A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity.

Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.

Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70.

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.

Investigating the phosphinic acid tripeptide mimetic DG013A as a tool compound inhibitor of the M1-aminopeptidase ERAP1.

ERAP1 is a zinc-dependent M1-aminopeptidase that trims lipophilic amino acids from the N-terminus of peptides. Owing to its importance in the processing of antigens and regulation of the adaptive immune response, dysregulation of the highly polymorphic ERAP1 has been implicated in autoimmune disease and cancer. To test this hypothesis and establish the role of ERAP1 in these disease areas, high affinity, cell permeable and selective chemical probes are essential. DG013A 1, is a phosphinic acid tripeptide mimetic inhibitor with reported low nanomolar affinity for ERAP1. However, this chemotype is a privileged structure for binding to various metal-dependent peptidases and contains a highly charged phosphinic acid moiety, so it was unclear whether it would display the high selectivity and passive permeability required for a chemical probe. Therefore, we designed a new stereoselective route to synthesize a library of DG013A 1 analogues to determine the suitability of this compound as a cellular chemical probe to validate ERAP1 as a drug discovery target.

Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release.

DHX8 is a crucial DEAH-box RNA helicase involved in splicing and required for the release of mature mRNA from the spliceosome. Here, we report the biochemical characterisation of full-length human DHX8 and the catalytically active helicase core DHX8Δ547, alongside crystal structures of DHX8Δ547 bound to ADP and a structure of DHX8Δ547 bound to poly(A)6 single-strand RNA. Our results reveal that DHX8 has an in vitro binding preference for adenine-rich RNA and that RNA binding triggers the release of ADP through significant conformational flexibility in the conserved DEAH-, P-loop and hook-turn motifs. We demonstrate the importance of R620 and both the hook-turn and hook-loop regions for DHX8 helicase activity and propose that the hook-turn acts as a gatekeeper to regulate the directional movement of the 3' end of RNA through the RNA-binding channel. This study provides an in-depth understanding of the activity of DHX8 and contributes insights into the RNA-unwinding mechanisms of the DEAH-box helicase family.

Synthesis and profiling of a 3-aminopyridin-2-one-based kinase targeted fragment library: Identification of 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one scaffold for monopolar spindle 1 (MPS1) and Aurora kinases inhibition.

Screening a 3-aminopyridin-2-one based fragment library against a 26-kinase panel representative of the human kinome identified 3-amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2(1H)-one (2) and 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one (3) as ligand efficient inhibitors of the mitotic kinase Monopolar Spindle 1 (MPS1) and the Aurora kinase family. These kinases are well recognised as attractive targets for therapeutic intervention for treating cancer. Elucidation of the binding mode of these fragments and their analogues has been carried out by X-ray crystallography. Structural studies have identified key interactions with a conserved lysine residue and have highlighted potential regions of MPS1 which could be targeted to improve activity and selectivity.

Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth.

Overgrowth syndromes comprise a group of heterogeneous disorders characterised by excessive growth parameters, often in association with intellectual disability. To identify new causes of human overgrowth, we have been undertaking trio-based exome sequencing studies in overgrowth patients and their unaffected parents. Prioritisation of functionally relevant genes with multiple unique de novo mutations revealed four mutations in protein phosphatase 2A (PP2A) regulatory subunit B family genes protein phosphatase 2, regulatory Subunit B', beta (PPP2R5B); protein phosphatase 2, regulatory Subunit B', gamma (PPP2R5C); and protein phosphatase 2, regulatory Subunit B', delta (PPP2R5D). This observation in 3 related genes in 111 individuals with a similar phenotype is greatly in excess of the expected number, as determined from gene-specific de novo mutation rates (P = 1.43 × 10(-10)). Analysis of exome-sequencing data from a follow-up series of overgrowth probands identified a further pathogenic mutation, bringing the total number of affected individuals to 5. Heterozygotes shared similar phenotypic features including increased height, increased head circumference and intellectual disability. The mutations clustered within a region of nine amino acid residues in the aligned protein sequences (P = 1.6 × 10(-5)). We mapped the mutations onto the crystal structure of the PP2A holoenzyme complex to predict their molecular and functional consequences. These studies suggest that the mutations may affect substrate binding, thus perturbing the ability of PP2A to dephosphorylate particular protein substrates. PP2A is a major negative regulator of v-akt murine thymoma viral oncogene homolog 1 (AKT). Thus, our data further expand the list of genes encoding components of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signalling cascade that are disrupted in human overgrowth conditions.

