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New Modalities, Technologies, and Partnerships in Probe and Lead Generation: enabling a Mode-of-Action centric paradigm Eric Valeur, and Patrick Jimonet J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00378 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

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Journal of Medicinal Chemistry

New Modalities, Technologies, and Partnerships in Probe and Lead Generation: enabling a Mode-ofAction centric paradigm Eric Valeur †*, Patrick Jimonet‡*. †

Medicinal Chemistry, Cardiovascular, Renal and Metabolic Diseases, IMED Biotech Unit,

AstraZeneca, Pepparedsleden 1, Mölndal, 431 83, Sweden ‡

External Innovation Drug Discovery, Global Business Development & Licensing, Sanofi, 13

quai Jules Guesde, 94400 Vitry-sur-Seine, France

Abstract With the rise of novel biology and high potential target identification technologies originating from advances in genomics, medicinal chemists are progressively facing targets of increasing complexity and often unprecedented. Novel hit finding technologies, combined with a wider choice of drug modalities, has resulted in a unique repertoire of options to address these challenging targets and to identify suitable starting points for optimization. Furthermore, innovative solutions originating from a range of academic groups and biotech companies require new types of collaborative models to leverage and integrate them in the drug discovery process. This perspective provides a guide for medicinal chemists covering contemporary probe and lead generation approaches, and discusses the strengths and limitations of each strategy. Moreover, the expansion of strategies to modulate proteins creates the opportunity of a modality-agnostic and mode-of-action centric hit finding paradigm. 1 ACS Paragon Plus Environment

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1. Introduction Over the last decade, the target landscape has dramatically changed. Many protein targets, now considered as the ‘low hanging fruits’, have been successfully drugged, progressively shifting focus to more challenging, often labelled as ‘undruggable’, target classes such as Protein-Protein Interactions (PPIs).1 The rise of novel target identification strategies, such as genomics,2 and genome-wide CRISPR screens,3 is also resulting in an increasing number of unprecedented targets. Furthermore, progress in the understanding of cell biology and of mechanisms involved in the regulation of protein levels is also further extending the target scope. Indeed, less than 2% of the human genome encodes protein, and noncoding RNAs play fundamental roles in regulating gene expression and other cellular processes, therefore representing a further reservoir of potential drug targets.4 To address this ever-changing target landscape, a broader variety of drug modalities is required. In addition to the opportunities presented by small molecules and antibody/protein therapeutics, a range of so-called New Modalities,5 including precise genome editing, modified peptides, oligonucleotides, macrocycles and various conjugates, have opened up novel ways to modulate targets. Medicinal chemists face the demanding responsibility of identifying starting points for these challenging and novel targets to either generate probe or lead molecules. Fundamentally, chemists also have a major role to play in selecting the right modalities. Interestingly, these various modalities can modulate proteins at different levels, including at the transcriptional and translational level (Figure 1), which triggers the opportunity to place the selection of the modeof-action (MOA) at the center of any drug discovery project, and apply the appropriate hit finding strategy based on this selection. Such a paradigm also fosters the possibility to be truly modality-

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agnostic by shifting the selection of modality after the choice of MOAs, and reinforces the step of building strong knowledge on targets for decision-making.6

Figure 1: MOAs affecting direct protein function and protein levels

Within a MOA-centric paradigm, DNA can be modified through precise genome editing such as CRISPR/CAS9.7,8,9 Many challenges exist with this emerging technology, likely restricting this MOA to few cases, such as rare genetic disorders, for now. Another MOA is to modulate proteins levels through up- or downregulation of RNA, including to affect splicing, using either oligonucleotides or small molecules approaches, or perhaps CRISPR.10 Oligonucleotides, in the form of small interfering RNA (siRNA) or antisense oligonucleotides (ASOs) can silence RNA targets and hence reduce protein levels, but also increase levels of downregulated proteins when used in the form of modified RNA.11,12 Small molecules that downregulate proteins at the translational level are also becoming a credible option, as exemplified with the PCSK9 protein.13 The next stage of intervention is to modulate protein levels through induction of degradation, for example with the so-called proteolysis targeting chimera (PROTAC).14 Finally, the classical MOA of modulating protein functions through agonism/antagonism is enabled through a choice of small molecule, peptide, antibody or even aptamer modalities.15,16,5 To enable probe and lead generation against these MOAs, a range of new hit finding technologies have been developed across modalities. For example, phage and RNA display have allowed production of large libraries of cyclic peptides containing natural and non-canonical amino 3 ACS Paragon Plus Environment


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acids.17 Progress in structural technologies is expanding the possibilities of structure-based lead generation through both protein structure mimetics and a range of small molecules diversity approaches such as virtual screening.18,19,20 The success of high throughput screening (HTS) using discrete screening collections can be optimized by new collaboration models, increasing the diversity and quality of such libraries.21,22,23 To enable a MOA-centric paradigm and with such a vast repertoire, the selection of the best approaches with their pros and cons, their issues and potential solutions, should be carefully made, knowing that access to innovation through external partners will often be necessary to succeed. With this prospect in mind, this review provides a contemporary guide to novel hit finding opportunities. Herein, the range of new hit finding strategies for directly modulating protein functions are broadly divided into structure-agnostic screens (Section 2), including through the extension of compound collections with partnerships (Section 3), and structure-enabled approaches (Section 4). The modulation of protein levels through RNA interference with oligonucleotides and small molecules, and through protein degradation is then covered (Section 5). All of these new approaches are brought into context with a guide to select the most suitable hit finding strategies within a MOA-centric paradigm (Section 6). Finally, an outlook on emerging hit finding technologies is provided (Section 7). All sections may be read independently, with the recommendation that medicinal chemists should first apply a MOA-centric selection for their target of interest before diving back into the relevant approaches. Due to the broad scope of this review, only representative examples of each strategy are presented.


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2. Structure-agnostic probe and lead generation for direct modulation of protein functions In the context of modulating protein functions through classical agonism or antagonism, medicinal chemists have access to a range of established and new technologies and modalities, regardless of the availability of structural information. Indeed, while the approaches described in this section are most relevant for proteins lacking structural data, these may still be applied to the cases where crystal structures are available, with the mindset of maximizing chances of identifying a starting point. This section focuses on novel approaches and modalities. Within the category of structure-agnostic technologies, the discovery of antibodies, in particular through phage display,24 is well-established and particularly well suited for challenging extra-cellular targets. Similarly, the so-called systematic evolution of ligands by exponential enrichment (SELEX) display and select method can robustly identify aptamers.25 The foundation and prospect for these two modalities are covered in details in selected reviews.15,16 2.1.Identification of stabilized peptides as starting points Phage display of peptides was developed several decades ago and represents a robust method to identify peptidic starting points against any protein. This strategy has historically only delivered linear peptides, with the associated challenges of limited stability and cell permeability.26 With the development and interest in next generation peptides to address challenging targets, novel technologies or adaptation of current technologies have emerged. In particular, phage display has now been extended to the generation of constrained cyclic peptides. After displaying random sequences containing three cysteine residues, the phage is exposed to a reactive small molecule, resulting in the formation of a bicyclic peptide (Figure 2a).27 The small molecule core and the length of the different loops (typically 3-6 amino acids) can be varied to induce conformational 5 ACS Paragon Plus Environment


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changes in the loop regions, resulting in increased library diversity.28,29 The constraint induces increased stability as well as higher affinity and specificity. For example, a library of around 109 bicyclic peptides underwent three rounds of selection against human plasma kallikrein (PKL).27 From the identified hits with binding affinities in the region of 100-400 nM, consensus sequences were identified and utilized for new library design which was applied to new rounds of selection. The best analogue 1 displayed a binding affinity of 20 nM for human PKL (

Figure 3). Interestingly, the hit was selective over the mouse orthologue, highlighting the very high selectivity that bicyclic structures can achieve. Overall, this approach has been applied to several target classes beyond proteases,30,31 including targets ‘undruggable’ to small molecules such as NOTCH132 and β-catenin,33 and is being pursued commercially by Bicycle Therapeutics.


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Figure 2: (a) modified phage display for the identification of bicyclic peptides. (b) modified mRNA display for cyclic peptides containing unnatural amino acids

Another modification consists in displaying β-hairpins, in essence via fusion of the two peptidic ends with the phage surface. Although the approach has been commercially advertised by Polyphor, explicit published examples have not been reported.34 Modified phage display approaches have also been developed from the inspiration of naturally occurring stabilized peptides. Lanthipeptides are a class of peptides produced by a range of bacteria and contain multiple thioether cycles. In an elegant approach, phage display was combined with modifying enzymes to generate post-translationally modified peptides and this de novo lanthipeptide library was applied to the identification of novel uPA inhibitors.35

Figure 3: Examples of stabilized peptides. Color coding for amino acids: Purple= D-amino acid

Another strategy has been aimed at enabling the incorporation of unnatural amino acids to increase diversity and stability. To generate any meaningful library size, display methods remain the approach of choice. Thus, the genetic code for natural amino acids and the translation machinery has been altered and reconstituted in vitro, significantly expanding the scope of mRNA display.17 To achieve this, tRNA are loaded with unnatural amino acids, switching the assigned codons normally encoding for a given natural amino acid to their non-proteinogenic replacement. Both modified and canonical tRNA are then mixed with ribosomes and with mRNA 7 ACS Paragon Plus Environment


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encoding for the peptides to be generated. Beyond the advantage of merely introducing unnatural amino acids, the major advantage of the system is its compatibility with chemoreactive unnatural amino acids. Thus, a chloroacetyl moiety can be post-translationally cyclized via reaction with a cysteine. This so-called RaPID system can generate cyclic peptides containing typically 4 to 25 residues with a library size of approximatively 1012, which are selected in an affinity setting, followed by amplification (Figure 2b).36,17 The technology has been applied to a range of targets including enzymes,37,38,39 and PPIs,40,41 and is commercialized by Peptidream. A recent example is the identification of selective histone demethylases inhibitors, where potent hits inhibited KDM4 family members in excess of 100 fold.42 In particular, peptide 2 bound the KDM4A substrate binding pocket leading to double digit nanomolar activity (IC50 = 42 nM,

Figure 3), and selectivity could be attributed to a specific interaction with an arginine residue. Further optimization led to peptides with increased stability and target engagement in cells, as demonstrated by cellular thermal shift assay (CETSA). Another significant example is the activation of the Met signaling pathway. Traditional drug discovery approaches are often aimed at inhibiting pathways and strategies to induce direct activation are scarce. Using the RaPID approach, monomeric Met binders where identified and turned into dimeric binders through cross-linking.43 This approach resulted in the recruitment of two Met proteins leading to formation of the homodimer and subsequent activation of the signaling pathway.


