Inhibiting Hedgehog - An update on pharmacological compounds and

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Inhibiting Hedgehog - An update on pharmacological compounds and targeting strategies Ilya Galperin, Lukas Dempwolff, Wibke Diederich, and Matthias Lauth J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00188 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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Journal of Medicinal Chemistry

Inhibiting Hedgehog - An update on pharmacological compounds and targeting strategies

Ilya Galperin 1, Lukas Dempwolff 2, Wibke E. Diederich 2,3, and Matthias Lauth 1,*

1

Philipps University Marburg, Center for Tumor- and Immune Biology (ZTI), Hans-Meerwein-Str.

3, 35043 Marburg, Germany.

2

Philipps University Marburg, School of Pharmacy, Center for Tumor- and Immune Biology (ZTI),

Hans-Meerwein-Str. 3, 35043 Marburg, Germany.

3

Philipps University Marburg, Core Facility Medicinal Chemistry, Hans-Meerwein-Str. 3, 35043

Marburg, Germany

*

Corresponding author

1 ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Important steps in embryonic development are governed by the Hedgehog (Hh) signaling pathway, an evolutionary conserved signal transduction cascade. However, Hh activity is not only crucial during embryo formation, but is also involved in adult tissue repair and in several malignancies. Particularly due to its link to cancer, small molecule Hh pathway inhibitors have been developed and the first compounds have been approved for use in Hh-driven basal cell carcinoma. Almost all advanced Hh inhibitors target the critical signaling component Smoothened (SMO), but pre-clinical research has identified additional compounds which can block the Hh pathway along its entire signaling cascade, which, in light of emerging drug resistance occurring with SMO inhibitors, is of high importance. Herein we give an overview on currently known Hh pathway inhibitors, delineating their respective strengths and weaknesses and describing potential drug targeting strategies to interfere with Hh signaling in different cancer settings.


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An introduction to mammalian Hedgehog signaling The Hedgehog (Hh) pathway is a developmentally important signaling cascade which is conserved throughout evolution from flies to men 1, 2. Exact Hh concentrations as well as signaling duration times are crucial for the correct development, specification and patterning of many tissues in our body and failures to execute proper Hh signal transduction during development are associated with severe embryonic malformations, such as holoprosencephaly 3. In the adult mammalian organism, Hh signaling is mostly inactive, with the exception of its reactivation in tissue repair processes and in stem cell compartments. In agreement with the concept that processes in tumors closely resemble those in wound healing and stem cell biology, it might not come as a surprise that active Hh signaling can be found in numerous malignancies. While certain cancers such as basal cell carcinoma (BCC, a non-melanoma skin tumor) or the Hh-subtype of medulloblastoma (MB, a pediatric cancer affecting the cerebellum) can be caused by the sole overactivation of Hh, other tumor types utilize the Hh system for modification of certain aspects of their biology. This includes the regulation of migration and metastasis, the development of chemo- or radioresistance or the establishment and activation of an abundant tumor stroma. Given the fact that Hh signaling is to a large degree absent in healthy adult tissue but selectively reactivated in malignant and repair conditions makes this pathway an ideal target in drug development as side effects should be minor. Indeed, large efforts have been undertaken by both pharmaceutical industry as well as academia to delineate the mechanisms of Hh signal transduction and to identify small molecule regulators. To date, an impressive number of compounds modulating the Hh cascade has been described. Of these, two substances have been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency 3 ACS Paragon Plus Environment


(EMA) for their use in advanced BCC. This review aims at summarizing and describing a selection of Hh pathway inhibitors, allowing the reader to get an impression of currently available subclasses of inhibitory molecules and their potential applications in cancer therapy.

Conveying the Hh signal: The signal transduction cascade Hh signaling relies on secreted ligands which are sensed by either the releasing cell itself (autocrine) or by neighboring cells (paracrine) (Fig. 1 graphically outlines the signaling cascade). Mammalian genomes contain three Hh genes (Sonic Hh (SHH), Indian Hh (IHH) and Desert Hh (DHH)), which are functionally highly similar but are expressed in different spatiotemporal patterns between tissues. Hh ligands are unusual signaling molecules in that they are subject to both palmitoylation and cholesteroylation, rendering them highly lipophilic. These posttranslational modifications are required for full functionality and for extracellular transport of the Hh ligands. Specialized proteins such as members of the Dispatched family (DISP1 and DISP2) and SCUBE2 are needed to release these lipophilic proteins from secreting cells. On the receiving cell, Hh proteins bind to 12transmembrane (12-TM) receptors of the Patched family (PTCH1 and PTCH2, with PTCH1 being the more dominant). PTCH1 resides in a specialized organelle, the primary cilium (a solitary microtubule-held protrusion of the cell membrane), which serves as an antenna to sense environmental cues. The ciliary compartment is continuous with the cell membrane/cytoplasm, yet it still possesses a unique composition of soluble and membrane proteins which is distinct from the rest of the cell. One example is the selective distribution of different phosphoinositides between the ciliary membrane and the cellular plasma membrane, which is established by the 4 ACS Paragon Plus Environment

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ciliary enzyme inositol polyphosphate-5-phosphatase E (INPP5E) and which impacts on the ciliary presence of signaling proteins. Import and export of proteins into the cilia compartment can be mediated via lateral membrane diffusion or via microtubule-based transport proteins, such as through the kinesin KIF7 in case of several Hh signaling components. In the absence of Hh ligands, PTCH1 antagonizes the activation of Smoothened (SMO), a 7-TM signaling protein belonging to the G-protein-coupled receptor (GPCR) superfamily. Without ligands, SMO is blocked from entering the primary cilium, preventing the activation of the canonical Hh signaling cascade. Upon Hh ligand binding, the PTCH1 receptor is exported from the cilium allowing SMO to enter and to transmit the signal to downstream elements of the pathway. Hh ligand perception by PTCH1 is further modulated by co-receptors acting in a positive (CDO, BOC, GAS1) or negative (HHIP) manner on the signal transduction. A central question in recent years has been the molecular mechanism how PTCH1 actually inhibits SMO. Earlier data had shown that this functionally negative interaction is non-stoichiometric and probably requires some catalytic pumping function of PTCH1 across the membrane 4. In light of recent data, the most likely candidate molecule to be pumped by PTCH1 is cholesterol 5 or a modified version of cholesterol (e.g. oxysterols). The close association between Hh signaling and cholesterol is stunning as not only the Hh ligands themselves are cholesterol-modified, but also DISP and PTCH proteins contain so-called sterol-sensing domains. PTCH is able to remove cholesterol from the inner leaflet of cellular membranes and, in turn 5, SMO requires cholesterol binding to its cysteinerich domain (CRD) for full activity (see later), suggesting that regulated cholesterol transport is a central feature of coordinated PTCH/SMO signal transduction. The current hypothesis envisions



