Structure-Based Optimization of a Series of Covalent, Cell Active Bfl-1 Inhibitors.

Bfl-1, a member of the Bcl-2 family of proteins, plays a crucial role in apoptosis regulation and has been implicated in cancer cell survival and resistance to venetoclax therapy. Due to the unique cysteine residue in the BH3 binding site, the development of covalent inhibitors targeting Bfl-1 represents a promising strategy for cancer treatment. Herein, the optimization of a covalent cellular tool from a lead-like hit using structure based design is described. Informed by a reversible X-ray fragment screen, the strategy to establish interactions with a key glutamic acid residue (Glu78) and optimize binding in a cryptic pocket led to a 1000-fold improvement in biochemical potency without increasing reactivity of the warhead. Compound (R,R,S)-26 has a kinact/KI of 4600 M-1 s-1, shows <1 µM caspase activation in a cellular assay and cellular target engagement, and has good physicochemical properties and a promising in vivo profile.

Fragment-Based Discovery of Novel MUS81 Inhibitors.

MUS81 is a structure-selective endonuclease that cleaves various branched DNA structures arising from natural physiological processes such as homologous recombination and mitosis. Due to this, MUS81 is able to relieve replication stress, and its function has been reported to be critical to the survival of many cancers, particularly those with dysfunctional DNA-repair machinery. There is therefore interest in MUS81 as a cancer drug target, yet there are currently few small molecule inhibitors of this enzyme reported, and no liganded crystal structures are available to guide hit optimization. Here we report the fragment-based discovery of novel small molecule MUS81 inhibitors with sub-µM biochemical activity. These inhibitors were used to develop a novel crystal system, providing the first structural insight into the inhibition of MUS81 with small molecules.

Design of a Lead-Like Cysteine-Targeting Covalent Library and the Identification of Hits to Cys55 of Bfl-1.

Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.

Identification and Evaluation of Reversible Covalent Binders to Cys55 of Bfl-1 from a DNA-Encoded Chemical Library Screen.

Bfl-1 is overexpressed in both hematological and solid tumors; therefore, inhibitors of Bfl-1 are highly desirable. A DNA-encoded chemical library (DEL) screen against Bfl-1 identified the first known reversible covalent small-molecule ligand for Bfl-1. The binding was validated through biophysical and biochemical techniques, which confirmed the reversible covalent mechanism of action and pointed to binding through Cys55. This represented the first identification of a cyano-acrylamide reversible covalent compound from a DEL screen and highlights further opportunities for covalent drug discovery through DEL screening. A 10-fold improvement in potency was achieved through a systematic SAR exploration of the hit. The more potent analogue compound 13 was successfully cocrystallized in Bfl-1, revealing the binding mode and providing further evidence of a covalent interaction with Cys55.

Discovery and Optimization of the First ATP Competitive Type-III c-MET Inhibitor.

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.

Fragment screening at AstraZeneca: developing the next generation biophysics fragment set.

Fragment based drug discovery is a critical part of the lead generation toolbox and relies heavily on a readily available, high quality fragment library. Over years of use, the AstraZeneca fragment set had become partially depleted and instances of compound deterioration had been found. It was recognised that a redevelopment was required. This provided an opportunity to evolve our screening sets strategy, whilst ensuring that the quality of the fragment set met the robust requirements of fragment screening campaigns. In this communication we share the strategy employed, in particular highlighting two aspects of our approach that we believe others in the community would benefit from, namely that; (i) fragments were selected with input from Medicinal Chemists at an early stage, and (ii) the library was arranged in a layered format to ensure maximum flexibility on a per target basis.

Discovery of a selective c-MET inhibitor with a novel binding mode.

The c-MET receptor tyrosine kinase has received considerable attention as a cancer drug target yet there remains a need for inhibitors which are selective for c-MET and able to target emerging drug-resistant mutants. We report here the discovery, by screening a DNA-encoded chemical library, of a highly selective c-MET inhibitor which was shown by X-ray crystallography to bind to the kinase in an unprecedented manner. These results represent a novel mode of inhibiting c-MET with a small molecule and may provide a route to targeting drug-resistant forms of the kinase whilst avoiding potential toxicity issues associated with broad kinome inhibition.

Surprising lipophilicity observations identify unexpected conformational effects.

