Biology subjects

London, N.

Publications and source records attributed to London, N..

18 recordsLinked to original sources

bioRxiv2026

Discovery of a molecular glue inhibitor that stabilises a non-productive Kalirin-Rac1 complex

Rho guanosine triphosphatases (GTPases) are molecular-switches implicated in neurodegenerative diseases, yet targeting them through competitive inhibition remains a challenge due to their high affinity for guanine nucleotides. Guanine nucleotide exchange factors (GEFs) catalyse GDP-to-GTP nucleotide exchange to activate GTPases, providing an alternative opportunity for GTPase modulation. Herein, we describe a complex-targeted strategy to inhibit nucleotide exchange with covalent molecular glues that engage the Kalirin-Rac1 GEF-GTPase complex at the nucleotide binding site are sequester the GEF Kalirin. Fragment hits were identified through XChem and in silico screening, and a fragment merging approach resulted in the generation of covalent inhibitors RS-009 and MC-278. Multiple analyses demonstrate our compounds inhibit nucleotide exchange both through competition with the nucleotides and by stabilising a ternary inhibitor-Rac1-Kalirin complex, thereby trapping Kalirin in a non-productive state and reducing GEF turnover. Biochemical selectivity screening and cellular activity-based protein profiling (ABPP) show that selectivity can be achieved across distinct GEF-GTPase complexes, which may result in improved spatiotemporal control over targeting the GTPase alone. This work provides evidence for targeting GTPase signalling via stabilization of the GEF-GTPase complex in a unique covalent molecular glue mechanism and provides the basis of a chemical probe or therapeutic.

biochemistry↗

bioRxiv2025

Discovery of orally bioavailable Zika and West Nile Virus antiviral compounds targeting the NS2B-NS3 protease

Flaviviruses are a class of pathogenic viruses with pandemic potential that are typically transmitted via infected arthropods. In addition, Zika virus is sexually transmissible and causes congenital malformations if infection occurs during pregnancy. Although over 1.5 million people were infected during the 2015-2016 Zika outbreak, to date, there are no clinical-stage vaccines or antivirals. Herein, we report the discovery of potent inhibitors of the Zika virus NS2B-NS3 protease that also show activity against the West Nile virus NS2B-NS3 protease. Starting from a crystallographic fragment screen, we employed a pharmacophore approach coupled with high-throughput library chemistry to elaborate fragments in the active site. Potent, metabolically stable, non-covalent, non-peptidomimetic inhibitors were identified with antiviral activity in vitro. The lead compound was progressed to a three-day Zika virus challenge study in AG129 mice, where it displayed significant viral RNA load reduction in both mouse plasma and spleen.

microbiology↗

bioRxiv2025

Open-science discovery of DNDI-6510, a compound that addresses genotoxic and metabolic liabilities of the COVID Moonshot SARS-CoV-2 Mpro lead inhibitor

The 2020 SARS-CoV-2 coronavirus pandemic highlighted the urgent need for novel small molecule antiviral drugs. (S)-x38 DNDI-6510 is a non-covalent SARS-CoV-2 main protease inhibitor developed by the open science collaboration COVID Moonshot. Here, we report on the metabolic and toxicologic optimization of the lead series previously disclosed by the COVID Moonshot Initiative, leading up to the selection of (S)-x38 DNDI-6510 as the preclinical candidate. We describe the thorough profiling of the series, identifying key risks such as formation of genotoxic metabolites and high clearance, which were successfully addressed during lead optimization. In addition, we disclose the in vitro and in vivo evaluation of (S)-x38 DNDI-6510 in pharmacokinetic and pharmacodynamic models, exploring multiple approaches to ameliorate rodent-specific metabolic clearance, and show that both co-dosing of (S)-x38 DNDI-6510 with an ABT inhibitor and utilizing a metabolically humanized mouse model (8HUM) achieve significant improvements in exposure. Through comparisons of ABT co-dosing and humanized mouse models in efficacy experiments, we demonstrate that continuous exposure over cellular EC90 is required for SARS-CoV-2 antiviral efficacy in vivo in an antiviral model using a mouse-adapted SARS-CoV-2 strain. Finally, (S)-x38 DNDI-6510 was assessed in maximum tolerated dose experiments in two species, demonstrating significant in vivo PXR-linked auto-induction of metabolism, leading to the discontinuation of this compound. In summary, we report the successful effort to overcome series-specific AMES liabilities in a lead development program. Downstream optimization of existing series will require in-depth optimization of rodent-specific liabilities and metabolic induction profile.

