mRNA Display Identifies Potent, Paralog-Selective Peptidic Ligands for ARID1B.

The ARID1A and ARID1B subunits are mutually exclusive components of the BAF variant of SWI/SNF chromatin remodeling complexes. Loss of function mutations in ARID1A are frequently observed in various cancers, resulting in a dependency on the paralog ARID1B for cancer cell proliferation. However, ARID1B has never been targeted directly, and the high degree of sequence similarity to ARID1A poses a challenge for the development of selective binders. In this study, we used mRNA display to identify peptidic ligands that bind with nanomolar affinities to ARID1B and showed high selectivity over ARID1A. Using orthogonal biochemical, biophysical, and chemical biology tools, we demonstrate that the peptides engage two different binding pockets, one of which directly involves an ARID1B-exclusive cysteine that could allow covalent targeting by small molecules. Our findings impart the first evidence of the ligandability of ARID1B, provide valuable tools for drug discovery, and suggest opportunities for the development of selective molecules to exploit the synthetic lethal relationship between ARID1A and ARID1B in cancer.

Optimization of Potent Ligands for the E3 Ligase DCAF15 and Evaluation of Their Use in Heterobifunctional Degraders.

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.

Design and Evaluation of a Low Hydrogen Bond Donor Count Fragment Screening Set to Aid Hit Generation of PROTACs Intended for Oral Delivery.

The development of orally bioavailable PROTACs presents a significant challenge due to the inflated physicochemical properties of such heterobifunctional molecules. Molecules occupying this "beyond rule of five" space often demonstrate limited oral bioavailability due to the compounding effects of elevated molecular weight and hydrogen bond donor count (among other properties), but it is possible to achieve sufficient oral bioavailability through physicochemical optimization. Herein, we disclose the design and evaluation of a low hydrogen bond donor count (≤1 HBD) fragment screening set to aid hit generation of PROTACs intended for an oral route of delivery. We demonstrate that application of this library can enhance fragment screens against PROTAC proteins of interest and ubiquitin ligases, yielding fragment hits containing ≤1 HBD suitable for optimizing toward orally bioavailable PROTACs.

Icolos: a workflow manager for structure-based post-processing of de novo generated small molecules.

SUMMARY: We present Icolos, a workflow manager written in Python as a tool for automating complex structure-based workflows for drug design. Icolos can be used as a standalone tool, for example in virtual screening campaigns, or can be used in conjunction with deep learning-based molecular generation facilitated for example by REINVENT, a previously published molecular de novo design package. In this publication, we focus on the internal structure and general capabilities of Icolos, using molecular docking experiments as an illustrative example. AVAILABILITY AND IMPLEMENTATION: The source code is freely available at https://github.com/MolecularAI/Icolos under the Apache 2.0 license. Tutorial notebooks containing minimal working examples can be found at https://github.com/MolecularAI/IcolosCommunity. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DockStream: a docking wrapper to enhance de novo molecular design.

Recently, we have released the de novo design platform REINVENT in version 2.0. This improved and extended iteration supports far more features and scoring function components, which allows bespoke and tailor-made protocols to maximize impact in small molecule drug discovery projects. A major obstacle of generative models is producing active compounds, in which predictive (QSAR) models have been applied to enrich target activity. However, QSAR models are inherently limited by their applicability domains. To overcome these limitations, we introduce a structure-based scoring component for REINVENT. DockStream is a flexible, stand-alone molecular docking wrapper that provides access to a collection of ligand embedders and docking backends. Using the benchmarking and analysis workflow provided in DockStream, execution and subsequent analysis of a variety of docking configurations can be automated. Docking algorithms vary greatly in performance depending on the target and the benchmarking and analysis workflow provides a streamlined solution to identifying productive docking configurations. We show that an informative docking configuration can inform the REINVENT agent to optimize towards improving docking scores using public data. With docking activated, REINVENT is able to retain key interactions in the binding site, discard molecules which do not fit the binding cavity, harness unused (sub-)pockets, and improve overall performance in the scaffold-hopping scenario. The code is freely available at https://github.com/MolecularAI/DockStream .