PTCH1 to pump away membrane inner leaflet-localized cholesterol, which would otherwise bind and activate SMO. The final effectors of canonical Hh signaling are the GLI transcription factors, of which there are three in mammals: GLI1, GLI2 and GLI3. GLI1 is a target gene of the pathway and cannot or only at very low levels be detected in unstimulated cells. GLI2 and GLI3 however are latent transcription factors and are regulated in their activity by Hh ligands. GLI3 is controlled through limited proteolysis by the proteasome. It is constantly synthesized as a full-length protein (GLI3FL) containing transcriptional activator and repressor domains. In the absence of ligands however, the activator domain is cleaved off rendering GLI3 a potent repressor (GLI3R) 6. In contrast, GLI2 is primarily regulated by complete and not by partial degradation as in the case of GLI3 7. The partial degradation process is triggered by PKA/GSK3β/CK1α-mediated phosphorylation and is supported by Suppressor-of-Fused (SUFU), a major negative regulator of the pathway. Besides PTCH1, SUFU is an important tumor suppressor affected by mutational inactivation in certain Hhdependent cancer types (e.g. MB) 8, 9. As protein kinase A (PKA)-mediated phosphorylation of GLI3 is central to Hh signal transduction, the pathway is significantly dependent on the levels of cAMP in/around the primary cilium. Without ligands, the ciliary compartment (and the basal body from which the cilium emanates) is enriched for cAMP generated through the action of the ciliary Gprotein-coupled receptor GPR161 10. Cyclic AMP can activate basal body-localized PKA, resulting in generation of GLI3R, which can enter the nucleus to block target gene expression. Hh ligands however, induce the SMO-dependent removal of GPR161 from the primary cilium, subsequently lowering the cAMP levels and PKA activity, thus stabilizing GLI3FL. Experimental small molecules


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(e.g. Forskolin, Eggmanone) affecting the production or degradation of cyclic AMP can therefore be used to modify Hh signaling 11. Full-length GLI3 and GLI2 can further be activated at the ciliary tip by a not well understood mechanism and can then promote target gene induction in the nucleus. Some target genes (e.g. GLI1, PTCH1, HHIP) serve to control the Hh pathway by feedback loops whereas others are more tissue- or cell type-specific (e.g. MYCN in MB).



Figure 1: Schematic representation of canonical Hh signaling in mammalian cells. Please refer to main text for details.


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It is important to note that the signal transduction cascade outlined above is considered the canonical Hh pathway as it was the first to be described and as it represents the most widely used mode of signal transduction during normal development. Nevertheless, studies mostly done in cancer settings have revealed that several non-canonical signal transduction mechanisms exist which cells are also utilizing 12, 13. These include, but are not limited to, SMO-dependent yet GLIindependent effects (or vice versa) or even primary cilia-independent effects of SHH 14-19.

The generally less-well understood non-canonical signaling modes often utilize a coupling of the GPCR SMO to G-proteins, in particular to the Gi family of small heterotrimeric G-proteins. While in some cell types the activation of Gi proteins contributes to GLI-mediated transcription, this is not the case in all cell types studied so far 20. However, G-protein stimulation mediates the effects of Hh ligands on Rho/Rac and the cytoskeleton, which is important for Hh-induced cell migration and endothelial tube formation

15, 21, 22.

G-protein-mediated non-canonical signaling by SMO is

much faster than GLI-mediated transcription and is also crucial for Hh regulation of cellular metabolism. Specifically, Hh promotes a shift towards increased glucose uptake and aerobic glycolysis (Warburg effect) by promoting a calcium influx, activation of CAMKK2 and subsequently of AMP-activated protein kinase (AMPK), the latter being a master energy sensor within the cell 19. The complexity and diversity of non-canonical signaling routes can be exemplified by these two

Gi-mediated cellular effects where Hh-mediated metabolic regulation requires the presence of a primary cilium whereas Rho/Rac activation does not 14, 15, 19, 23. In addition, G-protein-independent non-canonical mechanisms have also been explored, such as the SMAD-dependent activation of 9 ACS Paragon Plus Environment


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GLI2 transcription by TGFß without the need for upstream pathway elements such as PTCH1 or SMO 18. Furthermore, not only SMO is capable of eliciting non-canonical signaling, but also PTCH1. The Hh receptor has been proposed to function as a dependence receptor which initiates apoptosis as long as not being associated with its cognate ligand, in this case SHH. Overexpressed unliganded PTCH1 is able to recruit a pro-Caspase 9 complex by means of its intracellular C-terminal tail, followed by Caspase 3 activation and the induction of apoptosis

24, 25.

This GLI- and SMO-

independent process is blocked in the presence of Hh ligands binding to PTCH1. As small molecule inhibitors have been developed which target specific but distinct steps in the (canonical) Hh signaling cascade, the knowledge about the actual mode of signal transduction is crucial for the interpretation of the results obtained with the respective inhibitor. This also applies to the development of drug resistance, a common clinical problem with the use of e.g. SMO inhibitors. Mechanistically, this includes the outgrowth of SMO-mutant subclones which do not allow SMO antagonists to bind to their target or the activation of SMO-bypassing mechanisms such as downstream pathway activation, thereby phenocopying the outcome of some noncanonical mechanisms

26-28.

In general however, well-established non-canonical signaling

mechanisms (e.g. the coupling of SMO to Gi proteins) have so far not been considered for pharmacological targeting approaches but this might be worth doing so in the future.


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Small molecules targeting the Hh pathway In general, Hh pathway inhibitors can be broadly subdivided into a group of small molecules directly targeting a central Hh pathway component (e.g. SMO or GLI) and into another group not specifically targeting a core Hh pathway member, but Hh-modulating proteins (e.g. kinases, chromatin factors or cytoskeletal structures). The following text assembles examples from both categories but is instead structured based on the eventual target in the Hh pathway which will be affected by the therapeutic drug.

Compounds interfering with Hh ligand functions Many cancer types display an elevated level of Hh ligand expression with SHH being the most frequently encountered family member. A SHH-inactivating antibody (5E1) has been very informative in preclinical studies on the function of Hh ligands, but pharmacologically targeting the ligands directly has been difficult. Stanton and colleagues were the first to describe the discovery of a macrocyclic small molecule which they termed Robotnikinin ,1, (named after Dr. Robotnik, the opponent of the video game character Sonic the Hedgehog™) 29. A summarizing table encompassing all drug molecules discussed in this review can be found at the end of the article (table 1). 1 (Fig. 2) directly binds the biologically active N-terminal part of SHH with a Kd of 3.1 µM, blocks its function and inhibits Hh signaling in cell culture with an IC50 of 4 µM. Furthermore, it also proved active in a synthetic organotypic skin explant model system. So far, this experimental compound is the only known small molecule to directly inactivate Hh ligands.



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Another approach targeting Hh ligands aims at blocking the biologically important addition of palmitic acid to the Hh protein, a reaction catalyzed by the Hh acyl-transferase (HHAT, also known as Skinny Hedgehog (SKI1)). HHAT is inhibited by 5-acyl-6,7-dihydrothieno[3,2-c]pyridines

30-32

which were discovered through screening of 63,885 compounds from different, mostly commercially available compound libraries. Most published data is available on RU-SKI43, 2, (Fig. 2), one compound of this series 33, 34. 2 inhibits HHAT with an IC50 of 0.85 µM in vitro and interferes with tumor cell growth in cell culture and in vivo. An even more selective analog (RUSKI201 ,3, (Fig. 2)) with a lower IC50 (0.2 µM in vitro) has also been described 35. In the future, one might envision targeting additional Hh ligand-focused proteins such as for instance DISP1 36, against which there is currently no inhibitor available. In general, Hh ligandtargeting drugs could be very informative when deciphering the modes of Hh signaling or when drug resistance against other Hh inhibitors is a problem. It should be noted however that they might be ineffective in situations of pathway activation downstream of the ligands, as occurring for instance in the majority of BCC and MB cases with inactivating PTCH1 mutations 37-39.

