled for more advanced in vivo studies. 3.3. TGFβ β pathway inhibitors As indicated above, targeting TGFβ signaling in a selective fashion proved to be a useful tool to reveal a distinct role for TGFβ during specification of mesoderm towards cardiac mesoderm.59 From a medicinal chemistry and experimental pharmacology perspective, this story is far less developed than the above illustrated examples of Wnt and BMP inhibitors. However, similar approaches are underway in our group and key SAR-driven optimization of the annelated 1,4dihydropyridines, with 17 as the initial starting point, are summarized in Figure 8. A central obstacle has been the lack of knowledge regarding the cellular binding


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partner or concrete mechanism of TGFβ pathway inhibition via downregulation of the type II receptor. Hence, SAR studies rely on results from distinct cellular assays which typically aggravate devising a detailed SAR profile. The initial structure-activity information was quite steep with highly lipophilic derivatives as the most potent TGFβ inhibitors.64 However, since only the (+)-enantiomers of several DHPs were active and the first structural hints towards selectivity over the closely related Activin A pathway became clear, a specific mode of action was anticipated. A second SAR campaign was devoted to explore additional, more versatile substituents and functionalized derivatives and establish a 3D-QSAR model.127 The model was robust with a good predictive power, and therefore, reinforced the hypothesis that these compounds exert their TGFβ inhibitory activity through binding a distinct target. Unspecific activities that would derive from intrinsic properties of the compounds appear to play a negligible role, further underlined by a very poor correlation of TGFβ inhibition with their clogP values. Additional key findings included that the molecules tolerate derivatization with polar groups in the 2-position, paving the way for the design of more soluble, druglike compounds. However, chemical and metabolic liabilities typically associated with this compound class remain to be tackled before high-quality in vivo pharmacological studies can be performed.


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Figure 8. SAR-guided optimization of the novel TGFβ inhibitor 17.

In conclusion, this section established that several hit-to-lead programs have been realized or are ongoing for putative in vivo-regenerative small molecule agents. To date, mainly developmentally relevant signaling pathways are in the center of interest, and therefore most of these optimization campaigns originated from anticancer applications. Hence, the cardiac regenerative field can benefit and learn from oncology projects in both pharmaceutical and academic groups and should contemplate including such early lead candidates for advanced proof-of-concept studies in animals. Good examples in this regard would be the optimized 3-analogs 33 or 34, tankyrase inhibitors 36 or 37 and the porcupine inhibitors 38 or 39 (Fig. 6). In addition, beyond Wnt/β-catenin signaling, highly potent and selective BMP (e.g., 42) and TGFβ (e.g., ITD-1-class of compounds) inhibitors are available and provide valuable tools to probe efficacy in animal models of cardiac regeneration (Fig. 7,8). Finally, considering that even for exclusively ex vivo-


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intended cell culture protocols a given small molecule should meet a minimum degree of ‘drug-likeness’, many of the summarized compounds herein represent the current state-of-the-art for chemically-defined directed differentiation.


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4. ENDOGENOUS PROGENITOR CELLS FOR REGENERATION 4.1. Cardiac progenitor cells and their potential in cardiac repair An alternative strategy to generate new CMs following MI is through the proliferation and differentiation of endogenous multipotent cardiac stem and progenitor cells.128,

129

Cardiac progenitor cells (CPCs) help to maintain cardiac

homeostasis and once activated, such as following a MI, they have the ability to differentiate into all of the cardiac cells thought to be required to successfully regenerate an injured heart including CMs, endothelial cells and smooth muscle cells (Fig. 9a). In addition they have the potential to release paracrine factors, such as vascular endothelial growth factor-A (VEGF-A), which can act on other cardiac cells to stimulate repair.130 Hence, they are a promising cell population to understand and use for identifying molecules that can regenerate heart tissue in situ. A number of reputed progenitor populations have been identified in the adult heart.131-137 Different methods and markers have been used for identification and isolation of these populations, including specific stem cell selective culture systems and cell surface markers. The reported resident CPCs include c-kit+ CPCs, Sca1+ CPCs, side population CPCs and cardiosphere-derived cells. In addition, for the detection of early differentiation to the CM lineage, expression of transcription factors such as TBX5, NKX2.5, GATA4 and myocyte-specific enhancer factor 2C


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(MEF2C) are often used,138 where NKX2.5 emerges as a common marker across the various CPC populations.135, 139, 140 There is significant debate in the field surrounding the exact identity of these cell populations and how they are interrelated as well as the ability of all of these populations to form CMs. For example, there is controversy surrounding the cardiomyogenic activity of adult c-kit+ CPCs which exemplifies the challenges in this area.141, 142 In addition, due to the rarity of these cells in an adult mammalian heart, and as the rate that endogenous CPCs give rise to new CMs is thought to be very low, they are unlikely to be of physiological relevance.143 However, several studies have also shown that the replacement of CMs at the border zone of the infarct which are derived from progenitor and stem cells is greater than the number of new CMs being created from the division of pre-existing CMs.144, 145 Despite these debates it is clear that these cells exist and can play a role in cardiac repair. Hence, enhancing the population of CPCs and directing their differentiation towards CMs, endothelial cells and smooth muscle cells through pharmacological intervention could increase the regenerative capacity of the heart.146 To date, there is very limited understanding of the molecular mechanisms required for the proliferation and differentiation of CPCs and the key molecular signals required to control the specific cell fate of these cells. In addition, research in this area is hampered by the rarity and difficulty to isolate these cells. However,


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identification and understanding of the signaling pathways involved in the expansion and differentiation of CPCs to the specific cell types and the required timing of pharmacological intervention following an MI will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and raise the possibility that small molecule modulators of these signaling systems could redirect stem cell fate in the diseased adult heart leading to clinically effective in vivo cardiac regenerative therapies. 4.2.

Discovery

of

molecules

modulating

CPC

proliferation

and

differentiation 4.2.1. Hypothesis-based approaches Due to the limited knowledge of potential pathways and biological targets to modulate CPC proliferation and differentiation, different strategies have been taken to discover novel molecules and targets. These strategies include hypothesisbased approaches such as directly targeting known receptors present on CPCs, testing biological mechanisms which have been shown to provide a cardiac protective effect in vivo but where limited understanding of the underlying mechanisms exist and studying compounds with mechanisms which have been reported to enhance proliferation and/or differentiation of alternative stem or progenitor cell populations. Alternatively, unbiased screening approaches on relevant CPC populations can be employed to discover novel compounds and


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biological targets. This is a developing and new field but recent examples and potential future perspectives will be described. An example of a hypothesis-based approach includes that of the receptors for the growth factors insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF). IGF-1, a 72 kDa polypeptide binds to its tyrosine kinase receptor IGF-1R which is known to increase survival and proliferation of cardiac stem and progenitor cells,147 and HGF, a heterodimeric molecule consisting of a 69-kDa αchain and 34-kDa β-chain which signals via its tyrosine kinase receptor, c-Met, and known to have chemotactic properties,148 are both expressed on c-kit+ CPCs.149, 150 With this knowledge a combination of IGF-1 and HGF was administered as a single dose by an intracoronary injection to pigs after an acute MI.151 This treatment led, in a dose dependent manner, to significant activation and subsequent proliferation and differentiation of endogenous cardiac stem cells to form CMs and regenerate the microvasculature.


