The c-MET kinase is a driver of many cancers, and as such, there are a number of small molecule inhibitors of this kinase approved for clinical use. In this Microperspective, we provide a structural overview of the molecular basis by which these drugs inhibit c-MET, focusing on key features contributing to activity, selectivity, and drug resistance. Where necessary, relevant crystal structures not publicly available were determined and are discussed here alongside existing structural data.

Immunomodulatory imide drugs (IMiDs) like lenalidomide and pomalidomide are effective in treating multiple myeloma (MM) but pose hematotoxicity risks by degrading neosubstrates Ikaros (IKZF1) and Aiolos (IKZF3). When these IMiD scaffolds are integrated into proteolysis targeting chimeras (PROTACs), they can inadvertently lead to the degradation of these neosubstrates alongside the intended protein of interest (POI), raising safety concerns. This study profiles existing PROTACs and reveals instances of undesired degradation of IMiD-associated neosubstrates. We have developed in vitro hematopoietic assays to scrutinize the IMiD effects and describe the mechanistic insights on cell differentiation rewiring towards megakaryocytes together with an activation of the interferon response that is phenocopied by an Ikaros knock-out model. Moreover, we have identified a CRBN ligand that mitigates these safety liabilities and can be effectively incorporated into PROTACs. This advancement provides a promising path toward safer preclinical development of PROTACs, especially as the field expands into chronic disease treatments beyond oncology.

E3 ubiquitin ligases engage their substrates via ‘degrons’ - short linear motifs typically located within intrinsically disordered regions of substrates. As these enzymes are large, multi-subunit complexes that generally lack natural small-molecule ligands and are difficult to inhibit via conventional means, alternative strategies are needed to target them in diseases, and peptide-based inhibitors derived from degrons represent a promising approach. Here we explore peptide inhibitors of Cdc20, a substrate-recognition subunit and activator of the E3 ubiquitin ligase the anaphase-promoting complex/cyclosome (APC/C) that is essential in mitosis and consequently of interest as an anti-cancer target. APC/C engages substrates via degrons that include the ‘destruction box’ (D-box) motif. We used a rational design approach to construct binders containing unnatural amino acids aimed at better filling a hydrophobic pocket that contributes to the D-box binding site on the surface of Cdc20. We confirmed binding by thermal-shift assays and surface plasmon resonance and determined the structures of a number of the Cdc20-peptide complexes. Using a cellular thermal shift assay, we confirmed that the D-box peptides also bind to and stabilise Cdc20 in the cell. We found that the D-box peptides inhibit ubiquitination activity of APC/C Cdc20 and are more potent than the small-molecule inhibitor Apcin. Lastly, these peptides function as portable degrons capable of driving the degradation of a fused fluorescent protein. Interestingly, we find that although inhibitory activity of the peptides correlates with Cdc20-binding affinity, degradation efficacy does not, which may be due to the complex nature of APC/C regulation and effects of degron binding of subunit recruitment and conformational changes. Our study lays the groundwork for the further development of these peptides as molecular therapeutics for blocking APC/C as well as potentially for harnessing the APC/C for targeted protein degradation.

Unlocking novel E3 ligases for use in heterobifunctional PROTAC degraders is of high importance to the pharmaceutical industry. Over-reliance on the current suite of ligands used to recruit E3 ligases could limit the potential of their application. To address this, potent ligands for DCAF15 were optimized using cryo-EM supported, structure-based design to improve on micromolar starting points. A potent binder, compound 24, was identified and subsequently conjugated into PROTACs against multiple targets. Following attempts on degrading a number of proteins using DCAF15 recruiting PROTACs, only degradation of BRD4 was observed. Deconvolution of the mechanism of action showed that this degradation was not mediated by DCAF15, thereby highlighting both the challenges faced when trying to expand the toolbox of validated E3 ligase ligands for use in PROTAC degraders and the pitfalls of using BRD4 as a model substrate.

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

Supplementary Figures S1-5 show additional information regarding the Fgfr2-Tbc1d1 fusion, the effect of AZD4547 on established WESB and WESB-Fgfr2 tumors and tumor-derived cells, the causes of death observed with the different AZD4547 treatment schedules, differential gene expression analysis of multiple RTK family members and the overview of insertions in genes across all tumors used in this study.

Overview of the sequenced tumor samples, including sample IDs, treatment information and the type of transplanted material.

Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases.