O O O

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HN

O

O

O N

N H N

1 (Robotnikinin)

N

H N

S

S

Cl

O

2 (RU-SKI43)

3 (RU-SKI201)

Figure 2: Structural representation of compounds 1 (Robotnikinin), 2 (RU-SKI43), and 3 (RUSKI201) antagonizing Hh ligand function. 12 ACS Paragon Plus Environment

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The gold standard: SMO inhibitors The Smoothened protein represents a bottleneck in the canonical Hh signal transduction system and its genetic deletion or pharmacological blockade is associated with a complete abrogation of ligand-induced target gene expression. Intriguingly, many unbiased cellular screens for Hh pathway inhibitors revealed SMO (and not e.g. GLI) blockers, suggesting that SMO represents a well-suited structure for drug development approaches. This might be due to the fact that SMO belongs to the F-class of GPCRs and members of the GPCR-superfamily generally constitute very good drug targets due to structural reasons. Overall, SMO inhibitors currently represent the largest class of Hh pathway inhibitors and, at least in terms of their specificity and pharmacological properties, are also the most advanced compound class. On the molecular side, SMO contains two binding sites for small molecules: One in the extracellular cysteine-rich domain (CRD) and another one in the 7-transmembrane (7-TM) region. The CRD site possesses sterolbinding properties and cholesterol was in fact recently identified as an endogenous activator of SMO function 40, 41. In line with these findings, oxysterols (hydroxylated cholesterol variants which also occur endogenously) can modulate SMO in a positive or negative manner by binding to the CRD 42-44. In contrast, the heptahelical bundle (7-TM) site functions instead as the binding pocket for several synthetic SMO modulators which will be discussed below.

Cyclopamine and related compounds The first compound to be discovered as selective Hh inhibitor was the naturally occurring Cyclopamine (4, 11-deoxyjervine) from the plant Veratrum californicum (also known as false 13 ACS Paragon Plus Environment


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hellebore) 45, 46. 4 (Fig. 3) has a sterol backbone and can bind to both, the CRD as well as the 7-TM of SMO (Fig. 4). Paradoxically, it possesses opposing functions at these two sites: While it exerts antagonistic effects on Hh signal transduction through the 7-TM site, it displays partial agonistic effects through the CRD binding site. As a result, 4 induces the ciliary localization of SMO (which is controlled by the CRD and is considered a sign of active Hh signaling) but still blocks signal transduction and Hh target gene expression. 4 has never reached the clinical stage of drug development due to solubility issues, the acid sensitivity of the spirotetrahydrofuran ring system (E-ring), and, furthermore, unwanted side effects in mouse models. However, a series of Cyclopamine analogues was generated by semi-synthesis which showed improved pharmacological properties. Structural modification of the D-ring (homologue), reduction to the cis-decane (A-B-ring), as well as conversion of the 3-hydroxy-functionality into a methanesulfonamide finally resulted in IPI-926, 5, the most advanced compound (Saridegib, phase II) from Infinity Pharmaceuticals (Fig. 3b) 47, 48. 5 has been shown to induce significant tumor regression and extension of lifespan in a mouse model of medulloblastoma 49. 5 has also been the subject of clinical trials in several solid malignancies (phase I) and myelofibrosis (phase II).

H HN

HN H O

O H H

H H

H O O S N H

H

HO

H

H

H 5 (Saridegib)

4 (Cyclopamine)

Figure 3: Chemical structures of SMO antagonists 4 (Cyclopamine) and 5 (Saridegib).


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Figure 4: Crystallographically determined binding mode of 4 (Cyclopamine) to Smo (PDB code 4O9R) according to ref. 50 and visualized in PyMOL 0.99 (DeLano, W. L. (2002). The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA, USA). For clarity of presentation some residues of the 7TM have been omitted. The protein backbone is shown as cartoon (wheat), the ligand as well as important amino acids in stick representation, color coded by atom type, for the ligand: carbon, green; nitrogen, blue; oxygen, red; for the protein: carbon, wheat; nitrogen blue; oxygen, red. Potential hydrogen bonds are indicated by black dashes.

SMO-inhibiting compounds approved by the FDA Of all specific Hh inhibitors available, there are only two which have so far been approved by the FDA/EMA: Vismodegib (6, a.k.a. GDC-0449, marketing name Erivedge®, Fig. 3c) by Roche and Sonidegib (7, a.k.a. NVP-LDE-225, Erismodegib, marketing name Odomzo®, Fig. 3d) by Novartis. Both compounds are antagonizing the function of SMO by binding to the 7-TM site and both have been approved for use in locally advanced or recurrent/metastatic BCC. Numerous clinical trials 15 ACS Paragon Plus Environment


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(75 in case of 6; 40 in case of 7) with these drugs are currently underway. Chemically, 6 bears a N-(3-(2-pyridinyl)phenyl) benzamide core and is accessible via a four step synthesis starting from

2-chloro-5-nitroaniline 51. Screening of a combinatorial in-house library in a cell-based assay and further structural optimization of the initial screening hit, a biphenylcarboxamide, resulted in identification of 7 52. Despite having a low aqueous solubility and a moderate bioavailability, 6 proved very effective in a phase II BCC study (ERIVANCE) with an overall response rate (ORR) of 43 % in locally advanced BCC patients 53. Similarly, 7 demonstrated significant anti-tumor activity with comparable ORR to 6 in BCC studies

54.

Intriguingly, a Sonidegib/Docetaxel combination has recently shown

therapeutic benefit in patients with metastatic triple-negative breast cancer with one patient even demonstrating complete remission 55. The authors identified the Hh-activated tumor stroma as the prime target, which secondarily affected the mammary cancer stem cell compartment and led to improved chemotherapy sensitivity. Although the patient number was small in this study, the results were encouraging for the use of SMO antagonists in selected solid tumors other than BCC and MB. Another FDA-approved drug capable of inhibiting SMO and Hh signal transduction came from a repurposing approach and revealed the anti-fungal drug Itraconazole, 8,

56.

This orally

administered drug (Fig. 3) blocks the Hh pathway in reporter cells with an IC50 of 800 nM and has the major advantage that it is already clinically approved and shows only very little toxicity in patients. Competition experiments suggest that 8 has a distinct binding site on SMO compared to 4 (Cyclopamine) but the exact identity of this site has not been elucidated so far. 8 interferes with


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the import of SMO into the primary cilium and suppresses the Hh-dependent growth of allografted MB cells. A phase II trial has shown clinical efficacy in the treatment of BCC 57.

Additional pharmaceutical SMO antagonists Today, basically all major pharmaceutical companies have developed highly potent and selective SMO antagonists, which are involved in numerous clinical trials. These include BMS-833923 (9, a.k.a. XL-139, phase II, Fig. 5) from Bristol-Myers-Squibb or Pfizer’s PF-04449913 (10, a.k.a. Glasdegib, phase II, Fig. 5) and PF-5274857 (11, Fig. 5)

58, 59.

The latter compound is able to

efficiently penetrate the blood-brain barrier and is therefore a good candidate for use in brain malignancies such as MB or for the treatment of brain metastases 60. Cl

Cl

O

N

N H

O S O

6 (Vismodegib) F3CO

N

N

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N

Cl

8 (Itraconazole)

7 (Sonidegib)

CN Cl H N

N

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N O

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N H

N N N

NH H

HN N

9 (BMS-833923) 10 (Glasdegib)


S O O 11 (PF-5274857)

O


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Figure 5: Chemical structures of selected SMO antagonists 6 (Vismodegib), 7 (Sonidegib), 8 (Itraconazole), 9 (BMS-833923), 10 (Glasdegib), 11 (PF-5274857).