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Figure 9. Enhancing the activity of endogenous cardiac progenitor cells (CPCs) for heart regeneration. a) Different populations of CPCs exist and their activity enhanced in different ways. 1) Proliferation of resident CPCs will increase their number and lead to an enhanced population of CPCs which can differentiate to different cardiac cells. 2) Enhancing or directing the differentiation of CPCs toward cells required for cardiac regeneration: cardiomyocytes, endothelial cells and smooth muscle cells. 3) CPCs release paracrine factors which can act on other cardiac cells to stimulate repair. b) Chemical structures of small molecule CPC modulators.


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Agonists of cannabinoid receptor-type 2 (CB2) have been reported to modulate hematopoietic stem and progenitor cell mobilization and function as well as promoting proliferation of neural progenitor cells.152, 153 In addition, CB2 has been reported to be involved in cardioprotective effects on MI injury.154 However, the precise mechanism of action has not been described. Based on this knowledge, the selective CB2 agonist AM1241 (43) was tested in vivo to understand if this mechanism could promote endogenous regeneration and cardiac repair after MI via activation of CPCs (Fig. 9b).155, 156 43 was dosed by intraperitoneal (i.p.) injection for 7 days after MI and gave a significant improvement in cardiac function as measured by left ventricular ejection fraction and fractional shortening and a reduction in fibrosis. Interestingly, 43 treatment also increased the expression of ckit, a typical marker of CPCs, in the heart as well as increasing the number of CMs which had re-entered the cell cycle. Finally, treatment with 43 decreased the serum concentration of MDA, a biomarker of oxidative stress and the serum concentrations of the inflammatory cytokines TNF-α and IL-6. These results suggest that agonism of the CB2 receptor could cause proliferation of CPCs in vivo in the heart and contribute to cardiac regeneration. However, the potential effects observed with the CB2 agonist, 43, could be mediated via a variety of mechanisms or biological effects and it is not clear which is the driving factor in this case. It has been reported that inflammatory and growth cytokines can affect CPC activation


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and hence, modulation of the inflammatory response and oxidative stress as shown here could improve the post-MI microenvironment and benefit CPC activation and proliferation.151 Therefore, how much of the observed improvement is due to direct activation of the resident CPCs is not clear. It has been shown that increased cyclooxygenase (COX)-2 expression and increased levels of prostaglandin E2 (44, PGE2), a COX-2 downstream effector, in the heart induced by an MI provide a cardiac protective effect.157,

158

Hence, a

hypothesis was generated that the COX-2 pathway is important for endogenous stem/progenitor cell-driven CM replenishment and the effect of both COX-2 inhibitors and 44 was investigated (Fig. 9b).137 Treatment of mice with the panCOX pathway inhibitor indomethacin,159 or the selective COX-2 inhibitor celecoxib,160 significantly impaired CM replenishment in the border zone after an MI. When mice were treated with 44 (injected i.p. twice daily), a molecule which has been shown to be involved in stem cell mediated tissue regeneration,161,

162

increased numbers of CMs were observed at the border zone of the infarct. It was shown that 44 increased the expression of the CPC markers Sca-1 and Nkx2.5. Using a lineage tracing method this study showed that the Sca-1+ cells underwent differentiation to CMs. In addition, 44 elevated the number of M2 macrophage cells, suggesting a regulation of the inflammatory microenvironment after an MI. Finally, 44 was tested in aged mice (>18 months of age), which have a


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significantly reduced regenerative capacity, to test the impact of 44 treatment on CM regeneration under these challenging conditions. 44 treatment increased CM replacement as well as increased interleukin-10 (IL-10), an anti-inflammatory cytokine modulated by M2 macrophages.163,

164

Finally, additional mechanistic

studies revealed that the expression of the aging-associated marker gene transforming growth factor β-1 (TGFβ-1) declined in aged mice after 44 treatment. Subsequent treatment with RepSox (51, Fig. 9b),165 an inhibitor of TGF-β type 1 receptor kinase (ALK5) restored the replenishment of CMs suggesting that TGFβ1 activity may negatively regulate CM regeneration in aged hearts. In summary, this impressive study shows that 44 acts on Sca-1+ stem/progenitor cells, increases CM regeneration and acts on inflammatory cells to alter the M1 and M2 macrophage ratio, combining to give CM regeneration even in aged mice. However, the extent of which these results are due to the direct effect of 44 on the progenitor cells or an indirect effect via modulation of the inflammatory microenvironment of the infarct causing CPC differentiation will need further investigation. Further mechanistic studies suggest that the differentiation of cardiac Sca-1+ cells to CMs is mediated via the EP2 receptor. It will be interesting to see additional EP2 receptor agonists tested in mouse MI models as well as the more stable and longer-acting derivative of 44, 16,16-dimethyl-PGE2 which has shown


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superior activity in a different regenerative context (i.e., hematopoietic stem cells).166 Finally, the Wnt/β-catenin pathway modulator 7 (see Fig. 3), which selectively blocks the p300/β-catenin interaction, has been utilized for the ex vivo expansion of cardiac progenitor cells without differentiation and subsequently enhanced engraftment of multipotent progenitors into murine adult left ventricles.167, 168

4.2.2. Cell-based phenotypic screening approaches An alternative strategy to discover novel targets, pathways and chemical compounds in an unbiased manner is to screen large sets of compounds in an appropriate cell based phenotypic assay.169 Different cell systems can be used and all have pros and cons. The most physiologically relevant system would be human adult CPCs. However, the ability to isolate sufficient quantities of these cells prohibits compound screening of any size. From here, the cell population which can act as the best model system to mimic adult human CPCs and allow translation of endpoints in the phenotypic cellular assay to an in vivo setting needs to be selected. In addition, the number of cells, assay technology and measurement of relevant endpoints need to allow the testing of sufficient numbers of compounds to maximize the chances of discovering novel molecules and potential targets and biological pathways. An unbiased phenotypic screen can be performed with a set


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of tool compounds with known targets or with a diversity set. Screening of tool compounds is a cost efficient method to identify targets since fewer compounds need to be screened. As the potential targets are at least partially known, the results can be followed up by validating the targets using, for example, siRNA or CRISPR/Cas9 knockdown.170, 171 The drawback of this approach is that the chances of identifying novel drug targets are reduced. To increase the chances a diversity screen can be performed followed by medicinal chemistry optimization of the hit compounds and target deconvolution. A diversity screen should be large enough to cover a reasonable portion of chemical space. The hit compounds should then be optimized to reach a certain desired effect in the phenotypic assay. In addition, a role of the medicinal chemist is to secure that the compound set is supplemented with compounds which are known to modulate stem and progenitor cell proliferation and differentiation and that modulators of key signaling pathways are covered. For example, it will be of great interest to profile compounds described to differentiate either ESCs or iPSCs to CMs to understand how these compounds can affect cells at a later stage of development and which are already committed down the mesoderm lineage. For regenerative medicine approaches which monitor phenotypic events, high content biology is extremely useful as it is targetindependent and provides the opportunity to identify compounds that modulate novel targets or compounds that hit multiple pathways.169


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Interesting cell populations are emerging and screening assays being developed,172 which are giving opportunities to uncover novel biology and agents to discover and build understanding of new molecular pathways and targets important in CPC proliferation and differentiation. Traditionally, highly proliferative cell lines have been used. However, the relevance of these cells as a model system to translate to normal human cells and in vivo systems is questionable. Recent advances in iPSC technologies have the potential to produce hiPSC-derived CPCs to provide more relevant human disease modeling and assay readouts, on a large scale that can be used in screening campaigns.173 In addition to the primary assay the screening cascade needs to be built with the appropriate downstream assays and counter screens to ensure that the hit compounds give the required phenotypic effect and that the cells generated after compound treatment function as expected.