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Finally, a platform developed at Roche Nimblegen enables the production of high density peptides microarrays. Almost three millions peptides, including cyclic ones, can be synthesized on one single slide with high precision through digital, light-directed, controlled synthesis.44 The platform tolerates formation of cyclic peptides and incorporation of unnatural amino acids providing a broad diversity, which can be screened by surface plasmon resonance (SPR). As a proof of principle for the technology, novel binders to streptavidin were identified. While this approach provides far less diversity than display, the rapid synthesis and incorporation of a range of amino acids is attractive for hit optimization. Overall, these strategies are most relevant for challenging extracellular targets. In addition, when selectivity is deemed challenging to achieve, these stabilized peptides approaches may be more suitable than small molecules. Examples of intracellular targets remain scarce, which still places this field as a less favorable option for these targets, although further understanding of cellular uptake may eventually change this limitation.45,46,47,48 The process typically takes 3-4 months and requires building partnerships with specialized companies such as Bicycle Therapeutics, Polyphor or Peptidream. Importantly, follow-on functional assays are required to confirm the relevance of hits identified through these affinity-based assays. From an optimization standpoint, the cyclic nature of these peptides provides them with greater stability than linear peptides. However, when limited stability is observed during hit and lead optimization, in particular in vivo, several strategies are possible, including the introduction of D-aminoacids and other noncanonical derivatives.49 Half-life extension through, for example, lipidation is an additional possibility,50 which has demonstrated impact up to market with semaglutide.



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2.2.Screening libraries of peptidic natural products Compounds of peptidic nature are largely represented in natural products from various sources. These include the immunosuppressant cyclosporine originating from soil fungi and Kalata B1, a cyclotide isolated from plant extracts that has been used for labor acceleration through its uterotonic effect in Africa. Peptidic natural products possess complex properties often very difficult to mimic with synthetic analogs. Animal venoms are a representative example of such valuable peptides in the context of probe and lead generation. Venoms represent an enormous reservoir of bioactive compounds: 170,000 venomous species, each venom containing 100 to 1,000 compounds, and only a very small number already characterized (~3,000 compounds).51 This set of millions of natural products may be considered as a unique library of peptidic natural products of high interest for challenging targets, but also as valuable chemical tools to study biological mechanisms. Venom peptides have been the source of interesting new drugs for many years.52 From captopril originating from Pit viper for hypertension,53 to more recently ziconotide from Cone snail for chronic pain,54 and exenatide from Gila monster lizard for type 2 Diabetes,55 venoms have confirmed their value as source of drugs targeting various protein target types, as the three above mentioned compounds are respectively targeting an enzyme, an ion channel and a GPCR. Peptides found in venoms are generally highly structured with a diversity of tridimensional folds favorable for interaction with target proteins.56 The main target class of these compounds is clearly ion channels, as evolution has optimized venoms to immobilize preys generally through ion channel blockade. Importantly, this bioactive effect is however only observed for a very minor fraction of the peptide pool in venoms. The diversity of folds in venom peptides can therefore be considered as a library of cyclic folded peptides suitable for opportunistic interaction


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with any protein target, particularly when small molecules or antibodies are not a good option, especially for extracellular or membrane-bound targets, and when oral administration is not an absolute requirement.

Figure 4: Workflow for screening venoms

To perform their screening, venoms are fractionated into solutions containing about ten peptides (Figure 4). Active fractions are then further fractionated, until sub-fractions containing only one peptide are obtained for the next round of screening. The active peptides are then sequenced, and confirmed by chemical synthesis and folding (Figure 4). This process can be handled in parallel to classical compound collection screening but requires specialist knowledge, often sourced through an expert collaboration partner, to efficiently drive the whole process. One example of a specialized company in this field is Smartox. Limitations for the use of venoms in the hit finding process stem mainly from the process duration and the difficulty to confirm active venom fractions when single peptides are isolated and synthesized. However, these potential drawbacks are directly correlated with the quality of venom fractions that should contain a limited number of peptides within mixtures, devoid of any unwanted or reactive material that could negatively affect screening results. A comparison of 11 ACS Paragon Plus Environment


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head-to-head crude venoms and venom fractions in a primary screening underlined the significant added value of careful fractionation of crude venoms as an initial step. Many false positives are removed and false negatives using crude venoms revealed, therefore focusing efforts on high value active fractions to proceed towards peptide identification and synthesis for confirmation. The whole process is manageable within a few months but the outcome depends on the quality of the biological assays validating target engagement, and on the peptide hit structures, especially when considering potential folding difficulty if multiple disulfide bridges are present. Ion channels, GPCRs and other membrane proteins are of particular interest for this approach, as several successful peptide drugs have been reported recently.57,58,59 Interestingly, access to such venom-containing folded peptides, which may be considered as ‘mini-proteins’ covering a diversity of tridimensional structures, has recently been published using a very innovative approach. Knowledge from natural peptides originating from animal venoms, microorganisms and plants, has been coupled with high throughput de novo design of hyper stable constrained peptides, leading to a large number of novel and structurally-constrained peptidic structures.60 Overall, the strategy of screening collections of constrained peptides offers great potential towards the design of folded peptidic drugs for difficult targets with the significant advantage of chemical accessibility. It remains best suited for extracellular or membrane-bound targets as cell penetration of such cyclic peptides is still often difficult, even if examples of cell-penetrating peptides originating from venoms have been reported.61


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Figure 5: Principle of DELs

2.3. Screening large number of compounds with DNA-encoded libraries The concept of screening large libraries of compounds, particularly through mixtures, has been the basis of the split-and-pool methodology and combinatorial synthesis developed in the 1990’s. One of the main issues with this strategy was the identification of the active component(s) within a mixture. Various encoding methods were evaluated but practical application of the generally required solid-phase synthesis was quite cumbersome, including long library synthesis development and an uncertain hit identification process.62 The development of in vitro directed evolution methods for the synthesis of peptides is at the origin of the use of DNA sequences as encoding methodology associated with the combinatorial split-and-mix synthesis (Figure 5).63 Compared to other encoding methods, combinatorial synthesis is performed in solution and its use is typically limited to aqueous media and mild pH, due to the limitations associated with DNA solubility and stability in reaction conditions and solvents. The design and synthesis of large DNA-encoded libraries (DEL) of 106-109 members have been described using a rather limited set of reactions tolerated by the encoding tag.64,65 However, despite this limitation, several 13 ACS Paragon Plus Environment


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examples of successful hit finding, sometimes for challenging biological targets, have been published.66 As an example, DEL libraries were screened against the receptor interaction protein 1 (RIP1) kinase and led to nanomolar benzoxazepinone hits that were optimized into the clinical candidate 3 for inflammatory diseases (GSK2982772) (Figure 6).67 Noteworthy, an HTS was also performed in parallel to the DEL screening and provided another lead series possessing a different binding mode, but which was deprioritized due to lack of oral exposure. This example therefore demonstrates the complementarity of the DEL strategy.

Figure 6: Examples of hits and probes identified by DEL

The benefit of DEL relies primarily on the size of the libraries compared to regular HTS screening of about 106 diverse chemical structures. Enhancing diversity of DNA-compatible reaction types would strengthen the added-value of DEL, but library diversity may be increased with proprietary scaffolds and building blocks to build and decorate libraries. Novel strategies, such as the use of hexathymidine oligonucleotides, are also being developed to enable acidic catalysts and therefore increase diversity.68 Another limitation of DEL is the need to immobilize the biological target in order to identify potential binders within a mixture. Consequently, only soluble proteins can robustly be screened using affinity selection. Membrane proteins, such as GPCRs, known for their low expression, are less suited, although cellular systems have been reported.69 One strategy to circumvent this challenge is to produce membrane proteins in soluble formats by using either cell-free production 14 ACS Paragon Plus Environment

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systems or by cell lysis. In both cases, detergents, liposomes, nanodiscs or lipoparticles may be used to stabilize the protein in the absence of its membrane-stabilizing environment. Several technologies and know-how have been under development in academia and biotechnology companies with some success, including for GPCRs.70,71,72 Combination of cutting-edge technologies in affinity selection and protein production is therefore a winning combination to identify valuable binders for difficult proteins. Several biotechnology companies offer the DEL technology, including X-Chem, Nuevolution, Philochem, Vipergen, Dice or HitGen, using slightly different methods of encoding and chemical synthesis, but all with the objective to build large and diverse combinatorial libraries. The majority of large pharmaceutical companies have signed deals with at least one of the specialized DEL companies. Some have even internalized the technology, either by company acquisition (GSK with Praecis),73 or through technology transfer (Novartis with Nuevolution, and recently AstraZeneca with X-Chem), which highlights the recognized value of such a technology as part of the hit finding toolbox. DELs also offer the additional opportunity to identify allosteric sites through exposure to a large number of compounds. Several examples of new binding modes to known targets have been reported,74,75,76,77,78 which represents a particularly attractive way to identify functional effectors quite impossible to rationally design in the absence of useable structural knowledge of the target. An additional opportunity is the design of libraries of reactive molecules, often fragments, designed to covalently bind label free cysteines.79 In addition, the scope of DELs expands beyond lead generation and may be used in chemical biology for target validation and study of biological mechanisms.80 A recent example illustrating this use was the discovery of isoform-selective ATAD2 inhibitors and the chemical probe 4 (BAY-850) showing the unexpected property of