Put a ring on it: application of small aliphatic rings in medicinal chemistry.

Aliphatic three- and four-membered rings including cyclopropanes, cyclobutanes, oxetanes, azetidines and bicyclo[1.1.1]pentanes have been increasingly exploited in medicinal chemistry for their beneficial physicochemical properties and applications as functional group bioisosteres. This review provides a historical perspective and comparative up to date overview of commonly applied small rings, exemplifying key principles with recent literature examples. In addition to describing the merits and advantages of each ring system, potential hazards and liabilities are also illustrated and explained, including any significant chemical or metabolic stability and toxicity risks.

Supporting SARS-CoV-2 Papain-Like Protease Drug Discovery: In silico Methods and Benchmarking.

The coronavirus disease 19 (COVID-19) is a rapidly growing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Its papain-like protease (SARS-CoV-2 PLpro) is a crucial target to halt virus replication. SARS-CoV PLpro and SARS-CoV-2 PLpro share an 82.9% sequence identity and a 100% sequence identity for the binding site reported to accommodate small molecules in SARS-CoV. The flexible key binding site residues Tyr269 and Gln270 for small-molecule recognition in SARS-CoV PLpro exist also in SARS-CoV-2 PLpro. This inspired us to use the reported small-molecule binders to SARS-CoV PLpro to generate a high-quality DEKOIS 2.0 benchmark set. Accordingly, we used them in a cross-benchmarking study against SARS-CoV-2 PLpro. As there is no SARS-CoV-2 PLpro structure complexed with a small-molecule ligand publicly available at the time of manuscript submission, we built a homology model based on the ligand-bound SARS-CoV structure for benchmarking and docking purposes. Three publicly available docking tools FRED, AutoDock Vina, and PLANTS were benchmarked. All showed better-than-random performances, with FRED performing best against the built model. Detailed performance analysis via pROC-Chemotype plots showed a strong enrichment of the most potent bioactives in the early docking ranks. Cross-benchmarking against the X-ray structure complexed with a peptide-like inhibitor confirmed that FRED is the best-performing tool. Furthermore, we performed cross-benchmarking against the newly introduced X-ray structure complexed with a small-molecule ligand. Interestingly, its benchmarking profile and chemotype enrichment were comparable to the built model. Accordingly, we used FRED in a prospective virtual screen of the DrugBank database. In conclusion, this study provides an example of how to harness a custom-made DEKOIS 2.0 benchmark set as an approach to enhance the virtual screening success rate against a vital target of the rapidly emerging pandemic.

Targeting Cavity-Creating p53 Cancer Mutations with Small-Molecule Stabilizers: the Y220X Paradigm.

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability, and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N, and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket's accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutants as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therapy.

A structure-guided molecular chaperone approach for restoring the transcriptional activity of the p53 cancer mutant Y220C.

Aim: The p53 cancer mutation Y220C creates a conformationally unstable protein with a unique elongated surface crevice that can be targeted by molecular chaperones. We report the structure-guided optimization of the carbazole-based stabilizer PK083. Materials & methods: Biophysical, cellular and x-ray crystallographic techniques have been employed to elucidate the mode of action of the carbazole scaffolds. Results: Targeting an unoccupied subsite of the surface crevice with heterocycle-substituted PK083 analogs resulted in a 70-fold affinity increase to single-digit micromolar levels, increased thermal stability and decreased rate of aggregation of the mutant protein. PK9318, one of the most potent binders, restored p53 signaling in the liver cancer cell line HUH-7 with homozygous Y220C mutation. Conclusion: The p53-Y220C mutant is an excellent paradigm for the development of mutant p53 rescue drugs via protein stabilization. Similar rescue strategies may be applicable to other cavity-creating p53 cancer mutations.

SLMP53-2 Restores Wild-Type-Like Function to Mutant p53 through Hsp70: Promising Activity in Hepatocellular Carcinoma.