Other small molecule SMO inhibitors were developed by Novartis (NVP-LEQ506, 15, phase I, Fig. 6)

61

and by Eli Lilly (Taladegib, 16, a.k.a. LY-2940680, phase II, Fig. 7) 62, both compounds

being based on phthalazine as core structure. Based on the initial screening hit 12, first SARstudies led to the discovery of Anta XV, 13, which was further optimized regarding aqueous solubility, hERG activity as well as potency, rendering 14 and finally 15

61, 63.

Further structural

optimization of 16 resulted in 17 (originally termed Compound 23b), which proved to be 35-fold more potent than 16 and 23-fold more potent than 6 (Vismodegib) displaying an IC50 of 0.17 nM in a cellular Hh reporter assay 64. Another improved Taladegib derivative, 18 (originally termed Compound 21), could be developed following a structure-based approach (‘Dissection-thenEvolution’)

65.

Additional molecules were also described in the patent literature by various

pharmaceutical companies (reviewed e.g. in 66, 67).


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OH

H

12 initial screening hit

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N H

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OH

F N

N N

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HC

CH N

F

N N

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13 (Anta XV) solubility hERG potency

14

Figure 6: Optimization of the screening hit 12 leading to 15 (NVP-LEQ506).


N N

15 (NVP-LEQ506)


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F CF3 O HN N N N F

F3C O

HO

N

17

N N N

O

N N

F3C O

16 (Taladegib)

N

N N N N N 18

Figure 7: Further structural optimization of 16 resulting in compounds 17 and 18.


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Compounds binding to the CRD of SMO As mentioned above, SMO contains two major sites recognized by endogenous as well as synthetic small molecules. Besides the central 7-TM site region to which most synthetic compounds bind, SMO is controlled by the extracellular cysteine-rich domain (CRD). The CRD is crucial for high pathway activity and for ciliary translocation of SMO. Cholesterol has been identified as a potential endogenous molecule required for SMO activity and different hydroxylated variants of cholesterol (oxysterols, OHC) can modulate SMO activity through the CRD

41-43, 68.

Most oxysterols stimulate SMO activity, such as e.g. 20(S)-OHC (19, Fig. 8)

42.

However, an inhibitory sterol is also known (the Aza-sterol analog 22-NHC, 20, Fig. 8), which competes with 19 for CRD binding and suppresses SMO function

69.

The degree to which

oxysterols actually represent endogenous Hh modulators remains currently undefined and the micromolar concentrations required to impact SMO in combination with their high lipophility probably preclude their widespread use as pharmacological agents. Another class of SMO CRD-targeting compounds are Glucocorticoids 70 and stimulating as well as inhibitory Glucocorticoids have been identified 71. The latter include the two FDA-approved antiasthmatic drugs Budesonide (21, Fig. 8) and Ciclesonide, which interfere with SMO ciliary translocation and function also on Vismodegib-resistant mutant SMO. Although the crossreactivity with the Glucocorticoid receptor (GR) precludes a selective targeting of SMO at this point, it might lay ground for the future development of SMO-CRD specific glucocorticoids which have no or only little activity on the GR. Another reported compound which presumably binds to the SMO CRD and blocks Hh signaling as well as in vitro MB cell proliferation is Allo-1 (22, Fig. 8), bearing an imidazoline-2,4-dione 21 ACS Paragon Plus Environment


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(hydantoin) as core-structure 72. This compound was discovered in a cellular compound screen demonstrating that novel and synthetic molecules not binding to the 7-TM site can be identified. Where tested, CRD-binding antagonists were capable of inhibiting Vismodegib-resistant SMO because the responsible resistance-mediating mutations lie within the 7-TM site and it can be assumed that drug binding to the CRD site is not grossly affected. Hence, the development of clinically usable CRD-binding compounds is definitely of interest and a viable approach to combat SMO mutation-driven drug resistance.

Cl HO HO H

H H

NH

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HO

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20 (20-NHC)

21 (Budesonide)

22 (Allo-1)

Figure 8: Chemical structures of CRD-binding SMO modulators 19, 20, 21, and 22. All compounds except 19 function as antagonists.


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Some general considerations about SMO inhibitors SMO-selective drugs represent the most advanced group of Hh pathway inhibitors and have caused a lot of excitement when they first entered clinical trials, initially for treatment of BCC and MB. It was therefore very disappointing to see that acquired drug resistance is a major issue with this class of drugs 27. Approximately 30 % of patients with locally advanced BCC showed secondary resistance to SMO inhibitor therapy

73.

Mechanistically, this is brought about by selection of

tumor cell clones harboring point mutations in the drug binding pocket of SMO. The best studied example is a missense mutation of residue 473 of human SMO (D473H), which interferes with the binding of 6 (Vismodegib) to SMO. The side chain of Aspartate 473 is part of a hydrogen bond interaction network with two other residues (R400 and Q477) which keeps the SMO binding pocket in the correct shape and which stabilizes binding of 6 (Fig. 9). Mutation of D473 disrupts this interaction network, thereby compromising the binding pocket due to conformational changes affecting the orientation of the SMO transmembrane helices 74.



Figure 9: Crystallographically determined binding mode of 6 (Vismodegib) in Smo (PDB code 5L7I) according to ref.

75

and visualized in PyMOL 0.99 (DeLano, W. L. (2002). The PyMOL Molecular

Graphics System, DeLano Scientific, San Carlos, CA, USA). For clarity of presentation some residues of the 7TM have been omitted. The protein backbone is shown as cartoon (wheat), the ligand as well as important amino acids in stick representation, color coded by atom type, for the ligand: carbon, orange; nitrogen, blue; oxygen, red; chlorine, green; sulphur, yellow; for the protein: carbon, wheat; nitrogen, blue; oxygen, red. Potential hydrogen bonds are indicated by black dashes.

Various resistance-causing SMO point mutations have been identified so far and they can occur within the same tumor. Less frequent resistance mechanisms include the activation of signaling molecules downstream of SMO, such as genetic amplification of GLI2 or CCND1, activation of GLIenhancing SRF/MKL1 signaling or loss of SUFU

73, 76-78.

Furthermore, several kinases have been


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identified as druggable additional positive (e.g. atypical PKCι/λ, DYRK1B) and negative (e.g. CK1α) modulators downstream of SMO 79-82. The occurrence of drug resistance necessitates the identification of additional SMO antagonists capable of retaining their activity even on mutant SMO or of Hh pathway inhibitors which block signaling at another step in the cascade, preferentially inhibiting a downstream element. Alternatively, targeting pathways critical in drug-resistant cells such as PI3K-AKT signaling has helped to overcome resistance against SMO inhibitors in preclinical models 26, 83. Moreover, an activated RAS pathway was reported to suppress Hh signaling in MB, rendering tumor cells Hh inhibitor-independent and switching cellular dependencies away from Hh and towards the RAS signaling system 84, 85. Along these lines, resistance-associated cellular transdifferentiation in BCC has been shown to be associated with altered Wnt and Notch pathways

86, 87.

Therefore,

modulating additional pathways such as PI3K, RAS, Wnt or Notch might alleviate Hh inhibitor resistance in certain settings.