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inducing ATAD2 bromodomain dimerization (Figure 6Error! Reference source not found.).75 This chemical probe represents an invaluable tool to better understand ATAD2 biological role. Overall, every protein target difficult to drug using classical small molecule HTS represents a favorable application of DEL, particularly when dealing with a soluble protein and which can be effectively tagged and made suitable for immobilization on beads. Application is not limited to challenging targets, and the approach is also valuable for classical targets, representing a complementary strategy to other hit finding strategies. Importantly, this technology aims at identifying binders, and functional assays are therefore always needed to profile the hits. The process takes a few months from inception to the first hits. Historically, access to the technology has been restricted to multi-target partnerships, but recently some contract research organizations (CROs) have started to offer the approach as a fee-for-service, which will undoubtedly be attractive for small companies. 2.4. Screening libraries of non-peptidic macrocycles The size and molecular weight of macrocycles generally extend beyond the Lipinski rule-of-five boundary of 500 Daltons. This is clearly an advantage as it increases the possibility to bind a larger protein binding site compared to smaller size molecules, particularly considering the general flexibility of those large macrocycles favoring an induced-fit interaction.81 However, a balance between binding efficacy and favorable physico-chemical and pharmaco-kinetic properties needs to be found to select a suitable molecule for clinical development. A recent analysis of macrocyclic compounds on the market or in development has shown that oral bioavailability can be obtained in the molecular weight range of 500-1000.82 However, a careful management of properties, such as lipophilicity and solubility, is necessary to identify a balanced profile. Furthermore and importantly, a continuous progress in the knowledge of property- and structure-based modulation of membrane penetration is critical to produce new macrocyclic drugs 16 ACS Paragon Plus Environment

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for difficult targets that are not reachable or druggable with classical small molecules or antibodies.83 Access to macrocyclic structures has been enabled by DEL-specialized biotechnology and academic groups,84,85,86,87 but screening collections of discrete macrocycles is also a range of providers. Despite generally challenging chemistry, libraries of novel macrocyclic compounds, have been proposed by specialized companies such as Asinex, Analyticon, and Polyphor, or developed in house in major pharmaceutical companies as part of library enhancement initiatives to extend the chemical space of their screening sets. The promise of libraries has already been shown and much more is expected,88,89,90 with several macrocyclic compounds targeting challenging proteins moving to the clinic, either originating from macrocyclic hits, or designed during optimization. As a striking example, the macrocyclic inhibitor 5 (AMG-176) targeting the induced myeloid leukemia cell differentiation protein previously known as an undruggable protein of the Bcl-2 family, has been reported to be in clinical phase 1 for hematologic malignancies (Figure 7

Figure 7). It has previously shown efficacy at slowing down tumor growth following oral administration in multiple myeloma xenografts.91 Another example of macrocycle in clinical development is the third-generation ALK inhibitor lorlatinib 6 (PF-06463922), which displayed impressive efficacy in reversing



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resistance against crizotinib and other ALK inhibitors (Figure 7

Figure 7).92

Figure 7: Examples of macrocycles in clinical phase

Overall, macrocycles libraries are well suited for challenging targets, especially PPIs, and many examples of cell permeable macrocycles demonstrate they are an appropriate choice for intracellular targets. While macrocyclization restricts the number of conformations, probing in theory a smaller portion of the chemical space, this is mitigated by the adaptability of targets upon binding.81 For most companies, this screening strategy will have to rely on establishing partnerships or fee-for-service screening with specialized providers. 2.5. Identifying modulators of protein functions through phenotypic screening With lack of translation of target-based drug discovery still representing a major reason for attrition from preclinical to clinical phases,93 a renaissance of phenotypic approaches has been witnessed over the last years.94,95 Indeed, increased access to patient tissue samples offers the opportunity to screen libraries of compounds for new phenotypes. This lead generation strategy is in theory fully target-agnostic, although few companies tend to be prepared to optimize hits and eventually leads without target identification.94 Nevertheless, the approach can enable 18 ACS Paragon Plus Environment

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identification of novel targets as well as molecules driving a desired effect through polypharmacology. Phenotypic screening is crucial in the field of anti-infectives,96,97 and particularly relevant to the challenges faced with regenerative medicine, where biology is complex and often not well understood. In this respect, development of high content assays is enabling phenotypic assays, with multiple endpoints providing the opportunity to dissect phenotypes of interest.98 The increasing use of induced pluripotent stem (iPS) cells enables screening in a setting that may more accurately replicate specific disease phenotypes compared to, for example, immortalized cell lines,99 a consideration that can be further expanded to organoids, as illustrated with iPS-derived cardiac organoids.100 Moreover, more complex readouts such as quantitative polymerase chain reaction (qPCR) can also be incorporated.101 From a compound collection standpoint, the more translational approaches are typically lower throughput and consequently dictate screening a limited number of compounds, typically up to a few thousand. Thus, chemogenomic libraries consisting of annotated compounds known to interact with specific targets are often used.102,103 However, these approaches inherently reduce the opportunity of identifying novel targets since current knowledge in terms of probes represent only a few percent of the human proteome.104 A balance between translatability and throughput has therefore to be struck. In particular, the reliance of artificial stimuli to mimic disease state (for example chemically-induced) is often misleading and results in the identification of stimulusspecific hits which do not translate to patients. In addition, target deconvolution remains a major endeavor that hinders the relevance of screening large number of compounds to identify novel targets. Thus, acceptance of the lower throughput but more translational screens should still be embraced. Nevertheless, efforts have been directed at expanding the chemical space used in phenotypic screening including secreted proteins and natural products.105 A significantly different approach to identifying novel targets through phenotypic screening was developed by leveraging 19 ACS Paragon Plus Environment


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the ability of fragments to inherently have a higher probability of binding to proteins compared to larger entities.106 By functionalizing fragments with photoaffinity probes, and analyzing cells with proteomics, novel binders to proteins could be identified, and the approach was in particular applied to adipocyte differentiation.106 Overall, phenotypic screening offers many opportunities to identify modulators of protein function, but a focus on the most relevant approaches drives high costs. Importantly, the recognition that the scope of targets prosecuted through phenotypic screening is intrinsically wider is growing with the identification of target modulation at the RNA level, as will be discussed in section 5.2.

3. Expanding diversity for structure-agnostic screening through new partnerships High throughput screening remains a very valid and robust strategy to identify suitable starting points for drug discovery.21 Increasing diversity and quality of small molecules in screening collections is however required to offer the best target space coverage and the best lead-like compounds. 23,107,108,90 Historically, the enhancement of screening collections has been performed through compound acquisition from chemical suppliers and brokers.109,110 However, this model requires a large budget to cover a sufficiently large and diverse number of compounds.111 Another model exploited by several major pharmaceutical companies has been to outsource the synthesis of small molecule libraries designed by chemists of the ordering company. Here again, a very large budget is required to cover the cost of synthesis. Nevertheless, it allows internalization of compounds specifically designed for underexplored target classes or molecules that are mimetics of natural molecules, e.g. carbohydrates, nucleosides, generally poorly represented in screening collections, as well as in vendors’ offers. AstraZeneca exploited this


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model over a period of ten years in order to add more than 600,000 novel compounds to the compound collection.112,93 New business models aiming at accessing valuable chemicals bringing novelty and complementarity to internal compound collections have been developed recently. The objective has been to internalize large sets of novel compounds for screening at a much lower cost, and possibly more efficiently that classical purchasing. Several approaches, broadly covered under the umbrella of ‘open innovation’, have been evaluated and implemented as is illustrated in the rest of this section (Figure 8).

Figure 8: Overview of historical and new business models for expanding compound collections

3.1.Accessing larger collections through consortia The European Lead Factory (ELF) is a public-private partnership initiated in 2013, and involves seven pharmaceutical companies, five CROs and ten academic institutes over a 5-year period.113 The project is aimed at screening 300,000 industrial compounds and 200,000 newly designed 21 ACS Paragon Plus Environment


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academic compounds against academic and industry member targets.113 The first compound library was built from the pharmaceutical companies’ proprietary collections by selecting up to 50,000 molecules each, covering complementary diversity and lead-like properties.22 This initial 300,000 collection may be screened against up to 24 academic targets per year, and against an agreed number of internal targets for each pharmaceutical member. A second collection designed by academic and CRO members of the consortium has been implemented progressively during the five years of the project life thanks to an ambitious synthetic program at the CROs and with the end-goal of 200,000 compounds.114 The advantage for the pharmaceutical members has been twofold: a ‘give one – get nine’ chemicals to screen against internal targets, and an access to novel academic targets when public target programs are proposed to industrial partners following successful hit identification. For academics, the value of the consortium has been the access to a high quality screening collection, and the potential to identify an industrial partner for their innovative biology. Importantly, all screenings are structure-blinded and a structure-clearance process was defined at the start of the initiative to contractually frame IP rights on disclosed structures. The consortium is also beneficial to CROs, including through the development of a strong network with academic partners, the design and implementation of innovative chemistries, as well as an enhancement of the production of compounds necessary to build the new compound collection. The benefits gained by each partner therefore demonstrate the value of such a consortium. 3.2. Increasing the size of screening collections through compound swaps A next level of open innovation has been to swap a defined number of compounds and amount of material between two partners. This type of deal has been undertaken between two companies working in two different business areas, e.g. human health and crop protection, with each receiving freedom to patent and develop in their own field when interesting molecules originating 22 ACS Paragon Plus Environment

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from the partner are identified. This type of deal involves mainly chemoinformatic and logistic resources in order to select the best complementary chemical space and compounds adding value to the collection of the receiving party.115 Furthermore, and contrary to the previous consortium model, chemical structures are disclosed from the start and the amount of material to exchange is negotiated. This compound swap can reach an even higher stage of ‘openness’ by performing it between two competitors within the same business area. Such a deal was effectively signed in 2015 by Sanofi and AstraZeneca with the exchange of 200,000 compounds.116 The two partners accepted the risk of potential competition on the same targets as well as potential loss of IP for the compounds in the exchanged set. This apparent weakness is however outweighed by the opportunity to identify valuable hits from a collection dramatically enhanced by the partner’s set.