Structure-Enabled Discovery of a Stapled Peptide Inhibitor to Target the Oncogenic Transcriptional Repressor TLE1.

TLE1 is an oncogenic transcriptional co-repressor that exerts its repressive effects through binding of transcription factors. Inhibition of this protein-protein interaction represents a putative cancer target, but no small-molecule inhibitors have been published for this challenging interface. Herein, the structure-enabled design and synthesis of a constrained peptide inhibitor of TLE1 is reported. The design features the introduction of a four-carbon-atom linker into the peptide epitope found in many TLE1 binding partners. A concise synthetic route to a proof-of-concept peptide, cycFWRPW, has been developed. Biophysical testing by isothermal titration calorimetry and thermal shift assays showed that, although the constrained peptide bound potently, it had an approximately five-fold higher Kd than that of the unconstrained peptide. The co-crystal structure suggested that the reduced affinity was likely to be due to a small shift of one side chain, relative to the otherwise well-conserved conformation of the acyclic peptide. This work describes a constrained peptide inhibitor that may serve as the basis for improved inhibitors.

An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine-56.

The stress-inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine-targeted irreversible inhibitor. Using rational design, we adapted a validated 8-N-benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine-56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs.

Synthesis of a Ribose-Incorporating Medium Ring Scaffold via a Challenging Ring-Closing Metathesis Reaction.

A practical synthesis of a novel oxabicyclo[6.2.1]undecenetriol useful as a medicinal chemistry scaffold has been developed starting from l-ribose. The sequence involves an oxidation/Grignard addition sequence and a challenging ring-closing metathesis (RCM) reaction as the ring forming step. Exploration of the RCM substrate protecting groups revealed the key factor for successful nine-membered medium ring formation to be conformational bias of the reacting alkenes of the RCM substrate by very bulky silyl ether protecting groups. The synthesis also allowed access to an epimeric triol and saturated and unsaturated variants of the nine-membered ring. The medium ring conformation of the oxabicyclo[6.2.1]undecenetriol was determined by X-ray crystallography and correlated to the solution state conformation by NMR experiments.

Targeting the PPM1D phenotype; 2,4-bisarylthiazoles cause highly selective apoptosis in PPM1D amplified cell-lines.

The metal-dependent phosphatase PPM1D (WIP1) is an important oncogene in cancer, with over-expression of the protein being associated with significantly worse clinical outcomes. In this communication we describe the discovery and optimization of novel 2,4-bisarylthiazoles that phenocopy the knockdown of PPM1D, without inhibiting its phosphatase activity. These compounds cause growth inhibition at nanomolar concentrations, induce apoptosis, activate p53 and display impressive cell-line selectivity. The results demonstrate the potential for targeting phenotypes in drug discovery when tackling challenging targets or unknown mechanisms.

Erratum: iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells.

[This corrects the article DOI: 10.1016/j.isci.2023.107059.].

Scaffold-focused virtual screening: prospective application to the discovery of TTK inhibitors.