Contrary to expectation N-aryl pyrrolidinones (and isosteric imidazolinones and oxazolinones) are more lipophilic and less soluble than the corresponding piperidinones (tetrahydropyrimidinones and oxazinones). Exploration of the basis for these results uncovered a subtle interplay of steric and electronic effects that result in different conformations for the two classes of compounds which drive the observed effects.

Chemogenetics defines a short-chain fatty acid receptor gut-brain axis.

Volatile small molecules, including the short-chain fatty acids (SCFAs), acetate and propionate, released by the gut microbiota from the catabolism of nondigestible starches, can act in a hormone-like fashion via specific G-protein-coupled receptors (GPCRs). The primary GPCR targets for these SCFAs are FFA2 and FFA3. Using transgenic mice in which FFA2 was replaced by an altered form called a Designer Receptor Exclusively Activated by Designer Drugs (FFA2-DREADD), but in which FFA3 is unaltered, and a newly identified FFA2-DREADD agonist 4-methoxy-3-methyl-benzoic acid (MOMBA), we demonstrate how specific functions of FFA2 and FFA3 define a SCFA-gut-brain axis. Activation of both FFA2/3 in the lumen of the gut stimulates spinal cord activity and activation of gut FFA3 directly regulates sensory afferent neuronal firing. Moreover, we demonstrate that FFA2 and FFA3 are both functionally expressed in dorsal root- and nodose ganglia where they signal through different G proteins and mechanisms to regulate cellular calcium levels. We conclude that FFA2 and FFA3, acting at distinct levels, provide an axis by which SCFAs originating from the gut microbiota can regulate central activity.

Discovery of AZD8154, a Dual PI3Kγδ Inhibitor for the Treatment of Asthma.

Starting from our previously described PI3Kγ inhibitors, we describe the exploration of structure-activity relationships that led to the discovery of highly potent dual PI3Kγδ inhibitors. We explored changes in two positions of the molecules, including macrocyclization, but ultimately identified a simpler series with the desired potency profile that had suitable physicochemical properties for inhalation. We were able to demonstrate efficacy in a rat ovalbumin challenge model of allergic asthma and in cells derived from asthmatic patients. The optimized compound, AZD8154, has a long duration of action in the lung and low systemic exposure coupled with high selectivity against off-targets.

Fragment-Based Design of a Potent MAT2a Inhibitor and in Vivo Evaluation in an MTAP Null Xenograft Model.

MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.

Fragment-Based Discovery of Novel Allosteric MEK1 Binders.

The MEK1 kinase plays a critical role in key cellular processes, and as such, its dysfunction is strongly linked to several human diseases, particularly cancer. MEK1 has consequently received considerable attention as a drug target, and a significant number of small-molecule inhibitors of this kinase have been reported. The majority of these inhibitors target an allosteric pocket proximal to the ATP binding site which has proven to be highly druggable, with four allosteric MEK1 inhibitors approved to date. Despite the significant attention that the MEK1 allosteric site has received, chemotypes which have been shown structurally to bind to this site are limited. With the aim of discovering novel allosteric MEK1 inhibitors using a fragment-based approach, we report here a screening method which resulted in the discovery of multiple allosteric MEK1 binders, one series of which was optimized to sub-µM affinity for MEK1 with promising physicochemical and ADMET properties.

Structural Basis for Targeting the Folded P-Loop Conformation of c-MET.

We report here a fragment screen directed toward the c-MET kinase from which we discovered a series of inhibitors able to bind to a rare conformation of the protein in which the P-loop adopts a collapsed, or folded, arrangement. Preliminary SAR exploration led to an inhibitor (7) with nanomolar biochemical activity against c-MET and promising cell activity and kinase selectivity. These findings increase our structural understanding of the folded P-loop conformation of c-MET and provide a sound structural and chemical basis for further investigation of this underexplored yet potentially therapeutically exploitable conformational state.

Phenotypic high-throughput screening platform identifies novel chemotypes for necroptosis inhibition.