This deal

demonstrates the fundamental principles of open innovation: ‘to give to better receive’. Clearly, appreciating the level of risk and the benefit/risk ratio are cornerstones to the initiation of innovative business models. Another approach has been the reciprocal blinded access to the compound collection of two competitors. In this so-called ‘boomerang’ model, Bayer and AstraZeneca can screen a proprietary target on the compound collection of the partner, in exchange for the same in the opposite direction, essentially representing a ‘target swap’.117 A prerequisite for a screen to be endorsed, is for the nominated target not to be relevant to the partner. Considering the very small overlap between the two collections, approximatively 3%, this innovative model creates a pool of greater than 4.2 million compounds.118 3.3. Relaxed boundaries for compound sharing Systemic pressure to optimize R&D cost has pushed some pharmaceutical companies to outsource the management of their collection to specialized CROs possessing cutting-edge 23 ACS Paragon Plus Environment


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robotic equipment and facilities.119 This strategic evolution is facilitating the implementation of innovative business models by streamlining the flow of compounds between collaboration partners. A typical example is the deal between Sanofi and the academic French National Compound Library (‘Chimiothèque Nationale’).120 Compounds from this library originate from over 40 academic groups are now housed by Evotec, who manages and distributes the compounds for the academic members. In exchange, Sanofi has access to compounds from this library for structure-blinded screening under a risk-sharing contract.121 3.4. Risk-sharing and collection leasing partnerships Risk-sharing and library leasing partnerships represent another model of compound access, which usually relies on success-based milestones and redefines the traditional value chain. This approach rewards the success related to the identification of high value hits coming from structure-blinded screening against portfolio targets. Negotiated access fees are paid to chemical providers for a very small quantity of selected compounds bringing value to existing collections. These compounds are screened for a given period of time during which process and prenegotiated milestones related to high quality hit identification are executed following contractually-agreed terms. The benefit and value for chemical providers depends on an estimation of the number of screenings which will include the provider’s compounds and the expected hit rate. Screening of these external collections is usually conducted in parallel to internal collections depending on target types and capacities. An analysis of the outcome of seven partnerships at Sanofi has shown a similar hit rate to the internal diversity-based collection, but with the great advantage of identifying chemotypes complementary to internal ones. Importantly, competition between the various hit sources has required education of project teams to consider all the sources at a comparable level of potential value. The role of the ‘firewall’ scientists


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between partners is then critical to an efficient and successful implementation, as this risk-sharing model of compound access is definitely more a research collaboration than pure procurement. Importantly, this model is not limited to small molecules, and collections of other modalities can be considered. For example, natural products and mimetics, macrocycles, glycomimetics, glycoconjugates, nucleosides and others may be involved in such a model for screening against relevant biological targets, as is illustrated with AstraZeneca screening libraries of glycomimetics from Alchemia (now VAST Bioscience). 3.5. Crowdsourcing Another creative way to access innovation is to ask ‘the crowd’ for proposals around a specific need. A few companies focusing on crowdsourcing strategies have been developing dedicated portals or expert networks over the last decade, including Innocentive, NineSigma and Biowebspin. The objective is to make a community aware of a requester’s need in order to get proposals from solvers unknown to the requester or unexpected. Such a type of challenge can either be focused on a specific solution to solve a problem, generally financially rewarded, or a request for a partner possessing expertise, tools and/or technologies to initiate collaboration. This call for partners can be dedicated to a specific class of modalities such as macrocycles, as published on the Innocentive portal in April 2016.122 This innovative way to access expertise and solutions presents several advantages: 1) a quite short and simple process 2) a complementary approach to other sourcing methods based on scouting techniques such as the use of personal networks, participation to conferences and business meetings, or in silico queries 3) access to potential partners really willing to collaborate as they respond positively to a call. In this specific case of a call for partners on macrocycles, it was possible to identify innovation in academia, only partly published, but more importantly ready for industrialization through the creation of start-ups looking for pharma partners. 25 ACS Paragon Plus Environment


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Overall, the innovative partnerships presented in this section are aimed at accessing novel chemical matter and represent a complementary approach to the classical screening of internal collections. To select which collection to screen, the potential added-value of each set and their complementarity, as well as the capacity and cost of the screening, have to be considered in the context of each target. 4. Structure-enabled probe and lead generation for direct modulation of protein functions Possessing structural information on a target considerably widens the range of possible hit finding approaches. For example, the structure of ligand-receptor or more generally PPI interfaces can be used to quickly derive a peptidic starting point. More generally, structural information can be used for diversity-based approaches, in particular fragment-based screening, which has been a major pillar of structure-based lead generation, principally using NMR or XRay. Chemists are directed to detailed reviews in this field,

123,124

and this approach is

particularly well suited for classical targets. Virtual screening is also enabled through structure, and progress in in silico technologies is increasing its relevance as a potential systematic hit finding strategy. The need for structure generation is implicit and obvious for structure-enabled approaches. Therefore, developments in emerging technologies in this field, such as cryo-electron microscopy (cryo-EM) and free electron laser (FEL) are also discussed in this section due to their implication for challenging targets. 4.1. Generation of starting points through protein structure mimetics PPIs offer the unique opportunity to derive a starting point, by directly mimicking one of the binding partners and stabilizing it.125 The principle can leverage either secondary structures such as α-helices and β-sheets, or tertiary structures, by combining several units of secondary


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structures mimetics. In particular, motifs such as helix-loop-helix, loop-strand-helix-loop, loopstrand-helix or coiled coil are being increasingly investigated,126,127,128 although the approach may remain reduced to one helix of these tertiary motifs.129

Figure 9: Protein structure mimetics strategies. (a) stabilization of α-helices; (b) stabilization of β-hairpins; (c) stabilization of loops

α-helices represent about 60% of the key interactions at protein-protein interfaces making them a preferred motif to mimic.130 Two main approaches have been developed to stabilize such helices. Stapled peptides consist in cross-linking two amino acid residues located on the same side of the helix, usually through ring-closing metathesis performed on an α-methylated olefin located on the side-chain of amino acids (Figure 9a). The cross-link can either constitute a simple α-helix stabilizer, make additional contacts at the interface, or be leveraged for significant interaction at 27 ACS Paragon Plus Environment


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the interface.5 For example, starting from a fragment of the MAML1 protein, a known antagonist of NOTCH signaling,131 the stapled peptide 7 displaying 94% helicity bound the NOTCH transactivation complex with a KD of 120 nM and abolished NOTCH signaling, leading to inhibition of cell proliferation in T-ALL cells (Figure 10). Furthermore, in vivo efficacy through dose-dependent tumor regression was demonstrated in mice in a model of leukemia. Other examples of probes developed against notoriously difficult targets include β-catenin,132 and a range of PPIs.5 Further stabilization of α-helices can be achieved by a double cross-link,133 and are sometimes referred as ‘stitched’ peptides (Figure 9a).134

Figure 10: Examples of protein-structure mimetics. Color coding for amino acids: Light blue = β-amino acid; Purple = D-amino acid; Orange = unnatural amino acid used for ring closure metathesis. X indicates an amino acid whose identity has not been released.

Another approach utilizes α-helices inducers, such as proline. Other modifications relying, for example, on β-amino acids can achieve similar success (Figure 9a).135 In this particular case, the approach can be systematic, progressively scanning a sequence with repeats of an ααβ or αααβ pattern. For example, peptide hormones can be turned into so-called β-peptide foldamers. The


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repeat of an αααβ pattern was used in the sequence of parathyroid hormone PTH1(1-34) to generate a novel agonist of PTH1-R.136 This modification stabilized the α-helical structure of PTH1(1-34) and retained cAMP activity as exemplified by peptide 9 (Figure 10). In addition, the αααβ repeat resulted in increased proteolytic resistance and duration of action. Interestingly, in the context of GPCRs, introduction of β-amino acids can induce signaling bias as was shown in the case of the glucagon-like receptor 1 GLP1R.137 β-sheets are another secondary structure which can be effectively stabilized. The β-strand – turn – β-strand can be cyclized through interchain cross-links or through N-to-C terminus cyclization with a turn motif such as a D-Proline – L Proline-, biaryl-, and diketopiperazine-based systems (Figure 9b), and is sometimes referred as protein epitope mimetic (PEM).138 One prominent example is the mimicking of the antibacterial peptide protegrin I.139 The macrocyclization strategy followed by optimization led to potent peptides against several strains of Pseudomonas with strong selectivity against other bacteria as illustrated by peptide 8 (Figure 10). The overall profile enabled clinical evaluation, with a Phase III clinical study due to commence in 2018, demonstrating the strong potential of the approach. The β-hairpin stabilization has been applied to a range of biological targets, including proteases and chemokine receptors.140,141 While α-helices and β-sheets have been extensively pursued, loops represent an under-explored secondary motif. An analysis of interfaces revealed a propensity of loop motifs at several PPIs, which now offers the possibly to publicly search loop motifs with the LoopFinder algorithm.142,143 Loop motifs can be classically cyclized by forming a disulfide bridge (Figure 9c). One example is a cyclic peptide aimed at interacting with the TEAD transcription factor in the Hippo pathway, at the interface with one of its co-activator, YAP. Transcription factors are notorious for being ‘undruggable’ which calls for the use of different modalities. The PPI 29 ACS Paragon Plus Environment