Nevertheless, screening approaches have also revealed SMO inhibitors which could overcome drug resistance and still function on Vismodegib-resistant mutant SMO. Examples include the preclinical substances 23 (originally termed Compound 5, Fig. 10) 76, Chalcone-12 (24, Fig. 10) 88, Hh78 (25, Fig. 10) 89, MRT-92 (26, Fig. 10) 90, 15 (NVP-LEQ506, Fig.6) 61, the clinically tested TAK441 (27, Takeda Pharmaceutical, Fig. 10) 91 and the phthalazine derivative 16 (Taladegib, Fig. 7). With respect to pharmacological activity, the acylguanidine 26 is noteworthy as it displayed a very low nanomolar activity in cultured Hh-dependent cerebellar granule cells, making it one the most potent SMO inhibitors known. The high potency of this drug candidate is probably caused by its 25 ACS Paragon Plus Environment


Page 26 of 70

bridging of two distinct subsites within the 7-TM region of SMO. However, despite the fact that the compounds mentioned in this section can compensate for other SMO targeting drugs such as 6 (Vismodegib) in certain mutational settings, it remains open whether additional cancer mutations exist which would interfere with binding of these compounds as well.

H N

Cl

Cl

Cl N

O HN

N

O

O

O

O

O

N

O

N

O

N

24 (Chalcone-12)

N

N

HN

N H

25 (Hh78)

23

OH O

NH

O O

N H

N

N H

O

O O 26 (MRT-92)

N O

N

N H

HN O

O

O CF3 27 (TAK-411)

Figure 10: Examples of SMO inhibitors reported to retain activity against Vismodegib-resistant SMO.

In clinical trials, common adverse side effects encountered with SMO inhibitors were gastrointestinal disorders (nausea, vomiting and diarrhea), muscle spasms, fatigue, hair loss, and dysgeusia (taste sense distortion). At least some of these effects are likely to be on-target adverse effects caused by a systemic Hh blockade. For instance, Hh activity is known to play significant 26 ACS Paragon Plus Environment

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roles in hair follicle biology and in taste buds 92-94. Pregnancy represents a contraindication of Hh inhibitor use due to the widespread involvement of this pathway in embryogenesis. Moreover, certain postnatal processes such as bone growth are also affected by Hh and it was recommended that SMO antagonists may be used only in skeletally mature patients, which is of high importance in pediatric cancers such as SHH-driven MB 95-97. An unexpected mechanism seems to link the use of SMO inhibitors and the occurrence of muscle spasms and it was attributed to a selective partial agonism demonstrated for some drug molecules. Vismodegib, Cyclopamine and others allow SMO to enrich at the ciliary base, but prevent its trafficking to the tip. As a result, SMO is able to couple to Gi proteins at the base of cilia and to induce non-canonical signaling, leading to calcium influx and muscle contractions. Co-administration of L-type calcium channel blockers might solve this unforeseen problem. Despite the occurrence of acquired drug resistance, targeting Hh signaling in BCC and MB (both of which frequently harbor inactivating mutations in PTCH1, rendering them Hh pathway dependent) is per se a straight forward and reasonable approach. It was however quite unexpected to also observe a clinical failure of SMO antagonists in several additional types of solid cancer (for an excellent discussion on the reasons for failure see also 98). In one particular example, a pancreatic cancer trial had to be prematurely terminated because the cohort receiving the Hh inhibitor Saridegib displayed a worse survival compared to the control group (web page info from Infinity Pharmaceuticals, www.infi.com, Jan 2012). Other clinical trials were not that alarming but also could not manifest a significant benefit of using SMO antagonists in combination with conventional chemotherapy in pancreatic ductal adenocarcinoma (PDAC) 99, 100. What was going wrong? It turned out that pharmacological SMO inhibition targets primarily stromal Hh 27 ACS Paragon Plus Environment


Page 28 of 70

signaling in PDAC, which is elicited by tumor cell-derived secretion of Hh ligands

85, 101-103.

This

stromal cell population is activated by Hh signaling and, against previous expectations, exerts tumor-suppressive functions in PDAC, at least in mouse models of the disease

104-106.

The

depletion of stromal elements by Hh inhibition surprisingly favored tumor growth by enhancing tumor angiogenesis and infiltration with tumor-promoting immune-suppressive T-regulatory cells (Tregs). A tumor-restrictive role of Hh-activated stroma is not only limited to pancreatic cancer, but has also been reported in preclinical models of colon, prostate and lung cancer 107-109. Thus, it remains to be seen whether Hh inhibitors are suited for treatment in malignancies other than BCC and MB, tumor types which are known to be Hh-dependent. It could very well be that in other cancers, Hh pathway blockers will be used in the future only as combination therapy with drugs such as immune checkpoint inhibitors or anti-angiogenic therapies. These findings underscore the importance of knowing the exact modalities of Hh signal transmission (autocrine vs. paracrine, canonical vs. non-canonical) in a given tumor type in order to be able to appropriately design the best targeting strategy. As many epithelial tumors utilize both, canonical (mostly in the tumor stroma) and non-canonical (mostly in the tumor cells) modes of signal transmission, this task can be demanding and might explain the current lack of clear effectiveness of SMO antagonists in clinical trials. Finally, from a mechanistic point of view it should be mentioned that a nuclear pool of SMO has been described recently which also impacted on Hh target gene expression

110.

It is however

currently not fully clear what the exact physiological contribution of nuclear versus ciliary SMO is and to which extent available SMO inhibitors would target nuclear SMO in different cell types and cancer settings. Similar issues also apply to non-ciliary pools of SMO, which can function in a non28 ACS Paragon Plus Environment

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canonical manner to impact on cell migration 14 and which have even been described as being competent for some degree of signaling 111.

Strategies and compounds indirectly affecting SMO The Smoothened protein is a bottleneck in canonical Hh signaling and targeting SMO is therefore a logical approach. In light of the occurrence of drug resistance however, other targeting strategies might be envisioned. One such approach focuses on the primary cilium as a target structure. Regulated ciliary import and export are crucial for Hh signaling and SMO translocation is a key step in its signal transduction. Hence, compounds interfering with either ciliogenesis or ciliary trafficking will indirectly also affect canonical Hh signaling. One example of such a compound is the imidazopyridine derivate JK184 (28, Fig. 11)

112-114,

which is capable of

depolymerizing microtubules and therefore blocks ciliogenesis and Hh signaling. Other examples include compounds CA1 (29, Fig. 11) and CA2 which abrogate cilia formation and were discovered together with the preclinical compound series SA1 (30, Fig. 11) to SA7 which preserved cilia structure, but bound to SMO and interfered with ciliary SMO import 115. Many direct SMO binders interfere with cilia translocation. However, additional compounds inhibiting the SMO transport protein GPCR-associated sorting protein 2 (GPRASP2) have also been identified

116.

GPRASP2

binds to the ciliary targeting motif in SMO which is responsible for cilia translocation and the identified small molecules interrupt this protein-protein interaction, suppressing SMO transport. Intriguingly, many of these compounds are known for other mechanisms, such as targeting receptors for dopamine (D2: Bromopride, Droperidol), histamine (H1: Tripelennamin, Cyclizine), serotonin (5-HT3: Palonosetron), or opiods (µ: Tramadol) 116. It therefore comes as a surprise that 29 ACS Paragon Plus Environment


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these structurally diverse molecules all bind to the SMO-GPRASP2 interface and block SMO transport. However, this discovery might allow for a future development of SMO transportselective compounds not affecting other receptor systems.