pharmacology and toxicology↗

bioRxiv2025

State-of-the-art covalent virtual screening with AlphaFold3

Covalent inhibitors are a prominent modality for research and therapeutic tools. However, a scarcity of computational methods for their discovery slows progress in this field. AI models such as AlphaFold3 (AF3) have shown accuracy in ligand pose prediction, but their applicability for virtual screening campaigns was not assessed. We show that AF3 co-folding predictions and an associated predicted confidence metric ranks true covalent binders with near-optimal classification over property-matched decoys, significantly outperforming state-of-the-art covalent docking tools for a set of protein kinases. In a prospective virtual screening campaign against the model kinase BTK, we discovered a chemically distinct, novel, covalent small molecule that displays potent inhibition in vitro and in cells while maintaining marked kinome and proteomic selectivity. Co-crystallography validated the sub-angstrom accuracy of the predicted AF3 binding mode. These results demonstrate that AF3 can be practically used to discover novel chemical matter for kinases, one of the most prolific families of drug targets.

biochemistry↗

bioRxiv2025

Targeted Protein Degradation of NUDT5 Reveals an Unexpected Non-Enzymatic Role in 6-Thioguanine-Mediated Toxicity

6-Thioguanine (6-TG), an FDA-approved antimetabolite drug, is widely used in the treatment of leukemia. Its cellular effects require metabolic activation and are regulated through interactions with various proteins such as NUDT15, which catalyzes the hydrolysis of the active 6-TG metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP. Recent genome-wide CRISPR loss-of-function studies have identified another NUDIX hydrolase, NUDT5, as a crucial mediator of 6-TG toxicity. Here, we present the development and characterization of potent and selective NUDT5 degraders, guided by a cell-based assay screening strategy. These degraders, in conjunction with orthogonal CRISPR knock-out and reconstitution experiments, reveal a novel and unexpected, non-enzymatic role for NUDT5 in modulating the cellular response to 6-TG. Depletion of NUDT5 protein is antagonistic to NUDT15 inhibition, suggesting a distinct mode-of-action with potential implications for patient therapy.

cell biology↗

bioRxiv2025

A pharmacological modality to sequester homomeric proteins

Molecules that can perturb protein-protein interactions have an immense impact on chemical biology and therapeutics. However, such compounds typically rely on accessory proteins to function, such as E3 ligases in the case of targeted degradation, which may restrict their target and tissue scope or lead to resistance. Here we alleviate the need for accessory proteins with a novel pharmacological modality to knock-down protein function. Our strategy exploits protein symmetry as a selective vulnerability, and is widely applicable owing to the ubiquitous nature of homomeric proteins in cellular systems. We target homomeric proteins with PINCHs (Polymerization Inducing Chimeras) - bifunctional molecules composed of two linked ligands that act as bridges between homomers and trigger their supramolecular assembly into insoluble polymers. We design PINCHs that achieve efficient polymerization of three homomeric targets. In cells, we observed that a PINCH targeting Keap1 exhibited a longer duration of action compared to its monomeric inhibitor, and a PINCH targeting BCL6 displayed selective and improved B cell toxicity compared to its monomeric parent. Our results highlight PINCHs as a novel and general strategy to modulate and knock out protein function.

biochemistry↗

bioRxiv2024

Dual Targeting of Histone Deacetylases and MYC as Potential Treatment Strategy for H3-K27M Pediatric Gliomas

Diffuse midline gliomas (DMG) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in-vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in-vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.

cancer biology↗

bioRxiv2023

A simple method for developing lysine targeted covalent protein reagents

There is a growing demand for covalent tool compounds and chemical probes to investigate and perturb protein function and dysregulation. The combination of a covalent electrophile with a peptide or protein-based scaffold with an extended binding footprint enables the targeting of shallow protein surfaces, not typically addressable using small molecules. However, to fully exploit the potential of electrophilic proteins or peptides there is a need for versatile approaches to convert native peptide sequences into covalent binders that can target a broad range of residues. Here we report protein-based thio-methacrylate esters - electrophiles with a diverse reactivity profile that can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chains on the target. Guided by computational modeling, we designed and synthesized methacrylate phosphopeptides derived from 14-3-3-binding proteins and demonstrated these peptides irreversibly label 14-3-3{sigma} via either lysine or cysteine residues, depending on the position of the electrophile. Methacrylate peptides targeting a conserved lysine residue exhibited pan-isoform binding of 14-3-3 proteins, and efficiently labeled 14-3-3 proteins in lysates, as well as secreted 14-3-3 extracellularly. The irreversible binding to the predicted target lysines were confirmed by proteomics and X-ray crystallography of the complexes. Finally, we applied this approach to develop protein-based covalent binders. A methacrylate-modified variant of the colicin E9 immunity protein irreversibly bound to the E9 DNAse, resulting in significantly higher thermal stability relative to the non-covalent complex. Our approach offers a simple and versatile route to convert peptides and proteins into potent covalent binders.