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structure reveals the presence of an omega loop in YAP at the interface with TEAD, and this loop was selected for cyclization and optimization.144,145 Mutation of three key residues with noncanonical amino acids and disulfide cyclization afforded the lead peptide 10 with an IC50 of 25 nM (Figure 10). However, such cyclization induces an inherent metabolic soft spot through the disulfide. Therefore, an alternative strategy consists of cyclizing with reactive small molecule scaffolds, via covalent formation with cysteine and analogues, such as penicillamine (Figure 9c). The approach was applied to disrupt the interaction between stonin-2 and the epidermal growth factor substrate 15 (Ebs15) protein, which may reduce the Ebola virus infectivity. A hot loop from the stonin-2 containing ten amino acids was flanked with cysteine analogues and cyclized with a set of dibromomethyl aromatic systems to generate a range of macrocycles, including peptide 11.143 The best analogues bound Ebs15 with submicrolar affinity (compared with 18 µM for the uncyclized loop) (Figure 10). Pushing the concept of secondary structure stabilization further, emerging approaches are directed towards stabilizing tertiary structures. Such strategies are extremely attractive as they may deliver mini-proteins with antibody-like affinity but with much reduced molecular weight allowing for deeper tissue penetration and even potentially cell permeability in some cases. Using a computational approach, hyper stable peptides containing cross-links were designed to afford a range of tertiary protein structures.146 Remarkably, X-ray and NMR structures were accurately predicted by the in silico workflow, which will enable the design of tailored tertiary structures directed at specific biological targets. Overall, these approaches can systematically and rapidly deliver starting points, and are best suited for challenging extracellular and membrane-bound targets. They can also be effectively combined with other hit finding strategies.147 While intracellular delivery can in some cases be


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achieved, in particular with stapled peptides,148 this generally remains a challenge which still limits the potential for intracellular targets. As mentioned in section 2.1, an acceleration in the understanding of cellular uptake may progressively change this paradigm.45,46,47,48 Nevertheless, one fundamental point to consider in the context of probe and lead generation is the opportunity to use these protein structure mimetics as reporters for larger screens. Peptides can, for example, be tagged with a fluorophore, and then used in high throughput screening in a competition Fluorescence Polarization (FP) assay, potentially leading to novel small molecule hits with more optimizable properties. 4.2. Generation of stabilized peptidic starting points through scaffold grafting Alongside secondary and tertiary structure, unstructured peptide epitopes can also constitute starting points and be cyclized through a disulfide bridge as mentioned in section 4.1. An original alternative is to combine the binding affinity of the peptide epitope with a larger scaffold displaying high stability. This category of scaffolds is represented by cysteine-rich peptides present in many plants and organisms, and which display chemical, serum and proteolytic stability. Several scaffolds exist including cyclotides,149 sunflower trypsin inhibitor,150 and conotoxins.151 To leverage these scaffolds, parts of a sequence of one of the loops of a cysteinerich peptide is replaced by a peptide epitope sequence (Figure 11).152,153,154 This grafting process enables novel lead structures for a given biological target with both affinity and suitable properties. Typically, up to 10-15 amino acids can be grafted into a single loop. Beyond developing novel and stable binders, this strategy can also enable selectivity across isoforms. For example, a pentapeptide binder to the melanocortin receptor (MC-R) family, was grafted into the 6th loop of the cyclotide kalata B1 scaffold.155 Remarkably, analogue 12 displayed high affinity for the MC4 receptor with significant selectivity against the other isoforms 1, 2 and 5 (100-300 fold) (Figure 12). Cellular penetration remains a challenge, but grafting of a p53 peptide epitope 31 ACS Paragon Plus Environment


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into a cyclotide scaffold led to a 2 nM binder to HDM2 and displayed cellular activity in the micromolar range in a p53 deficient cell line,156 demonstrating the potential of the grafting concept for intracellular PPIs.

Figure 11: Principle of scaffold grafting

Figure 12: Example of cyclotide (Kalata B1) with a grafted MCR4 sequence

Overall, this approach is well suited to stabilize a known peptide epitope, derived for example from a ligand or binding partner. Due to the current limitation in cell permeability, the approach is best suited for challenging extracellular and membrane-bound targets, particularly when achieving selectivity is a key requirement. The approach can be demanding from a synthetic standpoint and requires specialist knowledge, which mostly comes from expert academic groups. 4.3.Massive virtual screening As a complementary strategy to HTS, virtual screening (VS) offers the promise of rapidly identifying potential de novo small molecule binders to a protein. VS can be broadly divided into structure-based VS (SBVS), which relies on docking putative ligands, and ligand-based VS 32 ACS Paragon Plus Environment

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(LBVS), which focuses on two-dimensional fingerprint or on three-dimensional pharmacophores and shape matching. For LBVS, only a ligand is required to perform the search. In the context of unprecedented targets, it therefore mainly applies to cases where a natural ligand, including cofactors and peptide is already known. Historically, SBVS has suffered from limited throughput (virtual screens in the region of 106 molecules maximum), partially as a consequence of limited computing power, as well as challenges in reliable scoring functions. With computing limitations progressively vanishing and the rise of machine learning algorithms,157 in addition to novel scoring methodologies,158,159 the question of library size and composition is becoming central. Since the size of small molecule chemical space is vast at over 1063 drug-like small molecules,160 rational selection approaches have to be considered,161 in particular since hits have to be synthesized to confirm their predicted activity. One approach consists in selecting commercially available compounds. For example, a large VS was performed on millions of molecules to identify inhibitors of EGFR kinase and BRD4.162 Another strategy consists in enumerating synthetically accessible de novo molecules,20 and libraries up to 1020, but typically more in the region of 1012-1015, can be generated today.163,164,165,166 Based on this strategy, scientists at Lilly designed a ‘proximal’ collection of molecules representing the in house rapidly synthesizable chemical space.167 The focus was put on robustness of chemical reactions and access from trusted vendors, leading to libraries typically in the region of 107-109, and up to 1011. Using this approach, selective inhibitors of the RIO2 kinase were identified. Specialized CROs and biotechnologies companies in the VS area include Exscientia, Relay Therapeutics and Nimbus Therapeutics. Overall, the VS strategy is still best suited for classical targets, with well-defined binding sites. In theory, a large screen can be performed in days to generate virtual hits. An important consideration with increased throughput and library sizes is the rapid access to hits from a 33 ACS Paragon Plus Environment


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synthetic standpoint. The power of the strategy can only be truly leveraged if many hits can be synthesized to confirm them. Therefore, synthetic technologies become an integral part of a massive VS paradigm. For example, establishing an expedited synthesis-purification platform can, beyond reducing hit optimization cycle time,168,169 be leveraged as a versatile approach to synthesize large numbers of hits generated from massive VS.170 While the ultimate development of VS coupled to machine learning algorithms should progressively reduce the number of false virtual hits, a burden on synthesis will remain as not every hit will likely be accessible through an automated platform. 4.4. Emerging structural technologies enabling structure-enabled lead generation As mentioned previously, the need for structure generation is implicit for all structure-enabled hit finding approaches. However, for numerous proteins, especially membrane proteins, generating structural information has proven challenging. Perhaps as a consequence, and despite the large numbers of GPCRs in the human genome, the number of GPCRs targets in the pipeline of pharmaceutical companies has tended to decrease over the last decade. While class A GPCRs have seen many successes with screening strategies for small molecules, delivering drugs for example for the adrenergic and opioid receptors, prosecuting class B and C GPCRs have resulted in limited success with small molecules. These receptors are often either orphan or in many cases their native ligands are large peptides, often calling for allosteric modulation when small molecules are chosen as a modality. Targeting allosteric modulation is extremely challenging, especially in absence of structure, but the development of new technologies over the last few years is shedding light on structural characteristics of such targets. For example, stabilization of the receptor with the StaR technology from Heptares Therapeutics has enabled the first crystal structure of prominent targets bound to agonists or antagonists such as the GLP1 class B GPCR.171172 In a fascinating example, a previously known antagonist of the Glucagon receptor 34 ACS Paragon Plus Environment

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was shown to bind to a novel allosteric binding site. The MK-0893 small molecule bound the receptor between the sixth and seventh transmembrane helix, likely resulting in prevention of Gprotein coupling and an antagonistic effect.173 The technology has brought stronger structural understanding on several other receptors, including CCR9,174 metabotropic glutamate receptors,175 the corticotropin releasing factor 1,176 and protease activated receptor 2.177 Beyond developing understanding, the approach opens up the possibility for fragment-based lead generation, although examples are yet to be published. Another strategy consists in stabilizing conformations linked to specific signaling pathways. The use of nanobodies has proven particularly successful to stabilize, for example, an active state of the β2 adrenergic receptor,178 and of its complex with the Gs protein,179 or for studying conformational changes during µ-opioid receptor activation.180 The approach of stabilizing pathway-specific conformations with nanobodies is being pursued by Confo Therapeutics to enable fragment-based screening of cryptic pockets, as exemplified with the β2-adrenergic receptor.181 In general, these structural approaches, aimed at generating starting points with bias towards a given signaling pathway through either experimental or virtual screening, may expand therapeutic opportunities of drugging GPCRs.182 With the pursuit of novel structures to enable fragment-based lead generation and more generally structure-based drug design (SBDD), novel technologies have emerged which expand the scope of tractable proteins from a structural standpoint. Notably, advances in cryo-EM over the past decade have increased the relevance of this technology to drug discovery. Structures of increasingly smaller protein size and better resolution have been enabled,19 for a range of biologically relevant targets including enzymes and ion channels.183,184,185,186,187 Typically, proteins with a lower limit of 64kDa and a resolution of around 3Å can be generated,19 which,