We describe and apply a scaffold-focused virtual screen based upon scaffold trees to the mitotic kinase TTK (MPS1). Using level 1 of the scaffold tree, we perform both 2D and 3D similarity searches between a query scaffold and a level 1 scaffold library derived from a 2 million compound library; 98 compounds from 27 unique top-ranked level 1 scaffolds are selected for biochemical screening. We show that this scaffold-focused virtual screen prospectively identifies eight confirmed active compounds that are structurally differentiated from the query compound. In comparison, 100 compounds were selected for biochemical screening using a virtual screen based upon whole molecule similarity resulting in 12 confirmed active compounds that are structurally similar to the query compound. We elucidated the binding mode for four of the eight confirmed scaffold hops to TTK by determining their protein-ligand crystal structures; each represents a ligand-efficient scaffold for inhibitor design.

Synthesis and evaluation of heteroaryl substituted diazaspirocycles as scaffolds to probe the ATP-binding site of protein kinases.

With the success of protein kinase inhibitors as drugs to target cancer, there is a continued need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses of substituted diazaspirocycles through ring-closing metathesis were demonstrated. Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient inhibitors of multiple kinases, suitable as starting points for further optimization. The binding modes of representative diazaspirocyclic motifs were confirmed by protein crystallography. Selectivity profiles were influenced by the hinge binder group and the interactions of basic nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The introduction of more complex substitution to the diazaspirocycles increased potency and varied the selectivity profiles of these initial hits through engagement of the P-loop and changes to the spirocycle conformation, demonstrating the potential of these core scaffolds for future application to kinase inhibitor discovery.

Determination of Ligand-Binding Affinity (Kd) Using Transverse Relaxation Rate (R2) in the Ligand-Observed 1H NMR Experiment and Applications to Fragment-Based Drug Discovery.

High hit rates from initial ligand-observed NMR screening can make it challenging to prioritize which hits to follow up, especially in cases where there are no available crystal structures of these hits bound to the target proteins or other strategies to provide affinity ranking. Here, we report a reproducible, accurate, and versatile quantitative ligand-observed NMR assay, which can determine Kd values of fragments in the affinity range of low µM to low mM using transverse relaxation rate R2 as the observable parameter. In this study, we examined the theory and proposed a mathematical formulation to obtain Kd values using non-linear regression analysis. We designed an assay format with automated sample preparation and simplified data analysis. Using tool compounds, we explored the assay reproducibility, accuracy, and detection limits. Finally, we used this assay to triage fragment hits, yielded from fragment screening against the CRBN/DDB1 complex.

Discovery of 2-(3-Benzamidopropanamido)thiazole-5-carboxylate Inhibitors of the Kinesin HSET (KIFC1) and the Development of Cellular Target Engagement Probes.

The existence of multiple centrosomes in some cancer cells can lead to cell death through the formation of multipolar mitotic spindles and consequent aberrant cell division. Many cancer cells rely on HSET (KIFC1) to cluster the extra centrosomes into two groups to mimic the bipolar spindle formation of non-centrosome-amplified cells and ensure their survival. Here, we report the discovery of a novel 2-(3-benzamidopropanamido)thiazole-5-carboxylate with micromolar in vitro inhibition of HSET (KIFC1) through high-throughput screening and its progression to ATP-competitive compounds with nanomolar biochemical potency and high selectivity against the opposing mitotic kinesin Eg5. Induction of the multipolar phenotype was shown in centrosome-amplified human cancer cells treated with these inhibitors. In addition, a suitable linker position was identified to allow the synthesis of both fluorescent- and trans-cyclooctene (TCO)-tagged probes, which demonstrated direct compound binding to the HSET protein and confirmed target engagement in cells, through a click-chemistry approach.

iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells.

To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented "hook effect" of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome.

Discovery of an In Vivo Chemical Probe for BCL6 Inhibition by Optimization of Tricyclic Quinolinones.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.

Benzimidazole inhibitors of the protein kinase CHK2: clarification of the binding mode by flexible side chain docking and protein-ligand crystallography.

Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature.

Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays.

By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein-protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.

Optimizing Shape Complementarity Enables the Discovery of Potent Tricyclic BCL6 Inhibitors.

To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.