Overview of insertions identified in the donor, vehicle- and AZD4547-treated tumors.

The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Mutations (SNVs) identified for RTKs and genes involved in the MAPK-ERK signaling pathway in the donor, vehicle- and AZD4547-treated tumors.

KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure-activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Numerous technical advances have made cryo-EM an attractive method for atomic structure determination. Cryo-EM is ideally suited for large macromolecular structures, while problems of low signal-to-noise prevent routine structure determination of proteins smaller than about 50 kDa. This size limitation excludes large numbers of important cellular proteins from structural characterization by this powerful technique, including many cell-signaling proteins of high therapeutic interest. In the present work, we use molecular engineering techniques to rigidify an imaging scaffold, based on a designed protein cage, to the point where 3 Å resolution can be achieved, even for very small proteins. After optimizing the design of the rigidified scaffold on test proteins, we apply this imaging system to the key oncogenic signaling protein KRAS, which represents an outstanding challenge in the area of structure-based drug design. Despite its 19 kDa size, we show that the structure of KRAS, in multiple mutant forms, and bound to its GDP ligand, can be readily interpreted at a resolution slightly better than 3.0 Å. This advance further expands the capability of cryo-EM to become an essentially universal method for protein structure determination, including for applications to small therapeutic protein targets.

Single-particle cryogenic electron microscopy (cryo-EM) has been elevated to the mainstream of structural biology propelled by technological advancements in numerous fronts, including imaging analysis and the development of direct electron detectors. The drug discovery field has watched with (initial) skepticism and wonder at the progression of the technique and how it revolutionized the molecular understanding of previously intractable targets. This article critically assesses how cryo-EM has impacted drug discovery in diverse therapeutic areas. Targets that have been brought into the realm of structure-based drug design by cryo-EM and are thus reviewed here include membrane proteins like the GABA(A)receptor, several TRP channels, and G protein-coupled receptors, and multiprotein complexes like the ribosomes, the proteasome, and eIF2B. We will describe these studies highlighting the achievements, challenges, and caveats.

Biophysical, affinity-based methods have been traditionally employed, and continue to have high impact, during the validation and characterization of the high-throughput screening (HTS) outputs, traditionally employed during the hit-to-lead phase. Isothermal titration calorimetry (ITC) directly measures the energy associated with a chemical reaction, usually initiated by the mixing of components. This chapter describes how to collect and exploit kinetic and thermodynamic data alongside structural data to improve success during hit to lead and subsequent lead optimization for biomolecular interactions involving potential drug molecules binding to their target proteins. The high information content of both ITC and surface plasmon resonance data allows for a unique combination providing a full thermodynamic and kinetic characterization of an interaction, including transition state analysis. The chapter also explains the considerations around the type of sensor surface, the attachment chemistry, and the introduction of linkers between the binding site and matrix of the sensor chip.

BACKGROUND:PLCγ enzymes are key nodes in cellular signal transduction and their mutated and rare variants have been recently implicated in development of a range of diseases with unmet need including cancer, complex immune disorders, inflammation and neurodegenerative diseases. However, molecular nature of activation and the impact and dysregulation mechanisms by mutations, remain unclear; both are critically dependent on comprehensive characterization of the intact PLCγ enzymes. METHODS:For structural studies we applied cryo-EM, cross-linking mass spectrometry and hydrogen-deuterium exchange mass spectrometry. In parallel, we compiled mutations linked to main pathologies, established their distribution and assessed their impact in cells and in vitro. FINDINGS:We define structure of a complex containing an intact, autoinhibited PLCγ1 and the intracellular part of FGFR1 and show that the interaction is centred on the nSH2 domain of PLCγ1. We define the architecture of PLCγ1 where an autoinhibitory interface involves the cSH2, spPH, TIM-barrel and C2 domains; this relative orientation occludes PLCγ1 access to its substrate. Based on this framework and functional characterization, the mechanism leading to an increase in PLCγ1 activity for the largest group of mutations is consistent with the major, direct impact on the autoinhibitory interface. INTERPRETATION:We reveal features of PLCγ enzymes that are important for determining their activation status. Targeting such features, as an alternative to targeting the PLC active site that has so far not been achieved for any PLC, could provide new routes for clinical interventions related to various pathologies driven by PLCγ deregulation. FUND: CR UK, MRC and AstaZeneca.

Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRASG12C mutant represents an "Achilles heel" and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine-quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRASG12C inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.

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.

Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.