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however, remains insufficient to enable SBDD in most cases. Going beyond the isolated examples of high resolution structures such as glutamate dehydrogenase,184 and higher throughput, will transform the relevance of cryo-EM to lead generation for virtual screening, epitope mimetics and even potential fragment screening. In particular, the technique being particularly well suited for membrane proteins, promises of opening new opportunities in this field are high. Free electron X-ray laser (FEL) is another emerging technology.18 The principle leverages short and intense flashes of X-ray radiation at room temperature and may enable not only high resolution structure for proteins but also study of binding events. Indeed, the brilliance, which relates to the concentration of photons, is significantly higher than the current most advanced synchrotron, by a factor of up to 109.18 Moreover, the technology enables data acquisition on small and weaker diffracting crystals compared to other X-ray methodologies. Overall, data is acquired rapidly (femtoseconds) on tens of thousands of crystals, allowing time-resolved investigation of conformational changes, which is not possible with cryogenic methods, which tend to freeze conformations.188 Examples of structures generated with this technology include the angiotensin II receptor with crystals almost 3000 times smaller than those used to acquire a structure with similar resolution with cryo methods,189 the CPV17 polyhedrin,190 as well as membrane proteins such as ATPases,191 proton channels,192 the smoothened receptor,193 and the serotonin 5-HT2B receptor.194 Since access to FELs remain very limited, this promising technology has yet to demonstrate concrete examples in lead generation, although it is being pursued commercially, with for example LeadXPro. Overall, these structural technologies are a field to monitor for medicinal chemists as advances against the current limitations are likely to progressively enable relevant structure and therefore expand the scope of structure-enabled targets and the associated lead generation strategies. 36 ACS Paragon Plus Environment

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5. Probe and lead generation to modulate protein levels Modulating proteins levels can be achieved through three different MOAs. At the earlier stage of protein production, DNA may now be targeted with precise genome editing, in particular through the CRISPS/CAS9 system (Figure 13).7,8,9 In short, an RNA sequence usually referred as ‘guide’ recognizes the gene to be modified which is then cleaved off by the CAS9 endonuclease leading to a knockdown. Novel development in the technology has also enabled subsequent insertion of a gene. Two key challenges need to be addressed for this modality: delivery/distribution and safety (specificity). In addition, concerns related to the innate immune response against the CAS protein have been raised recently.195 Delivery usually relies on viral vectors which have a capacity limitation and may therefore trigger immune response from the resulting high quantity of virus needed. Furthermore, this approach is to-date limited to few tissues: liver, muscle, eye. Despite being considered as cell therapy, medicinal chemists are encouraged to monitor developments and build awareness in this area since this modality may deliver very relevant probes for target validation.



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Figure 13: MOAs of modalities regulating protein levels At the second level, protein levels may be regulated through modulation of transcription and though up- or down-regulation of translation by interfering at different points of RNA regulation processes and modulate splicing (Figure 13). This may be achieved through several oligonucleotides approaches but also with classical small molecules, and more recently with CRISPR.10 At the third and last level, once the protein has actually been expressed, modulation of protein levels may be achieved by inducing protein degradation (Figure 13). Hit finding for these different MOAs is described in this section. 5.1. Modulation of transcription and translation with oligonucleotides From a lead generation perspective, oligonucleotides differ significantly from other modalities since developing a hit or lead originates from a targeted RNA sequence, incorporating several modified nucleotides to increase their stability (Figure 14). Oligonucleotides can be divided in 38 ACS Paragon Plus Environment

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several categories. Importantly, different types of oligonucleotides enable targeting of RNA at different points of the regulation machinery (Figure 13). Briefly, Antisense Oligonucleotides (ASOs) and small interfering RNA (siRNA) can be aimed at splicing correction by blocking a specific sequence, but also downregulation of a protein at the mRNA level, either via direct interaction with mRNA or via the RISC complex. ASOs and siRNA differ significantly: ASOs are single stranded oligonucleotides which are typically composed of 16 to 20 nucleotides, while siRNA are made of 20-25 oligonucleotides and are double stranded. In addition, siRNA act specifically via the RISC complex. The majority of ASOs target directly mRNA, but this modality may also be directed at or mimic the so-called micro RNAs (miRNAs). miRNA are non-coding RNA species which bind to the argonaut protein to form the RISC complex, which then recognizes a complementary mRNA sequence and is followed by degradation of the mRNA by RNAses (Figure 13). On one hand, when mimicking a miRNA, ASOs form part of the RISC complex in place of the miRNA and therefore promote mRNA degradation. On the other hand, and very interestingly, developing an ASO with a complementary sequence to a miRNA, blocks its function and therefore results in an up-regulation of mRNA and consequently of the encoded protein. Essentially, this means that ASOs can be used for both down- and up-regulating protein levels, although intervention at the miRNA remains an underdeveloped area of research. Several ASOs are either marketed, including fomivirsen, mipomersen, eteplirsen and nusinersen, or in clinical development. siRNA has experienced less success, although patisiran is due to become the first RNA inference drug to be approved. Separately, modified RNA (modRNA) brings a different paradigm to drug discovery by increasing levels of downregulated proteins. An oligonucleotide composed of 1000s of modified nucleotides is produced biosynthetically with RNA polymerases. The resulting RNA can be transfected and then be translated into a native or modified protein of interest intracellularly. The 39 ACS Paragon Plus Environment


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technology is still emerging although several programs have entered the clinic and the most advanced AZD8601 is due to enter Phase IIa in 2018, in heart failure patients undergoing cardiac bypass grafting (CABG) surgery. From a lead generation point of view, oligonucleotides are very different to modalities directly modulating protein functions. Being derived from the sequence of the targeted mRNA, they indeed afford systematically lead sequences through an established workflow, which aims at either accomplishing sequence complementarity via base-pairing or sequence mimicry. Essentially, a rule-based approach is used for ASOs to design compounds that must target unstructured regions. Indeed, the potential formation of secondary and tertiary RNA structure may render a particular sequence inaccessible to a complementary oligonucleotide sequence. For this reason, hundreds of sequences are typically screened, albeit also looking at safety markers. Interestingly, the approach is very fast with a typical target selection to clinical candidate cycle time of 18 months in the case of ASOs.

Figure 14: Examples of modified nucleotides. Modification of the carbohydrate: MOE = methoxyethyl; cEt = constrained ethyl; LNA = locked nucleic acid. The nucleobase is represented in green, and the replacement of the phosphate group by a thiophosphate is highlighted in light blue


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Overall, oligonucleotide approaches enable modulation of protein levels through different points of action. Lead generation follows a rule-based strategy, derived from sequences, and these approaches are particularly well suited for challenging intracellular targets. Importantly the MOA means that any scaffolding function is removed through the induced protein knockdown. Modified RNA is uniquely positioned for up-regulation of protein levels as well as for the expression of intracellular antibodies. Importantly, these technologies are highly complex and access is only possible through partnerships with specialized companies such as Ionis Pharmaceuticals (formerly known as Isis Pharmaceuticals), Alnylam, Moderna, Ethris or Curevac. One important limitation is tissue access through systemic delivery (typically liver, spleen and kidney) implying in some cases a need for local delivery. In addition, some cell types are refractory to oligonucleotide uptake. 5.2. Small molecule phenotypic approaches to modulate translation Historically, several small molecules have been known to interfere with DNA, including intercalants and alkylating agents. However, this approach is rather gene-unspecific and leads to general cytotoxicity or genotoxicity. A more precise field of intervention, which can directly affect protein levels is at the mRNA and non-coding RNA level. RNA can indeed form secondary, tertiary and quaternary structures, including loops, hairpins and bulges, which can be seen as pockets, much alike proteins. In addition, RNA interacts with protein creating proteinRNA interfaces and creating small molecule binding sites. Consequently, small molecules may well be a viable modality for interacting with RNA to control biological function.196,197,198 The first examples of lead generation for targeting RNA have predominantly been the results of phenotypic assays, directed to specific cellular events. For example, targeting the splicing process of the Survival of Motor Neurons (SMN) protein in the context of spinal muscular atrophy (SMA), a cell-based HTS was performed using a SMN2 gene reporter assay.199 Hits were 41 ACS Paragon Plus Environment


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confirmed by dose response and qPCR to confirm splicing activity, together with an ELISA assay to assess SMN protein levels. One scaffold, based on pyridazines, was optimized and led to compounds with a cellular activity of 5-20 nM, as exemplified by compound 13 (Figure 15). Further evaluation in vivo, in a model of SMA demonstrated dose-dependent increase in the levels of the SMN protein both in the brain and spinal cord. Using the most efficacious compound, the mode-of-action could be elucidated and was shown to involve specific splice variants. More specifically, the small molecule stabilized a protein-RNA complex consisting of the U1 small nuclear ribonucleic protein (snRNP) from the spliceosome, and the SMN2 premRNA. Interestingly, another small molecule scaffold was also reported to modify SMN2 splicing, although the mode of action was not elucidated at the time.200 In another example of phenotypic screening, a cell-based HTS was performed to identify modulators of PCSK9 secretion from CHO cells, a key target involved in the regulation of cholesterol levels with implication in cardiovascular diseases and obesity.13 While displaying only micromolar activity, the reported hit 14 was not cytotoxic and did not affect secretion of a control protein, pointing to a specific effect (Figure 15). The inhibitor was subsequently shown to affect the level of the PCSK9 protein by reducing its synthesis at the translational level, and could be linked to inhibition of the 80S ribosome. Interestingly, the inhibitor was also shown to be orally active in vivo in rats, reducing plasma PCSK9 and cholesterol levels.201