O H N

S N N

28 (JK184)

O

H N N

N H N

O

S

N S

N

Cl

N

N H

O

F

29 (CA1)

30 (SA1)

Figure 11: Examples of compounds indirectly affecting Hh signaling by interference with microtubules, primary cilia formation and/or SMO transport.

Another class of indirectly Hh suppressing drug molecules are the cholesterol-lowering statins (3Hydroxy-3-methylglutaryl-coenzyme-A reductase (HMG-CoA) inhibitors). Given that cholesterol is crucial for Hh signaling at several levels (Hh ligands, SMO function), interfering with cellular cholesterol availability does impact on Hh signaling 117-121. As such, statins might be used clinically to synergize with selective Hh pathway inhibitors 122.

Circumventing SMO: Downstream inhibition of Hh signaling In vitro studies on cultured cancer cells derived from different tumor types (e.g. colorectal, pancreatic, prostate, lung) have shown that, despite the fact that these cells frequently expressed the Hh target gene GLI1 (and therefore are considered to have active Hh signaling), they did not respond to either recombinant SHH protein or, even more importantly, to inactivating anti-Hh 30 ACS Paragon Plus Environment

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antibody or synthetic SMO antagonists 101, 123. This suggested that the tumor cells had lost their ability to respond to Hh ligands and/or that the intracellular signal transduction cascade was altered in a way precluding signal conveyance. Indeed, subsequent work revealed that several mechanisms in tumor cells block canonical Hh signaling, yet still allowing for the activation of GLI transcription factors by non-canonical means

80, 84, 85.

As one example, primary cilia are lost in

many epithelial tumor cells, such as e.g. in pancreatic, renal, prostate and breast cancer 124-127, interfering with proper reception and transmission of Hh signals. In contrast, cancer cells have activated a number of oncogenes promoting signaling elements which lie downstream of SMO. Most notably, the GLI1 and GLI2 transcription factors function to integrate many non-canonical inputs elicited by e.g. RAS, AKT, aPKC-ι/λ, DYRK1B, TGF-β, MRTF-A (MKL1)

18, 77-81, 84, 85, 103, 128.

These alternative and ligand-independent activation modes bypass upstream signaling components like PTCH1 or SMO and render inhibitors non-effective, at least when the aim is reduction of GLI-mediated transcription. Targeting GLI transcription factors can be achieved by blocking stimulating kinases with small molecule kinase inhibitors and a large plethora of potential candidates has been identified, but should not be the topic of this review.

The first small molecule antagonists of GLI function to be described were GANT58 (GLI-Antagonist 58, 31) (Fig. 8) and GANT61 (32, Fig. 8) which inhibit GLI1 and GLI2 with an IC50 of approx. 5 µM in cellular assays 129. These inhibitors function in in vitro and in vivo situations of elevated GLI1 expression which are insensitive to upstream pathway inhibition, demonstrating their use in cancer and in Hh-driven organ fibrosis 130. Due to the fact that by now 32 has become a widely used tool in the field, most of the available data are on this compound. 32 directly binds to GLI1/2 31 ACS Paragon Plus Environment


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between the zinc fingers 2 and 3, preventing DNA binding 131. Chemically, 32 is a hydrolysis-labile structure and as core the diamine moiety has been identified which is responsible for the GLIinhibitory effects

118, 132.

As the pharmacodynamics of 32 necessitate the use of relatively high

compound concentrations, numerous attempts were undertaken to identify additional GLI antagonists with sub-micromolar efficacy. Unfortunately however, to date none of these identified pre-clinical small molecules displayed a huge step forward in terms of potency and selectivity. This might be due to the fact that transcription factors typically possess rather shallow structures which serve as interfaces for protein-protein or protein-DNA interactions. Such structures are, in contrast to e.g. ATP-binding pockets in kinases, difficult to target with small molecules. However, virtual screening and structural docking revealed a GLI-associating molecule, the prenylated isoflavone Glabrescione B (GlaB) (33, Fig. 12), which binds to GLI1’s zinc finger domain and thereby interferes with GLI-DNA binding and is comparably potent to 32

133.

Intriguingly, other isoflavone structures (such as Genistein (34, Fig. 12) and Daidzein) have also been proposed as Hh pathway inhibitors, although their exact mode of action is currently not fully elucidated 134, 135. This suggests that the isoflavone core can potentially serve as a starting point in the development of novel Hh/GLI inhibitors. Surprisingly, specific substitutions at the isoflavone B-ring generated Hh inhibitory molecules which selectively targeted either SMO or GLI

136:

Isoflavones bearing a 4-(trifluoromethyl)phenoxy-substituent in 3’-position preferentially bound to the zinc finger of GLI1, 35 (Fig. 12), whereas compounds harboring this substituent in 4’position preferentially were proposed to bind to the heptahelical bundle of SMO, 36 (Fig. 12). Furthermore, a series of four small molecules (HPI-1 to HPI-4) were discovered which block GLI function in cells in the lower micromolar range 137. Interestingly, one compound (HPI-1, 37, Fig. 32 ACS Paragon Plus Environment

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12) was able to suppress exogenous GLI1/GLI2 activity, whereas another one (HPI-2, 38, Fig. 12) lacked an inhibitory effect on GLI1 but retained the capability to antagonize transfected GLI2, implying that it might be possible to discover GLI1/2-selective modulators. Collectively, the four compounds displayed different modes of action affecting GLI processing, trafficking and/or protein stability. This suggests that multiple physiological steps in the GLI biology are amenable to drug targeting. In addition, a large number of natural products has been described as GLI antagonists up to now, but the precise mechanisms of action of these molecules are often unresolved. These substances include the sesquiterpene Zerumbone (39, Fig. 12), the bisindole alkaloids Staurosporinone (40, Fig. 12) and Arcryaflavin C as well as Physalin B (41, Fig. 12) and Physalin F 138. Albeit being the most potent of this series, the Physalins are also challenging from a chemical point of view as they bear an unusual 13,14-seco-16,24-cyclo modification of the underlying steroidal core for which up to now no total synthesis has been reported 139. The same group also identified a series of pentacyclic triterpenes (Colubrinic acid, Betulinic acid, Alphitolic acid) as potential GLI inhibitors, although the reported IC50 values were quite high (approx. 30-40 µM) and drug optimization would be needed for future use 140. They also described additional natural compounds with GLI inhibitory potential and lower IC50 values, suggesting that natural substances can indeed be a viable source for new lead compounds 141-143. Molecular docking predictions have also identified naturally occurring substances such as the glycoalkaloid Solasonine (42, Fig. 12) as compounds capable of interfering with GLI-mediated transcription 144, 145. Although more work is required to elucidate the exact mode of action as well as the Hh/GLI specificity of Solasonine, the reported IC50 is in the lower micromolar range. A 33 ACS Paragon Plus Environment


comparable efficacy was reported for two natural epimeric compounds possessing a sterol backbone, isolated from the plant Cynanchum bungei Decne (Cynanbungeigenin C (CBC, 43, Fig. 12) and D (CBD)) 146. Interestingly, these compounds were able to cross the blood-brain barrier in mice and to inhibit signaling and cell growth in Hh-dependent MB cells. In line with their activity against GLI transcription factors, they could also block signaling in conditions of SMO inhibitor resistance. Although being quite different in overall chemical structure, Solasonine, Physalin B, and Cynanbungeigenin all display resemblance to a sterol scaffold, raising the putative question whether some forms of cholesterol-dependent processes might be involved at the GLI level of Hh signal transduction. Another natural product with GLI inhibitory potential (Taepeenin D, 44, Fig. 12) was isolated from Acacia pennata, a plant which can be found e.g. in Thailand 147. This terpenoid blocked GLI activity with an IC50 value in the low micromolar activity, but a thorough characterization of this compound awaits further studies. Several additional natural compounds have been described in the literature as Hh pathway inhibitors, but more work is required to faithfully assess their mode of action, specificity and toxicity 148-150.