As an example of a counter screen, in looking for

compounds which proliferate CPCs it would be good to secure that they do not indiscriminately proliferate many different cell types, such as cardiac fibroblasts. CPCs can also be generated from hiPSC as they differentiate to CMs, permitting examination of relevant human biology.140, 167 One of the methods to isolate these cells is the expression of the cell surface receptor KDR and lack of CKIT, and these cells are similar to their adult CPC counterparts in that they can generate CMs, endothelial cells, and smooth muscle cells.135, 140, 167, 174 Based on this data,


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the emerging hiPSC-derived CPCs could be used as a relevant and robust phenotypic screening platform to identify small molecules capable of enhancing CPC proliferation and differentiation to the cardiac lineages. Significant numbers of these cells can be produced and are now developed and commercially available (via Cellular Dynamics International, who offer iCell® Cardiac Progenitor Cells, derived from hiPSCs). As described in the previous chapter chemical tools have been reported which differentiate either ESCs or iPSCs to CMs. Some of these compounds have been shown to enhance differentiation to CMs when administered at a later stage, once the cells are committed down the mesoderm lineage. For example, inhibitors of the Wnt signaling pathway such as 4, an inhibitor of tankyrase 1 and 2, have been shown to differentiate CPCs to CMs. In addition, inhibition of Activin/Nodal/TGFβ and BMP signaling after induction of cardiac mesoderm from hiPSC has been shown to promote CM differentiation.41 In this work, treatment with the combination of the Nodal inhibitor 16,175 and the BMP inhibitor 15 at day 3 of differentiation, generated CMs positive for the cardiac marker Troponin T. Furthermore, 48 has been demonstrated to promote cardiac differentiation of ESCs (Fig. 9b),25,

176, 177

and used in several reported differentiation protocols of both

ESCs and iPSCs.30,

41, 178

However, the exact role and underlying mechanism of

ascorbic acid in the cardiac differentiation of iPSCs was not clear. Treatment of


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hiPSCs during day 2-6 of differentiation, a period for the specification of CPCs, was a critical time for ascorbic acid to take effect.179 48 also increased the expression of cardiovascular but not mesodermal markers. Ultimately, 48 treatment led to a 30-fold increase in the yield of iPSC-derived CMs. These effects were attributed to an increase in the proliferation of CPCs by promoting collagen synthesis via the MEK-ERK1/2 pathway. This result was further supported as 48 increased the proliferation of CPCs isolated from iPSCs. These results describe compounds which give rise to in vitro CPC proliferation and differentiation providing encouragement that screening larger compound sets, including additional compounds modulating these specific biological pathways, against isolated CPC populations will provide novel hits and targets to modulate the proliferation and differentiation of these cells. Ultimately, molecules identified in these screens could be either used directly to enhance proliferation and differentiation of adult CPCs or optimized through medicinal chemistry efforts to improve properties such as increased potency or efficacy, or physicochemical or PK properties necessary for effective in vivo experiments. This will provide compounds which can be used to address and answer the debate around whether understanding and controlling the proliferation and differentiation of CPCs is sufficient for the in situ regeneration of heart tissue.


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4.3. Potential of modulating epicardium-derived cells for cardiac repair Another population of CPCs exists in the epicardium, the epithelial cell sheet which covers the heart, known as epicardium-derived cells (EPDCs).180,

181

Epicardial cells play a major role in heart development. A portion of these cells undergoes epithelial-to-mesenchymal transition (EMT) and form multipotent EPDCs, which migrate into the myocardium, and differentiate into mostly fibroblasts and smooth muscle cells as well as a smaller percentage of endothelial cells. It has also been shown that after myocardial injury epicardial cells can be reactivated to generate EPDCs which can contribute to cardiac regeneration. In addition to contributing to the formation of new cells it has been demonstrated that EPDCs secrete paracrine growth factors, such as VEGF-A, FGF2 and PDGF-CC, which have been shown to promote the growth of blood vessels. In one elegant study the media taken from cultured EPDCs was administered to mice after an MI and after 1 week gave a reduced infarct size and improved heart function.182 Hence, it appears that the adult epicardium is a dynamic tissue and there is great interest in discovering ways to modulate epicardial function as well as expanding the population of EPDCs or directing their differentiation to enhance cardiac repair. Understanding the biological mechanisms and signaling pathways involved in activation of the epicardium, or proliferation and differentiation of the activated EPDCs after MI will allow novel strategies for endogenous cardiac repair.183


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Several molecules have been reported to enhance the population of EPDCs or change their cellular fate upon differentiation. Treatment of mice with the peptide thymosin β4 has been shown to activate EPDCs and give an increase in the number of cells positive for the epicardia1 progenitor marker, Wilm’s tumor gene 1 (Wt1+).184, 185 In a further recent study administration of a modified RNA molecule encoding human VEGF-A increased the pool of Wt1+ EPDCs.186 In this study differentiation of these progenitor cells to endothelial cells was enhanced. A third example revealed the role of Wnt signaling and specifically the importance of βcatenin/p300-mediated transcription for the contribution of epicardial progenitor cells to the myocardium.51 In this study treatment of rat epicardial mesothelial cells with 6 (see Fig. 3), a small molecule blocker of the CBP/β-catenin interaction, promoted EMT including showing an increase in the EMT marker Vimentin. In addition, treatment of mouse primary EPDCs with 6 led to a decrease in the levels of Wt1 protein and a change in cell morphology implying an increase in cell differentiation. Finally, 6 was dosed subcutaneously to female rats at a dose of 50 mg/kg/day for 10 days, starting directly after MI, and showed a significant improvement in cardiac function as measured by left ventricle ejection fraction. Finally, Sadek et al. designed a high throughput screen (HTS) assay using a reporter luciferase gene inserted into the Nkx2.5 locus in genetically engineered P19CL6 embryonal carcinoma cells.70 A diversity chemical library of 147000