Figure 15: Examples of small molecules binders to RNA


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Overall, the PCKS9 example is particularly exciting in the context of novel projects. Efforts to identify small molecule direct binders and effectors of PCSK9 have repeatedly failed, and the development of now marketed antibodies have been a more logical and successful approach. However, aiming at phenotypically modulating PCSK9 levels as was performed by the team at Pfizer, opens up the thinking that PPIs including secreted proteins may be targetable with small molecules via modulation of translation. Consequently, a paradigm-shifting hit finding strategy would be to regularly implement phenotypic screening for identifying different modulators of protein levels perhaps in parallel to target-based screening on that specific protein. 5.3. Modulation of translation with small molecules through direct RNA targeting The previous examples highlight the potential of interacting with some RNA species or complexes, as a result of phenotypic approaches. A range of approaches and assays has been developed to identify interactions of molecules with RNA, but this has to-date not being truly amenable to HTS of small molecules.202,203 Rationally targeting RNA requires novel approaches as robust HTS assays are lacking, although a new strategy has recently been published. One approach relies on so-called two dimensional combinatorial screen (2DCS) in a library-versuslibrary selection strategy (Figure 16).4 Agarose-coated small molecules microarrays are screened against libraries of RNA, and bound RNA can be isolated by gel excision and analyzed by reverse transcription PCR and sequencing. To deconvolute RNA sequences, a statistical method, called Structure-Activity Relationships Through Sequencing (StARTS) is applied, leading to small molecule-RNA secondary structure relationships. Importantly, this screening strategy is target-agnostic and can be seen as knowledge generation, which can be mined once a specific RNA sequence is selected as a target. The resulting database becomes a hit finding strategy dubbed Inforna.204 For example, the approach was leveraged to identify small molecule binders of a toxic CUG RNA repeat responsible for myotonic dystrophy type 1.205 A broader application 43 ACS Paragon Plus Environment


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has been developed to target miRNA, which are key regulators of coding RNA (Figure 13). Mature miRNAs are generated after processing by the Dicer and Drosha enzymes and pri- and pre-miRNA exhibit structures targetable by small molecules at the Dicer and Drosha cleaving sites. Following the Inforna approach, binders of the oncogenic miRNA18a resulted in upregulation of the STK4 kinase and subsequent apoptosis.206 Similarly, a miRNA210 binder could be identified and was shown to up-regulate the GDP1L enzyme and resulted in reduction in HI1a levels and induction of apoptosis in a breast cancer cell line. 207

Figure 16: principle of 2D combinatorial libraries and inforna Overall, the field of hit finding for direct RNA targeting is in its infancy. Other examples exist in the field for antibiotics, as illustrated by a specific screening strategy to target riboswitches.208 Considering the presumed highly flexible and dynamic structure of RNA, structure-based VS approaches are likely to remain challenging, although examples exist.209 There is a clear need for novel, especially high throughput, screening and structural approaches. Very recently, an assay based on affinity-selection mass spectrometry was developed specifically for RNA.210 Using this so-called Automated Ligand Detection System (ALIS) approach, 53,000 compounds were screened against a range of bacterial riboswitches and led to the identification of selective small


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molecules binders. More developments like this latest example are needed. In fact, more basic research is needed to leverage the potential of targeting RNA with synthetic molecules and develop novel lead generation approaches. Novel collaborations will therefore likely be required, as illustrated by the Chemical Genomics Centre of the Max Planck Society in Dortmund (Germany), which plans to investigate the druggability of RNA and RNA-Protein complexes from 2018 onwards. Beyond the interest in large pharmaceutical companies, several biotechnology companies have been created around this field including PTC Therapeutics, Arrakis Therapeutics, Nymirium, and very recently Expansion Therapeutics. Notably, the nature of the interactions with RNA may provide opportunities for small peptides and hybrid molecules to achieve higher level of selectivity.

Figure 17: principle of PROTAC 5.4. Direct modulation of protein levels through induction of degradation Inducing protein degradation to modulate protein levels represents an emerging MOA,211,212,213 and the main approach is based on small molecules forming a new modality called proteolysis targeting chimera (PROTAC). 214,215 Two ligands, one aimed at binding an E3 ligase and the other aimed at binding a protein of interest, are connected via a linker. Consequently, a PROTAC essentially brings together the two proteins, leading to ubiquitination of the targeted protein and its subsequent degradation by the proteasome (Figure 17). Two E3 ligase binders have repeatedly 45 ACS Paragon Plus Environment


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been used with thalidomide derivatives targeting the cereblon (CRBN) E3 ubiquitin ligase and small molecules ligands targeting von Hippel-Lindau (VHL) E3 ligase. For example, the VHL ligand was connected to binders of the TBK1 kinase, varying the length of the linker between the two entities 216. A 12 nM PROTAC 15 could be identified, which achieved degradation of TBK1, while achieving selectivity against related kinases (Figure 18). Considering the size of the chimera, cell permeability could be perceived as a challenge, but cellular activity has repeatedly been observed for many PROTACs. Since the degradation process is catalytic, only low intracellular concentrations are required to achieve degradation. One important consideration to keep in mind when selecting this MOA is the associated alleviation of any non-canonical and scaffolding function of the protein which is degraded. The attractive feature of PROTAC lies in the mere requirement of a binder rather than a functional inhibitor. Thus, the complete panel of structure-agnostic and structure-enabled lead generation strategies, especially DELs, can be considered to quickly identify a binder. Interestingly, the ligand to the protein of interest does not have to be limited to small molecules, and stabilized peptides were recently used to degrade the Estrogen Receptor α.217 Furthermore, PROTAC 16 based on a peptidic binding ligand to the KEAP1 E3 ligase has also been reported (Figure 18).218


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Figure 18: Examples of PROTACs. Color coding for amino acids: Purple = D-aminoacid. Overall, when considering direct protein degradation as MOA and PROTAC as the corresponding modality, the whole range of hit finding technologies described in section 2, 3 and 4 can be applied to identify binders to the protein of interest. Noteworthy, several biotechnology companies have been founded around this concept, including Arvinas, C4 Therapeutics and Kymera Therapeutics. An important additional step for identifying a probe or lead is however to successfully link the binder to an E3 ligase ligand. In this field, more research is required to robustly, rather than empirically, select the most effective linkers, and synergies with the field of drug conjugates for targeted delivery represent a vast opportunity to accelerate knowledge.

6. Selecting the ‘right’ probe and lead generation strategies 6.1. a MOA-centric framework The various structure-agnostic and structure-enabled hit finding strategies together with sequence-based approaches for oligonucleotides provide a vast repertoire of techniques to identify starting points against many targets. As mentioned at the beginning of this perspective, medicinal chemists are presented with the opportunity to place MOA at the center of any strategy. We believe that this modality-agnostic, MOA-centric paradigm maximizes the relevance and rationale of the chosen hit finding strategies. Within this paradigm and the framework summarized on Figure 19, biological knowledge on the target, pathway and disease has to drive selection of the MOA(s) by first considering at which level the target should/could be modulated: DNA, RNA or protein. In parallel, phenotypic approaches may be run in a target-agnostic or target-biased strategy. An associated consideration, is whether the target is located intra- or extra-cellularly and if the target product profile (TPP)



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exclusively requires oral administration. Furthermore, the tissue where the target is located has to be considered since delivery of some modalities can be challenging (for example, ASOs predominantly distribute to the liver, spleen and kidney). These parameters drive selection of the MOA(s), followed by selection of modalities, which clearly must be the result of a dialogue across the project team. Medicinal chemists must however be familiar with the different considerations in order to effectively influence decision-making. The next level of potential triaging is to determine whether structural information is available on the target or can be readily generated, either through homology models or through straightforward protein crystallization or NMR. This is of course most relevant for the MOAs based on protein binders. This stepwise approach unravels the range of possible hit finding strategies (Figure 19). Noteworthy, and as mentioned previously, structure-enabled targets may leverage all hit finding approaches including those that do not require structural information such as HTS or DELs. The decision on which strategy(ies) to select out of the hit finding tool kit is then project- and institution-specific. Nevertheless, an educated choice can be made based on the strengths and limitations of each approach, and these are summarized in the next sub-section.


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Figure 19: Mode-of-action centric hit finding. DNA is transcribed into RNA which is then translated into either an extracellular/membrane-bound protein, or an intracellular protein. When the target modulation is agnostic and/or the target is unknown, phenotypic screening may be used directly as illustrated within the first level oval.

Importantly, the stepwise guide presented above should only be seen as a pragmatic and modular framework rather than a ‘one size fits all’ process, since specific targets, corporate resources and capabilities, and competitive landscape are all factors influencing decision-making. Other factors driven by the disease to treat are also important for modality selection, including patient compliance and unmet medical need. 6.2. Strengths and limitations of hit finding strategies The pros and cons of each probe and lead generation approach have been discussed in their respective sections. In order to summarize these, a heat map to guide selection of the most suited strategies of a target-specific hit finding toolkit is provided on Figure 20. Some of the key criteria to take into account are: the time from project inception to the first hits; the cost of running the approach and/or to access it externally. In addition, the other criteria are directly aligned with the MOA-centric framework discussed in the previous sub-section, in particular target localization (intra-, extracellular or membrane-bound) and suitability for oral administration. Furthermore, proteins may be split into two categories: classical (many enzymes including kinases, to some extent ion channels, proteases, and class A GPCRs) and challenging (PPIs including E3 ligases and transcription factors, phosphatases, etc.). Taking DELs as an example (fifth row), the strengths and limitations are found from left to right on Figure 20. DELs take a few months for hit identification (orange). In terms of costs, it usually requires a partnership (red P), although fee-for-service is emerging (orange). It is well suited for most targets, both intracellular (green) and extracellular (green). Membrane-proteins are less


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desirable due to technical challenges (red). Challenging targets are a viable option (orange) depending on suitability of larger small molecules. It generates molecules that should be optimizable for oral delivery (green).

Figure 20: Heat map for the strength and limitations of hit finding strategies and their associated modalities. *cell and tissue specific. P: Requires partnership with specialized company.