biochemistry↗

bioRxiv2023

Direct recruitment of Mis18 to interphase spindle poles promotes CENP-A chromatin assembly

CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB LInker of Nucleoskeleton and Cytoskeleton (LINC) complex component Sad1. Mis18 recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1, but is independent of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity.

cell biology↗

bioRxiv2022

Fragment-Based Hit Discovery via Unsupervised Learning of Fragment-Protein Complexes

The process of finding molecules that bind to a target protein is a challenging first step in drug discovery. Crystallographic fragment screening is a strategy based on elucidating binding modes of small polar compounds and then building potency by expanding or merging them. Recent advances in high-throughput crystallography enable screening of large fragment libraries, reading out dense ensembles of fragments spanning the binding site. However, fragments typically have low affinity thus the road to potency is often long and fraught with false starts. Here, we take advantage of high-throughput crystallography to reframe fragment-based hit discovery as a denoising problem - identifying significant pharmacophore distributions from a fragment ensemble amid noise due to weak binders - and employ an unsupervised machine learning method to tackle this problem. Our method screens potential molecules by evaluating whether they recapitulate those fragment-derived pharmacophore distributions. We retrospectively validated our approach on an open science campaign against SARS-CoV-2 main protease (Mpro), showing that our method can distinguish active compounds from inactive ones using only structural data of fragment-protein complexes, without any activity data. Further, we prospectively found novel hits for Mpro and the Mac1 domain of SARS-CoV-2 non-structural protein 3. More broadly, our results demonstrate how unsupervised machine learning helps interpret high throughput crystallography data to rapidly discover of potent chemical modulators of protein function.

bioinformatics↗

bioRxiv2021

Chronic kidney disease alters Pin1 phosphorylation and parathyroid hormone mRNA binding proteins leading to secondary hyperparathyroidism

Parathyroid hormone (PTH) regulates calcium metabolism and bone strength. Chronic kidney disease leads to secondary hyperparathyroidism (SHP) which increases morbidity and mortality. In experimental SHP, the increased PTH gene expression is due to enhanced PTH mRNA stability, mediated by changes in its interaction with stabilizing AUF1 and destabilizing KSRP. Pin1 isomerizes target proteins and leads to KSRP dephosphorylation. In SHP, Pin1 isomerase activity is decreased and phosphorylated KSRP fails to bind PTH mRNA, resulting in high PTH mRNA stability and levels. However, the up- and down-stream mechanisms by which kidney failure stimulates the parathyroid remain elusive. We now reveal a pathway where kidney failure induces parathyroid Pin1 phosphorylation, linking Pin1, KSRP and PTH mRNA stability as key players for the high PTH expression in SHP. We identified by mass-spectrometry, changes in rat parathyroid proteome and phosphoproteome profiles induced by impaired renal function, including KSRP phosphorylation at Pin1 target sites. Furthermore, both acute and chronic kidney failure led to parathyroid-specific Pin1 Ser16 and Ser71 phosphorylation, which disrupts Pin1 activity. Accordingly, pharmacologic Pin1 inhibition, that mimics the decreased Pin1 activity in SHP, increased PTH expression ex-vivo in parathyroid organ cultures and in transfected cells, through the PTH mRNA protein-interacting element and KSRP phosphorylation at potential Pin1-binding motifs. Therefore, kidney failure leads to loss of parathyroid Pin1 activity by inducing Pin1 phosphorylation. This predisposes parathyroids to increase PTH production through impaired PTH mRNA decay that is dependent on KSRP phosphorylation. Pin1 and KSRP phosphorylation and the Pin1-KSRP-PTH mRNA axis thus drive SHP.