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compounds was screened leading to the discovery of a series of 3,5-disubstituted isoxazoles (Isx).71 Isx1 (45), a cyclopropyl-amide derivative, was selected for in vivo characterization and assessed using Nkx2.5-luc-BAC transgenic reporter mice, an equivalent animal model to the cells used in the original HTS (Fig. 9b). Once daily dosing (i.p.) for 1 week resulted in increased steady state luciferase activity in two tissues expressing Nkx2.5, the heart and the stomach. Significantly increased cell cycle activity was demonstrated in the heart, and in particular in the CM population, as shown by increased DNA synthesis and mitotic activity. 45 also dramatically altered the gene expression profile of Notch activated epicardiumderived cells (NEC), a cardiac resident progenitor cell population, towards CMlike precursors. 45 was subsequently administered immediately after induction of MI but effects on cardiac muscle genes was overridden by MI triggered activation of fibrosis genes. Administration of 45 3 days after MI resulted in significant improvement in cardiac ventricular function. However, this beneficial effect was not durable and disappeared completely 21 days after MI. Moreover, 45 treatment had no effect on early scar histology. The mode of action and molecular target of 45 was subsequently identified.72 Around 100 analogs of the Isx starting points were synthesized, enabling the discovery of more potent analogs such as 46 (Fig. 9b). The authors showed that Isx were implicated in the regulation of intracellular Ca2+ flux suggesting modulation


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of G(q)PCR signaling. A GPCR panel screen resulted in one hit, GPR68, an extracellular proton/pH sensing GPCR that was not known to have cardiac function and Isx act as direct agonists. Although 45 lacked in vivo efficacy, this study constituted an encouraging example of inducing cardiac resident progenitor cell proliferation and differentiation toward CMs using in vitro discovered small molecules as well as subsequent target deconvolution and validation. These studies highlight the possibilities to enhance the population and influence the differentiation of EPDCs and their potential benefits in cardiac repair. The search for new molecules and biological targets, both large and small, will be facilitated by applying innovative screening strategies such as the use of patientderived EPDCs for phenotypic screening purposes.187 To further enable the study of the role and function of the epicardium in cardiac repair a protocol for the generation of large numbers of iPSC-derived epicardial cells has been reported.42 The cells have been generated from hiPSC by stage specific activation of the BMP and Wnt signaling pathways. Access to these cells provides a further opportunity to study cells of the epicardial lineage with the potential to facilitate the identification of novel molecules and targets to modulate their activity.


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5. CARDIOMYOCYTE PROLIFERATION 5.1. Potential role of cardiomyocyte proliferation for heart regeneration Approaches to heart regeneration are not solely limited to using pluripotent or targeting multipotent (adult) stem cells but also include the stimulation of somatic cells. Proliferation of somatic cells is an important module in tissue homeostasis and also relevant to regeneration after injury, although strongly dependent on the respective organ. For example, significant proliferative activity is known for several tissues of the gut (e.g., intestine or liver) whereas the heart exhibits a very limited proliferative capacity. Nevertheless, it should be noted that it has been believed for a long time that CMs are exclusively post-mitotic and cannot re-enter the cell cycle. This dogma has since been confuted and a large body of evidence suggests a low but measurable renewal rate of adult CMs. Endogenous heart muscle cells therefore represent viable ‘targets’ to stimulate myocardial repair after injury. The questions are now which basic proliferative or renewal capacity we are dealing with and whether it can be enhanced by pharmacological means? The ‘renewal rate’ of CMs is characterized by the replacement of old cells without changes in their total number – a balance between apoptosis, proliferation of preexisting CMs and generation from progenitor cells. CM turnover rates have been estimated by different investigators and the numbers depend strongly on the


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techniques used.6, 8, 10 In 2009, an impressive study was reported by Bergmann et 14

al. who determined the annual turnover rate by retrospective quantification, taking advantage of an increase of

14

C isotope

C in the biosphere due to a

period of intense nuclear weapon testing during the Cold War.7 The authors showed that new CMs are generated at a rate of ca. 1-2% per year at 25 years of age which then drops to ca. 0.45% at the age of 75. In agreement with these findings, Mollova et al. determined an annual renewal rate of ca. 2% for 20 year old humans by fluorescence imaging-based quantification of the M-phase cell cycle marker phosphorylated histone H3.188 In rodents, quantifying the incorporation of 3H-labeled thymidine8,

189

or BrdU145 as well as

15

N metabolic

labeling techniques190 provided similar numbers ranging between ca. 1-4% turnover rates. Together, the majority of studies suggest very low turnover rates in the 1% range per year. Furthermore, this proliferative capacity drops during mammalian development as it is lost soon after birth and switches to hypertrophic growth. On the other hand it should be noted that the post-natal proliferative period appears to be longer in humans compared to rodents.188 Importantly, the above describes physiologic situations under homeostatic conditions. As far as CM proliferation is concerned, the key question is what happens after ischemic injury? Indeed, an increased proliferative activity of preexisting CMs has been observed in rodent models of MI,145, 190, 191 especially in the


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border zone after infarction.190 However, for ethical reasons it is difficult to obtain such mechanistic insights from studies in human failing hearts. But it is tempting to speculate that increased proliferation of pre-existing CMs observed after injury in lower mammals also takes place in humans. Since the vast majority of adult heart cells are terminally differentiated, the next question is what exactly speaks against those cells re-entering the cell cycle? It has been suggested that their complex contractile apparatus with its pronounced sarcomeric structuring, tight cell-cell contacts, bi- or polynucleated phenotypes and high number of mitochondria represent barriers for cell division.11, 192 Therefore, it appears likely that a certain degree of dedifferentiation would be required to allow for cell cycle entry. Indeed, such processes have been observed in zebrafish but also in rodents,190, 191, 193, 194 and growing evidence suggests that proliferation of pre-existing CMs happens through dedifferentiation under homeostatic and injury conditions. In fact, the Braun group has reported that mainly an intrinsic capacity of dedifferentiation provides CMs with the cellular plasticity needed to cope with tissue damage.11 In this context, they discovered increased levels of the cytokine oncostatin M (OSM) in heart tissue after infarction and revealed a key role of this protein for CM dedifferentiation and remodeling.195


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5.2. Discovery of small molecule stimulators of cardiomyocyte proliferation As outlined above, a large body of evidence suggest that CMs are not solely postmitotic but exhibit a proliferative activity under homeostatic and injury conditions and several mechanistic hypotheses exist to explain this capacity for renewal. Interestingly, several small molecule stimulators of CM proliferation have already been described, discovered from either hypothesis-driven approaches or screening campaigns. Among the first reports was a study by Engel et al. who discovered the p38MAPK inhibitor 9 (Fig. 10) as a pro-proliferative agent for adult rat CMs as p38 was shown to be a key negative regulator of mitosis in CMs.66 The same group also demonstrated that 9-treatment together with FGF1, a pro-angiogenic growth factor, enhanced cardiac regeneration after injury in vivo in rats.65 Another early and interesting finding was that chemical inhibition of GSK3β by the small molecule 13 promoted proliferation in neonatal and adult rat CMs by induction of their entry into the S-phase of cell cycle and up-regulation of several positive cell cycle modulators.196 In accordance with these studies, it has later been shown that genetic knockdown of GSK3β in vivo in mice leads to CM hyperproliferation (without hypertrophy) during embryonic development.197 Moreover, an inducible, CM-specific deletion of GSK3β in an adult mouse model of MI revealed that GSK3β knockdown protected against post-infarction