Half of human cancers harbor TP53 mutations that render p53 inactive as a tumor suppressor. In these cancers, reactivation of mutant p53 (mutp53) through restoration of wild-type-like function constitutes a valuable anticancer therapeutic strategy. In order to search for mutp53 reactivators, a small library of tryptophanol-derived oxazoloisoindolinones was synthesized and the potential of these compounds as mutp53 reactivators and anticancer agents was investigated in human tumor cells and xenograft mouse models. By analysis of their anti-proliferative effect on a panel of p53-null NCI-H1299 tumor cells ectopically expressing highly prevalent mutp53, the compound SLMP53-2 was selected based on its potential reactivation of multiple structural mutp53. In mutp53-Y220C-expressing hepatocellular carcinoma (HCC) cells, SLMP53-2-induced growth inhibition was mediated by cell cycle arrest, apoptosis, and endoplasmic reticulum stress response. In these cells, SLMP53-2 restored wild-type-like conformation and DNA-binding ability of mutp53-Y220C by enhancing its interaction with the heat shock protein 70 (Hsp70), leading to the reestablishment of p53 transcriptional activity. Additionally, SLMP53-2 displayed synergistic effect with sorafenib, the only approved therapy for advanced HCC. Notably, it exhibited potent antitumor activity in human HCC xenograft mouse models with a favorable toxicological profile. Collectively, SLMP53-2 is a new mutp53-targeting agent with promising antitumor activity, particularly against HCC.

Electrostatic Complementarity as a Fast and Effective Tool to Optimize Binding and Selectivity of Protein-Ligand Complexes.

Electrostatic interactions between small molecules and their respective receptors are essential for molecular recognition and are also key contributors to the binding free energy. Assessing the electrostatic match of protein-ligand complexes therefore provides important insights into why ligands bind and what can be changed to improve binding. Ideally, the ligand and protein electrostatic potentials at the protein-ligand interaction interface should maximize their complementarity while minimizing desolvation penalties. In this work, we present a fast and efficient tool to calculate and visualize the electrostatic complementarity (EC) of protein-ligand complexes. We compiled benchmark sets demonstrating electrostatically driven structure-activity relationships (SAR) from literature data, including kinase, protein-protein interaction, and GPCR targets, and used these to demonstrate that the EC method can visualize, rationalize, and predict electrostatically driven ligand affinity changes and help to predict compound selectivity. The methodology presented here for the analysis of EC is a powerful and versatile tool for drug design.

Aminobenzothiazole derivatives stabilize the thermolabile p53 cancer mutant Y220C and show anticancer activity in p53-Y220C cell lines.

Many cancers have the tumor suppressor p53 inactivated by mutation, making reactivation of mutant p53 with small molecules a promising strategy for the development of novel anticancer therapeutics. The oncogenic p53 mutation Y220C, which accounts for approximately 100,000 cancer cases per year, creates an extended surface crevice in the DNA-binding domain, which destabilizes p53 and causes denaturation and aggregation. Here, we describe the structure-guided design of a novel class of small-molecule Y220C stabilizers and the challenging synthetic routes developed in the process. The synthesized chemical probe MB710, an aminobenzothiazole derivative, binds tightly to the Y220C pocket and stabilizes p53-Y220C in vitro. MB725, an ethylamide analogue of MB710, induced selective viability reduction in several p53-Y220C cancer cell lines while being well tolerated in control cell lines. Reduction of viability correlated with increased and selective transcription of p53 target genes such as BTG2, p21, PUMA, FAS, TNF, and TNFRSF10B, which promote apoptosis and cell cycle arrest, suggesting compound-mediated transcriptional activation of the Y220C mutant. Our data provide a framework for the development of a class of potent, non-toxic compounds for reactivating the Y220C mutant in anticancer therapy.

The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant-reactivating drug with high clinical anticancer potential.

Mutant p53 as a therapeutic target for the treatment of triple-negative breast cancer: Preclinical investigation with the anti-p53 drug, PK11007.