Page 34 of 70

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N

N

N

S

N N

O

N

N

O

N O

N

31 (GANT58)

O

O

O 33 (Glabrescione B)

32 (GANT61)

O

O O

O

HO

O

O O

O OH O

O

OH

O

O

34 (Genistein)

O

O

CF3

35

CF3 36 N

OH

O O

O

O

O

N

N H

O

H N

N

O

O

O

O

37 (HPI-1)

38 (HPI-2)

39 (Zerumbone) HN

O H

O HO

O

O

HO O

O H

O

OH

HO

HO O

HO

O OH

O 41 (Physalin B)

O

NH

O

HN O O

O HO

O OH

40 (Staurosporinone)

OH O

O

42 (Solasonine)

O

O

O

OH OH

O

OH

O

HO

H

O O

43 (Cynanbungeigenin C)

44 (Taepeenin D)

Figure 12: Compounds inhibiting Hh signaling at the level of the GLI transcription factors. 35 ACS Paragon Plus Environment

NH


Page 36 of 70

Indirect GLI inhibitors with known mechanism of action With the exception of GANT61 and GlaB which have been shown to directly bind GLI transcription factors, the exact mechanism of action is currently unknown for the majority of Hh pathway inhibitors acting at the GLI-level of signal transduction. Nevertheless, specific compounds impacting on GLI-mediated gene transcription through indirect mechanisms are known. One such inhibitor category is represented by histone deacetylase (HDAC) inhibitors. Although HDACs are well appreciated for their transcriptionally repressive role, their impact on Hh signaling is opposite: HDACs deacetylate GLI1 and GLI2, retaining these factors in the nucleus and resulting in enhanced target gene transcription 151-153. Hence, downstream Hh pathway inhibition by HDAC inhibitors was able to block Hh-driven MB growth in murine cancer models and to overcome resistance against SMO antagonists

154, 155.

As HDAC inhibitors are far advanced in the drug

development pipelines and in clinical trials, it will be interesting to learn whether they can also be used in Hh-dependent malignancies. Another example of selective chromatin-directed Hh pathway blockers are BET-domain inhibitors such as (+)-JQ1 (only the (+)-enantiomer shows bioactivity)

156.

BET domains serve as reader

domains for acetylated histones and their association with acetylated histones (such as e.g. H3K27ac) makes them intimately involved in transcriptional activation. In terms of Hh signaling, BRD4 has been shown to be crucial for transcription of the GLI1 and GLI2 genes and blocking BRD4 function exerts strong negative impact on GLI-mediated target genes and cancer growth 156, 157.


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A preclinical compound targeting the class of Jumonji histone demethylases is JIB-04

158.

This

compound was shown to suppress Hh signaling by reducing the protein stability of GLI1. Intriguingly, this effect seems to be mediated by JIB-04 binding to the GLI1-interactor JMJD1A (encoded by the KDM3A gene) and appears to be independent of the inhibition of JMJD1A’s histone demethylase function, potentially implying structural changes evoked upon drug binding as a putative cause. A comparable scenario has for instance been described for Aurora kinase inhibitors impinging on N-MYC protein stability in an allosteric mechanism independent of their enzyme inhibitory function 159-161, suggesting novel approaches for GLI targeting. All of the above mentioned inhibitors of chromatin factors represent promising and potent Hh/GLI antagonists, but due to their multiple GLI-unrelated effects on chromatin (e.g. on MYC expression in the case of JQ1) they possess a certain degree of expected non-specificity. The big advantage of these compounds however, is that HDAC and BET inhibitors are far advanced in drug development and are already included in numerous clinical trials.

Downstream Hh inhibitors approved for clinical use As in the case of Itraconazole and SMO (see above), certain clinically approved medications have been identified as Hh inhibitors at the level of GLI. The fact that these compounds are already approved and that the safety profiles are well known makes them strong candidates for a straight forward and rapid inclusion into clinical trials. One of such compounds is Arsenic Trioxide (ATO), which is approved for the treatment of acute promyelocytic leukemia 162. ATO (chemically As2O3, the anhydride of arsenic acid (H3AsO3)) has been reported to directly bind to GLI1 and to interfere with the stability and the ciliary transport of GLI2, thereby blocking the central mediators of Hh37 ACS Paragon Plus Environment


induced gene activation

163.

Page 38 of 70

As such, ATO is functional in Vismodegib-resistant experimental

settings in vitro and in vivo 164. However, subsequent work revealed a more complicated picture of ATO function and it appears that this drug does not only antagonize the activator function of GLI transcription factors, but also of their repressor functions (contributed by GLI3) 165. Hence, ATO can also induce low-level Hh/GLI signaling, particularly in situations of weak intrinsic GLI activation in which GLI repressors play a dominant role 166. Another example of an FDA-approved drug with Hh-antagonistic properties is the anthelmintic substance Pyrvinium (45, Fig. 13), an allosteric activator of casein kinase 1 α (CK1α) 167. 45 is very potent and blocks cellular Hh signaling in the nanomolar concentration range, functioning also on Vismodegib-resistant SMO and in settings of downstream pathway activation upon SUFU depletion. Mechanistically, Pyrvinium-induced CK1α stimulation drives enhanced GLI1/GLI2 phosphorylation and proteasomal degradation. On the downside, 45 affects all other CK1αdirected processes unrelated to Hh, which however, can sometimes be beneficial as CK1α activation also blocks oncogenic Wnt signaling 168.

N N

+

N N

O 46 (Pirfenidone)

45 (Pyrvinium)

Figure 13: Clinically approved drugs with GLI-inhibitory ability.


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GLI2 stability is also targeted by Pirfenidone (46, Fig. 13), a 2-pyridone approved for use in idiopathic pulmonary fibrosis (IPF), a life-threatening fibrotic condition of the lung in which Hh signaling plays an important etiologic role 130, 169. 46 inhibits Hh signaling at a downstream level in human and murine fibroblasts by promoting the proteasomal degradation of GLI2 170. As GLI2 functions as a signaling platform connecting Hh with other pro-fibrotic pathways such as TGFβ, Pirfenidone treatment exerts strong anti-fibrotic effects. However, this drug needs to be given at relatively high doses, potentially causing difficulties in a future application as cancer therapeutic.