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remodeling and promoted CM proliferation.198 To further underline druggability of GSK3β inhibition (stimulation of Wnt/β-catenin signaling) for CM proliferation, it would be interesting to see more specific, potent and drug-like GSK3β inhibitors tested in vivo. Such small molecules are nowadays available (i.e., 49, Fig. 10)199 and should be favored over GSK3β inhibitors such as 13 or 14. Uosaki and colleagues used a high-content screening assay in mESC-derived CMs to search for small molecule stimulators of proliferation and also found that GSK3β (13) and p38MAPK (9) inhibitors were pro-proliferative.200 Moreover, the authors additionally discovered two Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors (52 (KN62), 53 (KN93)) and two ERK inhibitors (50 (SU1498), 51 (ZM336372)) that stimulated proliferation of mESC-derived CMs (Fig. 10). Notably, the authors elegantly validated all small molecules regarding developmental-stage specific modes of action. Along these lines, they found that only 13 and 9 were pro-proliferative in mESC-derived, E.9.5-mouse embryoderived, neonatal and adult CMs. The ERK inhibitors exhibited a similar profile but did not promote proliferation in adult rat CMs. The CaMKII inhibitor turned out be only effective on mESC- and E9.5 stage-derived CMs but neither on neonatal nor on adult CMs. These studies certainly stress the importance of considering the developmental state of CMs when performing screening campaigns for novel small molecule modulators.


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Finally, an in vivo screening approach in a transgenic zebrafish model to study CM proliferation was reported by Choi et al..201 They identified the IGF signaling agonist NBI-31772 (55) and the Smoothened agonist (i.e., Hedgehog signaling stimulator) SAG (54) which increased CM numbers during zebrafish embryo development (Fig. 10). Moreover, the authors could show that these compounds also stimulated CM proliferation during heart regeneration in adult zebrafish after injury.


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Figure 10. Small molecules and their targets that stimulate cardiomyocyte proliferation.

5.3. Potential strategies and targets for novel pro-proliferative agents In addition to the rational approaches and small molecules discovered by screening outlined above, approaches underpinned from basic understanding of


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cardiac biology will lead to the identification of additional targets, pathways and ultimately to novel chemical compounds. For example, several cell cycle checkpoints have been reported to stimulate proliferation from adult CMs as recently summarized by Senyo et al. and Zebrowski and Engel.192,

202

In this

regard, the homeodomain transcription factor Meis1 represents an interesting potential therapeutic target. The Sadek group revealed that Meis1 knockout in mice prolonged the postnatal, proliferative period of CMs and induced adult CMs to reenter the cell cycle without compromising heart function.203 It was further shown that Meis1 functions via transcriptional activation of the cyclin-dependent kinase inhibitors p15, p16 and most significantly p21. The Hippo pathway is a signaling cascade that has been heavily discussed to control CM proliferation. In vivo studies in developing mice showed that Yesassociated protein 1 (Yap1) –one of the targets of the Hippo signaling cascade – is important for the regulation of heart growth.204 Von Gise et al. used genetic gain and loss of Yap1 function to demonstrate that fetal activation of Yap1 promoted CM proliferation. Strikingly, they showed that Yap1 activation also increased postnatal CM cell cycle activity, although it seems that this Yap effect wanes with CM maturation.204, 205 The Olson group provided further details since Hippo/Yap exerts its growth-promoting activity via activation of IGF and Wnt signaling pathways.205,

206

Since most of the above outlined studies were performed in an


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embryonic development or postnatal context, it is interesting to see that Heallen et al. also showed that Hippo deficiency enhanced CM proliferation with functional recovery post-MI in adult mice.207 Considering that Hippo signaling is also discussed to contribute to the development of cancer, it will have to be shown whether targeting this pathway by pharmacological means is safe and effective in a regenerative context and may require selective targeting of the heart. In this regard, identifying direct Yap targets will bring this goal an important step closer as it may allow for selective, cellcontext specific perturbation of the pathway. Very recently, Lin and coworkers identified p110β, a catalytic subunit of PI3K, as a Yap effector and showed that it links Hippo-Yap and PI3K-Akt signaling to control CM proliferation.208 Moreover, several small molecule modulators of Hippo-Yap signaling have already been described as recently reviewed by Johnson, Halder and Santucci et al..209, 210 As discussed previously, neuregulin (NRG)-1β/ErbB signaling plays a significant role in cardiac development and specification of CM subtypes. NRG-1 binds to the ErbB4 receptor leading to its dimerization with the co-receptor ErbB2 and subsequent activation of various signaling cascades including the mitogenactivated protein kinase (MAPK) and PI3K/Akt pathways. Interestingly, endothelium-derived NRG-1 has also been implicated in CM proliferation where NRG-1 stimulated mononucleated (not binucleated) adult rat ventricular CMs to


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proliferate. These effects were supported further by mouse in vivo experiments supporting that administration of NRG-1 after MI promoted heart muscle regeneration.211 In summary, several small molecules with distinct targets and modes of action have been reported to exert pro-proliferative activity on CMs, and notably, a number of which have been discovered by unbiased phenotypic screening. In this regard, it should be noted that an important technical question is also how to address true pro-proliferative activity? Common proliferation markers include EdU (or BrdU), pHistone H3 or Ki67. However, these are surrogates for DNA synthesis or nuclear division but not for actual cytokinesis.5,

8, 212

Phenotypic readouts

addressing this question will be needed to capture true proliferative activity, especially for more mature CM states. Moreover, many of the described compounds were derived from studies in developing rodents which have a greater regenerative capacity compared with adult animals and where the biological mechanisms required to regenerate the adult heart may be different. It will have to be shown which and to what extent the key signals identified in developing rodents can be transferred and utilized for adult heart regeneration. However, several promising biological targets and signaling pathways have already been disclosed to play vital roles in adult CM proliferation, supporting the notion that pre-existing CMs can indeed be exploited as a source for regeneration of the myocardium after


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injury. Such targets eventually will provide rational starting points for (small molecule) pharmacological intervention.


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6. CARDIAC FIBROBLASTS, FIBROSIS & REPROGRAMMING

In this chapter we would like to stress that heart regeneration and the respective possible therapeutic interventions are not limited solely to de novo cardiogenesis from stem cells of diverse sources or from pre-existing CMs. Regeneration also includes acute and chronic processes of wound healing and tissue repair for which cardiac fibroblasts play a central role due to their high abundance, strategic location, phenotypic plasticity and ability to secrete cytokines and extracellular matrix (ECM) components.12 However, for detailed information on cardiac fibrosis, hypertrophy and remodeling the reader is referred to more specific reviews.12, 13, 213, 214 6.1. Role of cardiac fibroblasts and fibrosis in cardiac repair Following ischemic injury, lost CMs are replaced by fibrotic (scar) tissue during a highly concerted repair process. In principle, cardiac repair after MI can be divided into three stages in which fibroblasts are capable of exerting multiple important functions by phenotypically responding to changes in their microenvironment: 1) the “acute phase” of inflammation that deals with the massive necrotic CM loss, 2) the “proliferative phase”, in which fibroblasts become the dominant cell type in the infarct region and are “activated” to become myofibroblasts that express contractile proteins, such as α-SMA, and secrete ECM proteins to build a collagen-based matrix scaffold (i.e., scar tissue), 3) the ACS Paragon Plus Environment