The identification of a targeted therapy for patients with triple-negative breast cancer (TNBC) is one of the most urgent needs in breast cancer therapeutics. The p53 gene is mutated in approximately 80% of patients with TNBC, and is a potential therapeutic target for patients with this form of breast cancer. The 2-sulfonylpyrimidine compound, PK11007, preferentially decreases viability in p53-compromised cancer cell lines. We investigated PK11007 as a potential new treatment for TNBC. IC50 values for inhibition of proliferation in a panel of 17 breast cell lines by PK11007 ranged from 2.3 to 42.2 µM. There were significantly lower IC50 values for TNBC than for non-TNBC cell lines (p = 0.03) and for p53-mutated cell lines compared with p53 WT cells (p = 0.003). Response to PK11007 however, was independent of the estrogen receptor (ER) or HER2 status of the cell lines. In addition to inhibiting cell proliferation, PK11007 induced apoptosis in p53 mutant cell lines. Using RNAseq and gene ontology analysis, we found that PK11007 altered the expression of genes enriched in pathways involved in regulated cell death, regulation of apoptosis, signal transduction, protein refolding and locomotion. The observations that PK11007 inhibited cell proliferation, induced apoptosis and altered genes involved in cell death are all consistent with the ability of PK11007 to reactivate mutant p53. Based on our data, we conclude that targeting mutant p53 with PK11007 is a potential approach for treating p53-mutated breast cancer, including the subgroup with TN disease.

2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells.

The tumor suppressor p53 has the most frequently mutated gene in human cancers. Many of p53's oncogenic mutants are just destabilized and rapidly aggregate, and are targets for stabilization by drugs. We found certain 2-sulfonylpyrimidines, including one named PK11007, to be mild thiol alkylators with anticancer activity in several cell lines, especially those with mutationally compromised p53. PK11007 acted by two routes: p53 dependent and p53 independent. PK11007 stabilized p53 in vitro via selective alkylation of two surface-exposed cysteines without compromising its DNA binding activity. Unstable p53 was reactivated by PK11007 in some cancer cell lines, leading to up-regulation of p53 target genes such as p21 and PUMA. More generally, there was cell death that was independent of p53 but dependent on glutathione depletion and associated with highly elevated levels of reactive oxygen species and induction of endoplasmic reticulum (ER) stress, as also found for the anticancer agent PRIMA-1(MET)(APR-246). PK11007 may be a lead for anticancer drugs that target cells with nonfunctional p53 or impaired reactive oxygen species (ROS) detoxification in a wide variety of mutant p53 cells.

An in silico algorithm for identifying stabilizing pockets in proteins: test case, the Y220C mutant of the p53 tumor suppressor protein.

The p53 tumor suppressor protein performs a critical role in stimulating apoptosis and cell cycle arrest in response to oncogenic stress. The function of p53 can be compromised by mutation, leading to increased risk of cancer; approximately 50% of cancers are associated with mutations in the p53 gene, the majority of which are in the core DNA-binding domain. The Y220C mutation of p53, for example, destabilizes the core domain by 4 kcal/mol, leading to rapid denaturation and aggregation. The associated loss of tumor suppressor functionality is associated with approximately 75 000 new cancer cases every year. Destabilized p53 mutants can be 'rescued' and their function restored; binding of a small molecule into a pocket on the surface of mutant p53 can stabilize its wild-type structure and restore its function. Here, we describe an in silico algorithm for identifying potential rescue pockets, including the algorithm's integration with the Dynameomics molecular dynamics data warehouse and the DIVE visual analytics engine. We discuss the results of the application of the method to the Y220C p53 mutant, entailing finding a putative rescue pocket through MD simulations followed by an in silico search for stabilizing ligands that dock into the putative rescue pocket. The top three compounds from this search were tested experimentally and one of them bound in the pocket, as shown by nuclear magnetic resonance, and weakly stabilized the mutant.

Harnessing Fluorine-Sulfur Contacts and Multipolar Interactions for the Design of p53 Mutant Y220C Rescue Drugs.