Conclusion Mammalian Hh signaling can be activated by canonical and non-canonical mechanisms. The former mode of pathway activation is predominantly utilized during normal embryonic development, in the Hh-dependent tumors BCC and MB as well as in the mesenchymal tumor stroma. In contrast, non-canonical modes of signal transduction can often be encountered in epithelial tumor compartments of various solid cancers. The plasticity in signaling cascades becomes obvious in cases of drug resistance against small molecule inhibitors targeting the signaling bottleneck SMO (see fig. 14 for a graphical overview of Hh inhibitors and supplemental table S1 for a summary of all inhibitors discussed). SMO represents a formidable drug target with 39 ACS Paragon Plus Environment


structurally deep pockets to accomplish efficient and selective drug binding and highly potent antagonists are now approved for clinical use. However, the selective outgrowth of resistant clones harboring SMO mutations or the induction of functions bypassing the need for SMO necessitate the detailed knowledge about cancer-specific non-canonical activation forms and the development of additional Hh pathway targeting drugs. Unfortunately, as of now non-SMO drug targets in the Hh pathway have proved difficult to inhibit, but pre-clinical proof-of-concept studies have strengthened the principle of pharmacological inhibition of Hh downstream elements. Along these lines, the drug development process as well as the introduction of new substances for Hhdriven malignancies might be accelerated by repurposing approaches utilizing approved drugs with a known safety profile which also target the Hh system. In general, it will be important to generate a toolbox of inhibitors which can be combined to overcome resistance and which can be utilized in a personalized manner based on the signaling mode in the specific tumor type and patient. This said, it would mean that future Hh drug development activities would have to include as well as go beyond the well-established SMO.


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Number in manuscript

Name in literature

Target

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Robotnikinin RU-SKI43 RU-SKI201 Cyclopamine IPI-926 (Saridegib) Vismodegib (GDC-0449) Sonidegib (NVP-LDE-225) Itraconazole BMS-833923 (XL-139) PF-04449913 (Glasdegib) PF-5274857 Compound 12 Anta XV Compound 13 NVP-LEQ506 Taladegib (LY-2940680) Compound 23b Compound 21 20(S)-OHC 22-NHC Budesonide Allo-1 Compound 5 Chalcone-12 Hh78 MRT-92 TAK-441 JK184 CA1 SA1 GANT58 GANT61 Glabrescione B Genistein Compound 12 Compound 13 HP-1 HP-2 Zerumbone Staurosporinone Physalin B Solasonine Cynanbungeigenin C Taepeenin D

SHH HHAT HHAT SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO SMO Microtubules (Cilia) Ciliogenesis SMO GLI1, GLI2 GLI1, GLI2 GLI1, GLI2 GLI ? GLI1 SMO GLI1, GLI2 GLI2 GLI GLI GLI GLI ? GLI ? GLI


References 29 33, 34 35 45, 46 47-49 51, 53 52, 54 56, 57 58, 59 58, 59 58-60 61, 63 61, 63 61, 63 61 62 64 65 42 69 70, 71 72 76 88 89 90 91 112-114 115 115 129 129, 131, 132 133 134, 135 136 136 137 137 138 138 138 144, 145

146 147


45 46

Pyrvinium Pirfenidone

CK1α GLI2

Table1 42 ACS Paragon Plus Environment

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167, 168 170

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Figure 14: Schematic outline of an active Hh pathway and the points at which known small molecule inhibitors interfere with signal transduction.



Abbreviations used AKT, AKT serine/threonine kinase 1 (PKB); BCC, Basal cell carcinoma; BRD, Bromodomaincontaining protein; CCND1, Cyclin D1; CK1α, Casein kinase 1 α; CRD, Cysteine-rich domain; DHH, Desert Hedgehog; DISP1, Dispatched RND transporter family member 1; GLI, GLI family zinc finger; GLI3FL, GLI3 full-length protein; GLI3R, GLI3 repressor form; GPRASP2, GPCR-associated sorting protein 2; GSK3β, Glycogen synthase kinase 3β; HDAC, Histone deacetylase; Hh, Hedgehog; HHAT (=SKI1), Hh acyl-transferase (Skinny Hedgehog); IHH, Indian Hh; INPP5E, Inositol polyphosphate-5-phosphatase E; JMJD1A, Jumonji domain containing 1A; KIF7, Kinesin family member 7; MB, Medulloblastoma; MYCN, Gene encoding N-MYC; MKL1 (=MRTF-A), Myocardin related transcription factor A; N-MYC, N-myc proto-oncogene protein; PDAC, Pancreatic ductal adenocarcinoma; PI3K, Phosphatidylinositol-4,5-bisphosphate 3-kinase/ Protein kinase B; PKA, Protein kinase A; PTCH, Patched family; SCUBE2, Signal peptide, CUB domain and EGF like domain containing 2; SHH, Sonic Hh; SMO, Smoothened; SRF, Serum response factor; SUFU, Suppressorof-Fused; TREGs, Immune-suppressive T-regulatory cells.


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Biographies Ilya Galperin studied biology with focus on human and molecular biology at the Saarland University (Germany), where he obtained his diploma title in 2012. From 2012 to 2017 he worked at the Institute of Food Chemistry and Food Biotechnology at the Justus Liebig University in Giessen (Germany), where he obtained his Ph.D. (2018). Since 2018 he started to work at the Center for Tumor and Immune Biology at the Philips University in Marburg (Germany) with a focus on the reprogramming of pancreatic tumor cells. Lukas Dempwolff studied chemistry at the Leibniz University Hannover (Germany), where he received his B.Sc. (2012) and M.Sc. degree (2014), in addition to an Erasmus internship at the University of Stockholm, Sweden. He obtained his Ph.D. (2019) on ligand-based drug design of novel transcription modulators under the guidance of Prof. Dr. Wibke Diederich at the Philipps University Marburg. Wibke E. Diederich studied pharmacy at the Philipps University of Marburg (Germany) where, in 1999, she also obtained her Ph.D. in Pharmaceutical Chemistry under the guidance of Prof. Manfred Haake. After a two-year post-doctoral stay in the group of Prof. Gunda Georg at the University of Kansas (USA), she returned to the Philipps University of Marburg, where she initially became assistant professor and in 2010 was appointed full professor of Medicinal Chemistry. Since 2013 she also heads the Core-Facility of Medicinal Chemistry at the Center for Tumor and Immune Biology. Her research focuses on the structure-based design and synthesis of small molecules especially in the field of neglected diseases and cancer.



Matthias Lauth studied biochemistry at the Goethe University of Frankfurt (Germany), followed by a PhD period at the Max-Planck Institute for Neurobiology (Martinsried, Germany) and the University College London (UK). After completion of his PhD thesis in 2003, he went to the Karolinska Institute (Stockholm, Sweden) for a postdoctoral period in the lab of Prof. Rune Toftgård, focusing on Hedgehog signaling and the development of small molecule inhibitors. In 2009, he returned to Germany to start his own research group at the University of Marburg. The research interests of his group are on Hedgehog signaling and the pharmacological modulation of tumor-stroma interactions. ML also coordinates the activities of a DFG-funded research consortium focusing on the pancreatic cancer microenvironment (DFG-KFO325).


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Acknowledgements We apologize to all the numerous colleagues who have contributed excellent work to the field, but whose work we could not cite due to space constraints. We are grateful for funding obtained from the German Research Foundation (DFG-KFO325, DFGLA2829/9-1, DFG-DI827/5-1, DFG-DI827/6-1), the German Cancer Aid and the University Hospital Giessen-Marburg (UKGM).

Corresponding author information Matthias Lauth, PhD Philipps University Marburg, Center for Tumor- and Immune Biology (ZTI) Hans-Meerwein-Str. 3, 35043 Marburg, Germany Email: [email protected]

Keywords Hedgehog, Smoothened, SMO, Vismodegib, GLI, Tumor stroma.



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