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“maturation phase” of the scar tissue formed during the earlier phases which is marked by changes in collagen scaffold quality (i.e., matrix cross-linking) and a progressive loss of cellular components.12, 13 While repairing the damage caused by the infarct warrants structural integrity and function of the myocardium an excessive continuing fibrosis (i.e., deposition of ECM proteins) and scar maturation predispose the development of heart failure. Cross-linking of ECM proteins and the continuous activation of contractile proteins-expressing myofibroblasts contribute to the process of scar contraction, rendering a higher hemodynamic burden to the remaining heart muscle. These processes cause a number of changes, including alterations in the heart’s geometry and function that lead to chamber enlargement, hypertrophy and remodeling.213, 215, 216

Moreover, mounting evidence suggests that cardiac fibroblasts could also

contribute to arrhythmia following infarction.217 Therefore,

fibroblast-targeted,

anti-fibrotic

pharmacological

intervention

represents an important therapeutic handle to treat cardiac fibrosis, hypertrophy, remodeling, and consequently, heart failure to further improve the regenerative responses to ischemic injury post-MI. Aside from the infarcted myocardium, pathological persistence of myofibroblasts and excessive ECM deposition also appears in hypertension and diabetes leading to reduced ventricular function and heart failure. Such patients would similarly benefit from anti-fibrotic therapies.214


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Given their many key functions in the infarcted and remodeling heart, cardiac fibroblasts are attractive target cells for anti-fibrotic strategies. In this regard, therapeutic interference with the renin-angiotensin-aldosterone system (RAAS) is a well-recognized mechanism. Treatment with angiotensin converting enzyme (ACE) inhibitors and angiotensin II (AngII) receptor 1 (AT1) antagonists as a classic therapeutic regimen for hypertension and heart failure has a very good long-term outcome and reduces mortality.218 Importantly, RAAS inhibition also shows beneficial effects on heart failure development after MI and has been associated with reduced fibrosis.12, 218 Along these lines, the “protective arm” of RAS during tissue injury and regeneration should not go unmentioned which includes AT2 receptor-mediated cardioprotective and anti-fibrotic effects.219 Furthermore, TGFβ/Smad signaling plays a key role for fibroblast activation and proliferation after injury.220 In fact, TGFβ appears to act as a downstream effector of AngII and has been proposed as a “master switch” of cardiac fibrosis and remodeling.220 Therefore, TGFβ inhibition is an attractive therapeutic strategy but difficult to implement for this indication considering the various cell/tissuedependent (patho)physiological roles of this signaling pathway. Several biological and chemical agents are available as TGFβ/Smad inhibitors, from which many have already shown promising results in animal models of MI and cardiac fibrosis.221-224

However,

in-depth

knowledge

about

the

spatio-temporal


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characteristics of TGFβ signaling and critical pathway components in the failing heart is required to guarantee a safe and effective anti-fibrotic therapy via TGFβ inhibition.220 Another central contributor to the pathogenesis of chronic heart failure is endothelin-1 which exerts vasoconstrictive and mitogenic properties via interaction with its two receptor subtypes (e.g., ET receptor A and B). Endothelin-1 is also tightly linked to AngII and TGFβ signaling cascades and has been proposed as a downstream effector of TGFβ/AngII driving fibroblast activation and fibrosis.214, 225, 226

ET receptors have proven to be druggable targets as the dual ET-A/B

antagonist bosentan is approved for the treatment of pulmonary arterial hypertension. In addition, the AngII/TGFβ/ET-1 axis has also been linked to connective tissue growth factor (CTGF) and platelet-derived growth factor (PDGF) signaling.214 It will be interesting to see whether pharmacological agents that act beyond direct targeting of the RAAS also prove beneficial for cardiac fibrosis and remodeling. In summary, regeneration of heart muscle after a MI is also a matter of wound healing and how the quality of the formed scar tissue changes further during disease. This process has tremendous impact on re-establishing the functionality of the myocardium and determines to which extent pathological compensatory mechanisms such as hypertrophy and remodeling further advance the progression


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of heart failure. Since cardiac fibroblasts fill a central role in this process, they represent attractive targets for pharmacological perturbation. However, many important questions need to be addressed in order to make this approach safe and effective. For example, the actual source of fibroblasts during early and late cardiac wound healing should be identified to nominate a specific subset of fibroblasts that should be targeted. Further insights into molecular mechanisms of fibroblast activation and proliferation will possibly produce novel druggable targets to control excessive fibrosis that is counterproductive to functional heart regeneration. Basic research is also required to clarify whether the persistence of scar tissue and its change in quality over time is a process that can be pharmacologically manipulated to regress scarring.213 6.2. Towards therapeutic fibroblast reprogramming With the above said, reversing part of cardiac fibrosis (a degree will be needed to prevent cardiac rupture) and replacing scar tissue by functional CMs and vasculature would be the most desirable therapeutic aim to regenerate heart muscle after infarction. In the following we will discuss that this approach may indeed be feasible in the future. Spurred by milestone discoveries in reprogramming fibroblasts to iPSCs using specific transcription factors,15 miRNAs and small molecules,227 a next logical step has been to partially reprogram fibroblasts without transitioning through a


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pluripotent state. Following this idea, Efe et al. utilized viral transduction of the pluripotency genes Oct4, Sox2 and Klf4 (i.e., Yamanaka factors) together with a small molecule inhibitor of JAK/STAT signaling (i.e., JAK inhibitor I, JI1) to transdifferentiate murine fibroblasts to CMs without passing through a cardiac progenitor or pluripotent state.228 Additional research groups have subsequently demonstrated partial reprogramming of murine fibroblasts through a cardiac progenitor state by forced expression of only Oct4 together with a defined cocktail of small molecules.229 This cocktail comprised of the TGFβ/Smad inhibitor 16, the GSK3β inhibitor 14, the monoamino oxidase (MAO) inhibitor tranylcypromine (also “parnate”), here used for its inhibiting effect on the lysine-specific demethylase 1 (LSD1, KDM1) and the adenylyl cyclase activator forskolin. Moreover, transdifferentiation of human fibroblasts has also already been achieved by overexpression of Mesp1 and Ets2.230 As opposed to the above described transdifferentiation by partial reprogramming, direct reprogramming to CMs does not involve the induction of a dedifferentiated state. Building on early successes by Davis et al. who overexpressed MyoD, a master regulator of skeletal muscle formation, to generate muscle cells from fibroblasts,231 Ieda et al. showed that cardiac fibroblasts can be directly reprogrammed to CM-like cells by ectopic expression of genes related to cardiac development, i.e. Gata4, Mef2c and Tbx5 (i.e., GMT cocktail).232 The Olson and