6.3.Phasing hit finding approaches Notably, several hit finding technologies and several modalities may be relevant for a given target. In some companies, there has been a tendency to either run multiple approaches in parallel or to only run one single approach. Rather than considering a once-for-all process selection but also a single modality track, there is an opportunity for a more agile and dynamic strategy,



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running multiple waves of hit finding at different time points. This may foster speed and maximize impact from knowledge generation, with each wave of hit finding informing the next one.219 Furthermore, one initial modality, for example a modified peptide, may represent a chance to build further knowledge on a target by affording a probe for target validation and then be pursued as a front runner track. It may also be used to generate a reporter molecule, for screening another modality such as small molecules. This overall integrated hit finding and integrated modality landscape approach maximizes the chances of finding relevant and multiple chemical series. To illustrate this concept, an example is presented on Figure 21. Overall, following the guiding principles of this modality-agnostic MOA-centric paradigm, novice and experienced medicinal chemists have the opportunity to make an informed decision for the right approaches and to enable robust probe and lead generation. Importantly, with knowledge across science expanding fast, regular updating of the state-of-the-art for each approach and modality has also to be captured and implemented to constantly adopt the most relevant strategies.

Figure 21: Example of phased hit finding waves integrating multiple strategies and modalities 52 ACS Paragon Plus Environment

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7. Outlook: additional opportunities for probe and lead generation Armed with the knowledge from the previous sections, medicinal chemists may further expand their range of opportunities by considering the upcoming wave of novel hit finding approaches and technologies. In this respect, this final section illustrates the continuous flow of novel ideas to systematically find more and better starting points. As was discussed in section 3, the question of the ideal proprietary compound collection size resurfaces regularly. An HTS can typically screen 106 compounds, and DNA-encoded libraries 109. In comparison to the estimated drug-like chemical space of 1063, this does not even ‘scratch the surface’. Accessing different chemical space is therefore a recurrent theme, and progress in synthetic biology is enabling novel molecules to be generated. For example, Nature is able to synthesize complex molecules including peptides without relying on the classical ribosomal machinery. The field of natural products typically results from the expression of enzymes carrying synthetic transformations, which are combined in a sequence. These so-called biosynthetic gene clusters can be mined to either exploit novel compounds synthesis from organisms, or reshuffled and recombined to generate novel entities. For example, LifeMine Therapeutics is exploiting fungi and Lodo Therapeutics bacteria from soil. GyreOx is also generating collections of macrocyclic compounds by leveraging specific enzymes based on academic research.220 Excitingly compound synthesis can be combined with a readout assay (linked to survival), also called biosensor. One specific example is the discovery of novel Brome mosaic virus inhibitors at the company Evolva.221 Selection and recombination of biosynthetic gene clusters including alkaloid, flavonoid, polyunsaturated fatty acid was performed to generate artificial chromosomes in yeast. Following transfection, yeast cells were subjected to the virus and survival clones were selected for



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deconvolution. Upon characterization, 74 new lead-like compounds could be identified and 28 showed activity in a secondary assay. Another powerful example for the discovery of cyclic peptides starting points is the intracellular, traceless method referred as SICLOPPS (split intein synthesis of proteins and peptides), which can display in the region of 106 cyclic peptides, typically 5- to 7-mers. SICLOPPS can be associated with the so-called reverse two-hybrid system (RTHS), which links cell survival to inhibition of a PPI of interest.222 The principle, which can also be extended to unnatural amino acids,223 has been applied to several PPIs, including to the hypoxia-inducible factor 1 (HIF-1) transcription factor heterodimer with the identification of a 124 nM binding affinity inhibitor.224 In order to broadly explore chemical space governed by a biological target of interest, one proposal is to link mutation to the synthesis and assaying of the compound, in essence leveraging directed evolution.225 In such a paradigm, cells such as yeast are engineered to synthesize small molecules or peptides and are subjected to a selection pressure linked to a biological target of interest. Surviving cells are selected and subjected to mutations to improve the synthesized inhibitor, and this cycle of pressure, selection and mutation is repeated until a potent inhibitor can be identified. While major advances will have to be seen, emerging examples in the field of proteins are illustrating the potential of the approach.226 Such a paradigm is likely to revolutionize hit finding for challenging targets. On the screening side, HTS is still widely used but presents the disadvantage of a labor intensive and generally over reductionist assay systems, typically not representative of the complexity of a pathological three-dimensional cell environment. Microfluidic cell chips are under development to mimic those environments at the microscale level. Several technical challenges on material, miniaturized devices, still need to be overcome to make this technology the future screening process closer to the targeted disease.227 54 ACS Paragon Plus Environment

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Finally, artificial intelligence (AI) is likely to significantly impact probe and lead generation in the coming years. Beyond the hype of the technology, the opportunity to broadly mine data to identify hits and also the possibility to further leverage virtual screening should provide a further extended set of hit finding strategies. AI is also being increasingly leveraged for synthesis planning, which should facilitate rapid synthesis of virtual hits.228,229 In addition, machine learning may enable the design of bifunctional molecules of great value for many diseases. The company Exscientia, in collaboration with Evotec, has recently presented the discovery of a small molecule simultaneously antagonizing the Adenosine 2A receptor and inhibiting CD73.230

8. Conclusion With the rise of genomics and other target identification strategies, the need for probes and leads to validate and drug these targets is increasing dramatically. In this respect, the expanding range of approaches both for random screening and rational design are providing medicinal chemists with the opportunity to effectively identify starting points. Acceleration of knowledge across disciplines is also further expanding the hit finding toolkit, illustrated, for example, with the recently published expansion of the genetic code with two additional nucleobases which would further increase the high diversity of genetic encoding technologies including phage and mRNA display.231 Additional opportunities exist, including bringing together modalities, as illustrated by drug-conjugates.232 From a decision-making standpoint, the opportunities to leverage protein degradation, to stabilize protein-protein interactions and to activate pathways also trigger a need to revise screening strategies against novel targets. The possibility to address these additional MOAs, together with the opportunity to use other New Modalities, calls indeed for a shift from an almost ‘all inhibition’ mindset around targets with systematic hit finding strategies, to a modality-agnostic, MOA-centric hit finding paradigm. In order to exploit these expanded probe 55 ACS Paragon Plus Environment


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and lead generation opportunities, new models of collaboration and partnerships are required. Apart from very large pharmaceutical companies, it is unlikely that many companies can develop all capabilities in house. Even in large pharmaceutical companies, boundaries are relaxing, with a redefinition of the pre-competitive concept, illustrated by the exchange of compounds between AstraZeneca and Sanofi, an approach still inconceivable in company cultures focused on inwards innovation. Enabling this MOA-centric paradigm requires to overcome several challenges, predominantly cultural. First, an outwards-looking and networked innovation corporate culture is necessary, embracing the fact that expertise in specific areas likely sits outside the company, and represents an opportunity to combine internal and external knowledge. Second, the expansion of drug modalities may require some level of reskilling for drug discovery scientists to become fluent with these new types of molecules. Finally, the broadening of MOAs further blurs the boundaries between medicinal chemistry and chemical biology with the associated need of building a broader skillset beyond drug design. Seeing these challenges as an exciting opportunity to foster creativity, with new modalities, new technologies, and new partnerships, medicinal chemists are equipped like never before to tackle the challenges of answering increasingly complex biological questions and developing starting points towards new drugs.

Corresponding author information: Eric Valeur: Telephone number: +46 706384056; email address: [email protected]; Patrick Jimonet: Telephone number: +33158938738; email address: [email protected]

Biographic sketch: Eric Valeur


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Eric Valeur is Director of New Modalities & Innovation, Medicinal chemistry at AstraZeneca, Sweden. Over his 15 years Drug Discovery experience gained at Merck, Novartis and AstraZeneca, he has led medicinal chemistry teams across several therapeutic areas and across modalities, including small molecules, macrocycles, peptides, oligonucleotides, drug conjugates and PROTACs, delivering clinical candidates and novel technologies. He is passionate about challenging targets and disruptive drug discovery, including outwards-looking innovation partnerships, such as the AstraZeneca - Max Planck Institute Satellite Unit he currently leads, and which is based in the group of Prof. Herbert Waldmann. Eric is co-author/co-inventor of over 50 publications, patents, posters and oral presentations, and holds a PhD from the University of Edinburgh (under supervision of Prof. Mark Bradley) and an MBA from Imperial College London. Patrick Jimonet Patrick Jimonet’s present role at Sanofi is to bring external innovation to the early portfolio and the Lead Generation process; his particular focus is on modalities, technologies and innovative biology through various and novel business models and partnerships. Previous assignments were the identification of key areas for improvement in early phases of discovery, leadership of a global interdisciplinary team responsible for the identification of novel GPCR targets and validated tool compounds, manager of multi-cultural teams of PhDs, project leader of projects from hit finding to clinical candidate. All activities have been performed at Sanofi and predecessor companies in France and in the US. Patrick received his PhD in chemistry at the Institut de Chimie des Substances Naturelles of the CNRS in Gif-sur-Yvette, France. Acknowledgement



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The authors are grateful to Dr. Alleyn Plowright, Dr. Peter Hamley and Dr. Andrew Davis for critical review of the manuscript, and to Dr. Graham Smith and Dr. Samantha Hughes for providing critical inputs for the RNA and VS sections respectively.

Abbreviation ALIS, Automated Ligand Detection System; ASO, antisense oligonucleotide; cEt: constrained ethyl; CETSA, cellular thermal shift assay; CRO, contract research organization; CRISPR, clustered regularly interspaced short palindromic repeats; DEL, DNA-encoded library; EM, electron microscopy; FEL, free electron laser; HTS, high throughput screening; iPS, induced pluripotent stem (cell); MOA, mode-of-action; modRNA, modified RNA; PEM, protein epitope mimetic; PPI, protein-protein interaction; PROTAC, proteolysis targeting chimera; qPCR, quantitative polymerase chain reaction; RaPID, random nonstandard peptide integrated discover; RTHS, reverse two-hybrid system; SBDD, structure-based drug design; SELEX, systematic evolution of ligands by exponential enrichment; SICLOPPS, split intein synthesis of proteins and peptides; siRNA, silencing RNA; SMA, spinal muscular atrophy; SPR, surface plasmon resonance; StARTS, structure-activity relationships through sequencing; TPP, target product profile; VS, virtual screening. References (1)

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