Regulated necrosis or necroptosis, mediated by receptor-interacting kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like protein (MLKL), contributes to the pathogenesis of inflammatory, infectious and degenerative diseases. Recently identified necroptosis inhibitors display moderate specificity, suboptimal pharmacokinetics, off-target effects and toxicity, preventing these molecules from reaching the clinic. Here, we developed a cell-based high-throughput screening (HTS) cascade for the identification of small-molecule inhibitors of necroptosis. From the initial library of over 250,000 compounds, the primary screening phase identified 356 compounds that strongly inhibited TNF-α-induced necroptosis, but not apoptosis, in human and murine cell systems, with EC50 < 6.7 µM. From these, 251 compounds were tested for RIPK1 and/or RIPK3 kinase inhibitory activity; some were active and several have novel mechanisms of action. Based on specific chemical descriptors, 110 compounds proceeded into the secondary screening cascade, which then identified seven compounds with maximum ability to reduce MLKL activation, IC50 >100 µM, EC50 2.5-11.5 µM under long-term necroptosis execution in murine fibroblast L929 cells, and full protection from ATP depletion and membrane leakage in human and murine cells. As a proof of concept, compound SN-6109, with binding mode to RIPK1 similar to that of necrostatin-1, confirmed RIPK1 inhibitory activity and appropriate pharmacokinetic properties. SN-6109 was further tested in mice, showing efficacy against TNF-α-induced systemic inflammatory response syndrome. In conclusion, a phenotypic-driven HTS cascade promptly identified robust necroptosis inhibitors with in vivo activity, currently undergoing further medicinal chemistry optimization. Notably, the novel hits highlight the opportunity to identify new molecular mechanisms of action in necroptosis.

Industrial scale high-throughput screening delivers multiple fast acting macrofilaricides.

Nematodes causing lymphatic filariasis and onchocerciasis rely on their bacterial endosymbiont, Wolbachia, for survival and fecundity, making Wolbachia a promising therapeutic target. Here we perform a high-throughput screen of AstraZeneca's 1.3 million in-house compound library and identify 5 novel chemotypes with faster in vitro kill rates (<2 days) than existing anti-Wolbachia drugs that cure onchocerciasis and lymphatic filariasis. This industrial scale anthelmintic neglected tropical disease (NTD) screening campaign is the result of a partnership between the Anti-Wolbachia consortium (A∙WOL) and AstraZeneca. The campaign was informed throughout by rational prioritisation and triage of compounds using cheminformatics to balance chemical diversity and drug like properties reducing the chance of attrition from the outset. Ongoing development of these multiple chemotypes, all with superior time-kill kinetics than registered antibiotics with anti-Wolbachia activity, has the potential to improve upon the current therapeutic options and deliver improved, safer and more selective macrofilaricidal drugs.

ProSAR: a new methodology for combinatorial library design.

A method is introduced for performing reagent selection for chemical library design based on topological (2D) pharmacophore fingerprints. Optimal reagent selection is achieved by optimizing the Shannon entropy of the 2D pharmacophore distribution for the reagent set. The method, termed ProSAR, is therefore expected to enumerate compounds that could serve as a good starting point for deriving a structure activity relationship (SAR) in combinatorial library design. This methodology is exemplified by library design examples where the active compounds were already known. The results show that most of the pharmacophores on the substituents for the active compounds are covered by the designed library. This strategy is further expanded to include product property profiles for aqueous solubility, hERG risk assessment, etc. in the optimization process so that the reagent pharmacophore diversity and the product property profile are optimized simultaneously via a genetic algorithm. This strategy is applied to a two-dimensional library design example and compared with libraries designed by a diversity based strategy which minimizes the average ensemble Tanimoto similarity. Our results show that by using the PSAR methodology, libraries can be designed with simultaneously good pharmacophore coverage and product property profile.

Effect of mutations involving charged residues on the stability of staphylococcal nuclease: a continuum electrostatics study.

A continuum electrostatics model is used to calculate the relative stabilities of 117 mutants of staphylococcal nuclease (SNase) involving the mutation of a charged residue to an uncharged residue. The calculations are based on the crystallographic structure of the wild-type protein and attempt to take implicitly into account the effect of the mutations in the denatured state by assuming a linear relationship between the free energy changes caused by the mutation in the native and denatured states. A good correlation (linear correlation coefficient of approximately 0.8) is found with published experimental relative stabilities of these mutants. The results suggest that in the case of SNase (i) charged residues contribute to the stability of the native state mainly through electrostatic interactions, and (ii) native-like electrostatic interactions may persist in the denatured state. The continuum electrostatics method is only moderately sensitive to model parameters and leads to quasi-predictive results for the relative mutant stabilities (error of 2-3 kJ mol(-1) or of the order of k(B)T), except for mutants in which a charged residue is mutated to glycine.