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Srivastava groups later demonstrated successful reprogramming in vivo in mice, using the GMT cocktail or a GMT plus Hand2 cocktail of transcription factors, respectively.233, 234 Intriguingly, this treatment improved heart function implying its potential therapeutic value. Meanwhile, additional reprogramming protocols have been reported, also for human fibroblasts, as reviewed by Srivastava and Berry.235 Of note, Jayawardena and colleagues avoided the need for ectopic expression of specific transcription factors and instead successfully used a cocktail of cardiac miRNAs, which they discovered from a screen for cardiac reprogramming of murine fibroblasts.236 In addition, they showed a 10-fold increased reprogramming efficiency using the JI1 small molecular JAK/STAT inhibitor. Injection of this miRNA cocktail into ischemic myocardium also showed evidence of successful reprogramming in vivo, very recently further substantiated by the same group demonstrating functional improvement of the heart after treatment.237 Taken together, promising progress has been made in the field of reprogramming somatic cells to functional CMs. But as with other approaches to heart regeneration, many obstacles will have to be overcome before heart failure patients can possibly benefit from such a therapy. For example, current shortcomings are the relatively low efficiency of reprogramming, greater understanding of the functional and epigenetic characteristics of the newly created CMs, the problematic targeted delivery of reprogramming factors and with that the potential safety


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concerns. Safety regarding the use of viral vectors has been a general concern with many gene therapy approaches. However, it has recently been demonstrated that a solely small molecule-based reprogramming strategy is sufficient for generating iPSCs from mouse fibroblasts, even replacing Oct4 which was up to that point thought to be essential.238 Hou et al. conducted several screening campaigns to identify “chemical substitutes” for Oct4. They were eventually able to induce pluripotency from mouse fibroblasts with the six compounds-comprising small molecule cocktail “VC6TFZ” consisting of valproic acid, 14 (GSK3β inhibitor), 616452 (TGFβ inhibitor), tranylcypromine (LSD1 inhibitor), forskolin (adenylyl cyclase activator) and 3-deazaneplanocin A (DZNep, a histone methyltransferase inhibitor). This study encourages hope that a similar approach will also be possible for direct reprogramming. However, for these discoveries to develop into therapeutic strategies for human heart failure this work will need to be extended to human cardiac fibroblasts which is considered far more challenging and discovering new compounds to reduce the number of molecules required in the treatment cocktail. Furthermore, similar to transplantation approaches using PSCderived CMs, the quality and characterization of the induced CMs will be important to ensure non-arrythmogenic functional integration into the myocardial syncytium. In addition, the first attempts towards CM subtype-specific reprogramming have been undertaken and were reported recently.239


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7. CONCLUSIONS AND OUTLOOK

Restoring heart function after ischemic injury represents a major therapeutic aim as the available treatment options post-MI are stretched to the limit, largely dealing with the symptoms and not treating the cause, i.e. regenerating lost heart muscle cells. Different strategies are being performed for therapeutic endogenous regeneration of an adult heart and early examples and exciting data are emerging in both cellular and pre-clinical animal models. These include enhancing proliferation of pre-existing CMs, increasing the population of CPCs and stimulating their differentiation to the cell types required for cardiac repair including CMs and cells of the vasculature, or reprogramming cardiac fibroblasts to CMs. Significant questions remain including which of these strategies will be best-suited and sufficient to repair an injured adult heart, will the newly formed CMs successfully mature and electrically couple to the surrounding myocardium and can the emerging biological mechanisms and targets be safely targeted therapeutically? As we have summarized in this review, there are now a number of cardiomyogenic small molecules available, many of which are being widely used for in vitro applications to efficiently generate CMs from PSCs. Several of these address key cardiopoietic pathways that may also be of in vivo relevance to stimulate responses post-MI. An example is the Wnt/β-catenin signaling pathway


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which can be inhibited by small molecules to drive cardiac differentiation from CPCs. However, the pathway can also be stimulated to promote proliferation of existing adult CMs. This example highlights that a precise spatio-temporal administration of such compounds post-MI will be important to harness their therapeutic utility.

Hence, a greater understanding of the biological pathways and mechanisms driving in vivo relevant regenerative events needs to be developed to help address these obstacles. Knowledge of potential pathways is emerging from the reported actions of growth factors acting on cell surface receptors. In addition, in the absence of this hypothesis-based approach, phenotypic screening against physiologically relevant human cell types provides an unbiased approach to discovering novel molecules and biological targets, both unknown and known, which can provide the desired phenotype, such as proliferation of CMs or CPCs. It should be highlighted that the quality of the discovered hits from such efforts as “chemical tool compounds” is critical. A high degree of target and pathway selectivity would be desirable in order to draw meaningful mechanistic conclusions. As an example, we highlighted a finding by Laco et al. who reported that the cardiogenic activity of the p38MAPK inhibitor 9 on hPSCs was primarily due to interference with the well-known Wnt/β-catenin pathway.


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On the other hand, phenotypic screening approaches also enable the discovery of polypharmacology, where the actions of a compound on multiple targets may provide the phenotype of interest, or increased efficacy compared with modulating a single biological target. Once hits have been discovered which provide the phenotype of interest, the compounds will need to be improved through medicinal chemistry optimization and with an effective screening cascade in place to ensure the hit compounds are having the desired effect through the required biology and not just assay artifacts, or are more specific for the cell type of interest, for example, not proliferators of multiple cell types. Optimization of hit compounds will include improvements in potency and efficacy in the in vitro cellular systems and improvements in physicochemical and PK parameters such as clearance and half-life, to provide chemical probes suitable for in vivo testing in the most relevant animal models to translate to adult human disease. This is a key step in the discovery of novel small molecules to translate in vitro results to an in vivo setting to understand the impact of the molecule and molecular mechanism on cardiac repair and improvements in cardiac function. The field of regenerative medicine is often dealing with “Janus-faced” signaling pathways such as Wnt/β-catenin, TGFβ or Hippo-Yap. Perturbation of these pathways may be beneficial in a regenerative context but, on the other hand, are potentially pro-oncogenic. Consequently, modulation of these signaling pathways


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have been of high interest for anti-cancer drug development and a number of small molecule modulators have already undergone extensive hit-to-lead optimizations (e.g., TNKS inhibitors, porcupine inhibitors). Such compounds may therefore serve as good in vivo regenerative tools to explore applicability for heart regeneration and could accelerate transformative efforts. In this regard, we would like to stress again that concerns have been raised over the safety of regenerative therapies, including the potential for genetic instability, leading for instance to proliferative disorders including tumor formation and growth and the potential for undesirable epigenetic changes, which could have profound effects on cells. It will be important to understand the required dosing regimen, and effects of the compound on tissues outside of the heart will need careful monitoring. The development of methodologies to identify potential safety hazards, such as quantitative preclinical and clinical imaging technologies, will be fundamental to gain insight into the safety implications of stimulating repair of damaged tissue from endogenous stem cell sources. In addition, detailed gene expression profiling and bioinformatic analyses will be important to monitor gene expression patterns; this knowledge can also be utilized to improve drug candidates to accomplish desirable attributes, while eliminating or minimizing side effects. Small molecules have the potential to be delivered precisely to the heart and to be modified to provide varying PK profiles to stimulate and optimize the