Many oncogenic mutants of the tumor suppressor p53 are conformationally unstable, including the frequently occurring Y220C mutant. We have previously developed several small-molecule stabilizers of this mutant. One of these molecules, PhiKan083, 1-(9-ethyl-9H-carbazole-3-yl)-N-methylmethanamine, binds to a mutation-induced surface crevice with a KD = 150 µM, thereby increasing the melting temperature of the protein and slowing its rate of aggregation. Incorporation of fluorine atoms into small molecule ligands can substantially improve binding affinity to their protein targets. We have, therefore, harnessed fluorine-protein interactions to improve the affinity of this ligand. Step-wise introduction of fluorines at the carbazole ethyl anchor, which is deeply buried within the binding site in the Y220C-PhiKan083 complex, led to a 5-fold increase in affinity for a 2,2,2-trifluoroethyl anchor (ligand efficiency of 0.3 kcal mol(-1) atom(-1)). High-resolution crystal structures of the Y220C-ligand complexes combined with quantum chemical calculations revealed favorable interactions of the fluorines with protein backbone carbonyl groups (Leu145 and Trp146) and the sulfur of Cys220 at the mutation site. Affinity gains were, however, only achieved upon trifluorination, despite favorable interactions of the mono- and difluorinated anchors with the binding pocket, indicating a trade-off between energetically favorable protein-fluorine interactions and increased desolvation penalties. Taken together, the optimized carbazole scaffold provides a promising starting point for the development of high-affinity ligands to reactivate the tumor suppressor function of the p53 mutant Y220C in cancer cells.

Exploiting Transient Protein States for the Design of Small-Molecule Stabilizers of Mutant p53.

The destabilizing p53 cancer mutation Y220C creates an extended crevice on the surface of the protein that can be targeted by small-molecule stabilizers. Here, we identify different classes of small molecules that bind to this crevice and determine their binding modes by X-ray crystallography. These structures reveal two major conformational states of the pocket and a cryptic, transiently open hydrophobic subpocket that is modulated by Cys220. In one instance, specifically targeting this transient protein state by a pyrrole moiety resulted in a 40-fold increase in binding affinity. Molecular dynamics simulations showed that both open and closed states of this subsite were populated at comparable frequencies along the trajectories. Our data extend the framework for the design of high-affinity Y220C mutant binders for use in personalized anticancer therapy and, more generally, highlight the importance of implementing protein dynamics and hydration patterns in the drug-discovery process.

pROC-Chemotype Plots Enhance the Interpretability of Benchmarking Results in Structure-Based Virtual Screening.

Recently, we have reported a systematic comparison of molecular preparation protocols (using MOE or Maestro) in combination with two docking tools (GOLD or Glide), employing our DEKOIS 2.0 benchmark sets. Herein, we demonstrate how comparable settings of data preparation protocols can affect the profile and AUC of pROC curves based on variations in chemotype enrichment. We show how the recognition of different classes of chemotypes can affect the docking performance, particularly in the early enrichment, and monitor changes in this recognition behavior based on score normalization and rescoring strategies. For this, we have developed "pROC-Chemotype", which is an automated protocol that matches and visualizes ligand chemotype information together with potency classes in the pROC profiles obtained by docking. This tool enhances the understanding of the influence of chemotype recognition in early enrichment, but also reveals trends of impaired recognition of chemotype classes at the end of the score-ordered rank. Identifying such issues helps to devise score-normalization strategies to overcome this potential bias in an intuitive manner. Furthermore, strong perturbations in chemotype ranking between different methods can help to identify the underlying reasons (e.g., changes in the protonation/tautomerization state). It also assists in the selection of appropriate scoring functions that are capable to retrieve more potent and diverse hits. In summary, we demonstrate how this new tool can be utilized to identify and highlight chemotype-specific behavior, e.g., in dataset preparation. This can help to overcome some chemistry-related bias in virtual screening campaigns. pROC-Chemotype is made freely available at www.dekois.com.