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regenerative capacity of the heart. Building a thorough understanding of the PKPD relationship of the molecule will allow the establishment of the most effective dosing regimen that results in the required drug concentration in the heart and ultimately the most effective response. Lack of functional efficacy and even safety concerns may be a matter of timing of compound treatment. For example, administering a drug immediately after MI may enhance the efficacy of cardiac muscle regeneration and limit the fibrotic response. However, the strength of the heart wall needs to be sufficient to withstand the pressure of a beating heart and hence some fibrotic repair may be required so a suitable balance needs to be found. Moreover, effectively translating from animals to humans also requires a thorough understanding of signaling pathways across species. It is known that differences in manipulating signaling cascades can give rise to opposite effects, which may complicate translational understanding, so a thorough understanding will be required. Even within a given species, it could become difficult to transform certain findings from a model system to a regenerative interrogation. For instance, many studies have been performed in the developing heart (embryo) or within the early postnatal period. Of course, knowledge gained from these studies may potentially unravel targets and mechanisms that could subsequently be utilized to develop therapeutic modalities.4 However, examples from endeavors towards identifying pro-proliferative small molecules have demonstrated that many


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results cannot be easily translated to adult organisms. While GSK3β and p38MAPK inhibitors appeared to be pro-proliferative on adult CMs, similarly identified CaMKII and ERK inhibitors only promoted proliferation in very early, immature CMs.200 A strategy to reduce the potential of adverse events in non-cardiac tissues is to target the heart specifically and perhaps even selectively at the border zone (myocardial tissue of intermediate injury adjacent to the infarct) following a MI.240 Several techniques can be used, including direct intramyocardial injection during open-heart surgery or using catheter-based methods.241 However, for longer-term therapies, less invasive treatment will be necessary. Interesting examples are appearing, including bispecific conjugates and immunoliposomes, and novel techniques, such as biopolymer devices. More specifically, following a MI the injured heart expresses unique proteins that differentiate it from the surrounding healthy tissue. Conjugation of a molecule to a ligand for one of these proteins can then be used to target the molecule to the injured heart. One example is the stromal cell derived factor-1 (SDF1)-GPVI (a recombinant form of the soluble platelet collagen receptor glycoprotein VI) fusion protein, where the GPVI protein is the targeting molecule which delivers the chemokine SDF1 to the damaged vasculature contributing to its regeneration.242 These techniques will allow exciting opportunities for targeting small molecules to the heart after systemic


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administration. Many of these advances will be driven by highly productive collaborations between scientists in academia and industry. Only a few examples of small molecules impacting heart regeneration in vivo have been reported to date, such as 6, 9, 43 and 44. However, this is likely to increase in the coming years as more relevant biological targets are discovered and early in vitro active hit compounds are optimized to provide suitable in vivo properties. Once molecules have been discovered through phenotypic screening a major requirement and challenge will be to understand the target(s) which are being modulated. Target hypotheses can be generated through testing the compounds against panels of biological targets or based on knowledge of the targets that the small molecule, or related compounds, are known to modulate. The hypotheses can subsequently be tested by profiling diverse chemical structures that are known to modulate the same targets, building understanding of activity of the compound on the target in a biochemical assay and relating this to the effects in the cellular system or via knocking out or knocking in the suspected target, for example, using siRNA and CRISPR/Cas9 technologies to recapitulate the phenotypic effect. In addition, target deconvolution techniques such as chemoproteomics can also be used to pull down novel targets which then need subsequent confirmation.243 The Isx story (45) provides a good example of the discovery of novel compounds via in vitro screening and the discovery and


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validation of a potential novel target for cardiac regeneration. While target deconvolution is a developing area, it is clear that new techniques will be required to expedite this process and these methods will undoubtedly provide additional understanding of biological pathways and novel biological targets relevant to endogenous heart regeneration. As reported herein, there is evidence to suggest that different strategies to achieve therapeutic heart regeneration are feasible. Many questions and significant challenges remain and much needs to be learned. However, the regenerative strategies and approaches to discover new small molecule therapeutics discussed herein could ultimately solve the limited availability of heart donors and human CMs, and, most importantly, offer a major impact for the treatment of heart failure.


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ACKNOWLEDGEMENTS We would like to thank Dr. Qing-Dong Wang for helpful discussions and critical reading of the manuscript. This work was supported by the German Federal Ministry of Education and Research (BMBF) grant 131605.

ABBREVIATIONS USED ALK, activin A receptor-like kinase; BMP, bone morphogenetic protein; CB2, cannabinoid receptor-type 2; CBP, CREB-binding protein; CMs, cardiomyocytes ; COX-2, cyclooxygenase 2; CPCs, cardiac progenitor cells; CK, casein kinase; CVDs, cardiovascular diseases; EB, embryoid body; ECM, extracellular matrix; EHT, engineered heart tissue; EMT, epithelial-to-mesenchymal transition; EPDCs, epicardium-derived cells; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; GSK3β, glycogen synthase kinase 3-beta; HCS, highcontent screening; HEK cells, human embryonic kidney cells; hESCs, human embryonic stem cells; HGF, hepatocyte growth factor; hiPSCs, human induced pluripotent stem cells; IC50, half-maximal inhibitory concentration; HTS, highthroughput screening; IGF-1, insulin-like growth factor 1; LE, ligand efficiency; LipE, lipophilic efficiency; MI, myocardial infarction; PD, pharmacodynamics; PDGF, platelet-derived growth factor; PK, pharmacokinetic; p38MAPK, p38 mitogen-activated protein kinase; PPIs, protein-protein interaction inhibitors;


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PSCs, pluripotent stem cells; RA, retinoic acid; SAR, structure-activity relationship; TCF, T-cell factor (transcription factor); TGFβ, transforming growth factor beta; TNKS, tankyrase; VEGF, vascular endothelial growth factor.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]. Phone: +49-231-7557083 Biographies Alleyn Plowright obtained his PhD in organic chemistry with Professor Gerald Pattenden at the University of Nottingham in 1999, and continued with postdoctoral studies with Professor Andrew Myers at Harvard University in 2000– 2001. In 2002, he joined AstraZeneca UK as a medicinal chemist working in the Oncology and then Metabolic Disease research areas. He moved to AstraZeneca, Sweden, in 2006 and in 2012 became Senior Principal Scientist, Medicinal Chemistry in the Cardiovascular and Metabolic Diseases Innovative Medicines unit. His research interests include drug design, phenotypic screening and drug discovery for cardiovascular and metabolic diseases including regenerative medicine. Dennis Schade is a pharmacist by training (2005) and received his PhD in medicinal chemistry at the Christian-Albrechts-University of Kiel (2009). He


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stayed there shortly and went on for postdoctoral studies as a Research Fellow of the German Research Foundation (DFG) at the Sanford-Burnham Medical Research Institute and the Human BioMolecular Research Institute (2010-2011). Funded by the German Federal Ministry of Education and Research (BMBF), he became “Group Leader Medicinal Chemistry” at the Technical University Dortmund (2012) for independent research in the field of small molecule stem cell modulators. His current research is focused on the development of “Chemical Tools for Cardiac Regeneration” with a general interest in drug design and development, cardiovascular diseases, stem cells and regenerative medicine, phenotypic assays and pharmacokinetics.


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Medicinal Chemistry Approaches to Heart Regeneration - American

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