molecular biology↗

bioRxiv2021

Covalent Flexible Peptide Docking in Rosetta

Electrophilic peptides that form an irreversible covalent bond with their target have great potential for binding targets that have been previously considered undruggable. However, the discovery of such peptides remains a challenge. Here, we present CovPepDock, a computational pipeline for peptide docking that incorporates covalent binding between the peptide and a receptor cysteine. We applied CovPepDock retrospectively to a dataset of 115 disulfide-bound peptides and a dataset of 54 electrophilic peptides, for which it produced a top-five scoring, near-native model, in 89% and 100% of the cases, respectively. In addition, we developed a protocol for designing electrophilic peptide binders based on known non-covalent binders or protein-protein interfaces. We identified 7,154 peptide candidates in the PDB for application of this protocol. As a proof-of-concept we validated the protocol on the non-covalent complex of 14-3-3{sigma} and YAP1 phosphopeptide. The protocol identified seven highly potent and selective irreversible peptide binders. The predicted binding mode of one of the peptides was validated using X-ray crystallography. This case-study demonstrates the utility and impact of CovPepDock. It suggests that many new electrophilic peptide binders can be rapidly discovered, with significant potential as therapeutic molecules and chemical probes.

biochemistry↗

bioRxiv2020

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor.

Designing covalent inhibitors is a task of increasing importance in drug discovery. Efficiently designing irreversible inhibitors, though, remains challenging. Here, we present covalentizer, a computational pipeline for creating irreversible inhibitors based on complex structures of targets with known reversible binders. For each ligand, we create a custom-made focused library of covalent analogs. We use covalent docking, to dock these tailored covalent libraries and to find those that can bind covalently to a nearby cysteine while keeping some of the main interactions of the original molecule. We found ~11,000 cysteines in close proximity to a ligand across 8,386 protein-ligand complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In prospective evaluation against a panel of kinases, five out of nine predicted covalent inhibitors showed IC50 between 155 nM - 4.2 M. Application of the protocol to an existing SARS-CoV-1 Mpro reversible inhibitor led to a new acrylamide inhibitor series with low micromolar IC50 against SARS-CoV-2 Mpro. The docking prediction was validated by 11 co-crystal structures. This is a promising lead series for COVID-19 antivirals. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.

biochemistry↗

bioRxiv2020

PRosettaC: Rosetta based modeling of PROTAC mediated ternary complexes

Proteolysis-targeting chimeras (PROTACs), which induce degradation by recruitment of an E3 ligase to a target protein, are gaining much interest as a new pharmacological modality. However, designing PROTACs is challenging. Formation of a ternary complex between the protein target, the PROTAC and the recruited E3 ligase is considered paramount for successful degradation. A structural model of this ternary complex could in principle inform rational PROTAC design. Unfortunately, only a handful of structures are available for such complexes, necessitating tools for their modeling. We developed a combined protocol that alternates between sampling of the protein-protein interaction space and the PROTAC molecule conformational space. Application of this protocol - PRosettaC - to a benchmark of known PROTAC ternary complexes results in near-native predictions, with often atomic accuracy prediction of the protein chains, as well as the PROTAC binding moieties. It allowed the modeling of a CRBN/BTK complex that recapitulated experimental results for a series of PROTACs. PRosettaC generated models may be used to design PROTACs for new targets, as well as improve PROTACs for existing targets, potentially cutting down time and synthesis efforts.

biochemistry↗

bioRxiv2020

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

biophysics↗

bioRxiv2020

Sulfopin, a selective covalent inhibitor of Pin1, blocks Myc-driven tumor initiation and growth in vivo

The peptidyl-prolyl cis-trans isomerase, Pin1, acts as a unified signaling hub that is exploited in cancer to activate oncogenes and inactivate tumor suppressors, in particular through up-regulation of c-Myc target genes. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to discover covalent inhibitors targeting Pin1s active site nucleophile - Cys113, leading to the development of Sulfopin, a double-digit nanomolar Pin1 inhibitor. Sulfopin is highly selective for Pin1, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement, and phenocopies genetic knockout of Pin1. Although Pin1 inhibition had a modest effect on viability in cancer cell cultures, Sulfopin induced downregulation of c-Myc target genes and reduced tumor initiation and tumor progression in murine and zebrafish models of MYCN-driven neuroblastoma. Our results suggest that Sulfopin is a suitable chemical probe for assessing Pin1-dependent pharmacology in cells and in vivo. Moreover, these studies indicate that Pin1 should be further investigated as a potential cancer target.

cancer biology↗

bioRxiv2018

Rapid covalent-probe discovery by electrophile fragment screening

Covalent probes can display unmatched potency, selectivity and duration of action, however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening. Such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge, and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2, and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile fragment screening as a practical and efficient tool for covalent ligand discovery.

biochemistry↗

bioRxiv2017

Overcoming insecticide resistance through computational inhibitor design

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could therefore restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent computational design to produce nano/pico-molar boronic acid inhibitors of the carboxylesterase E7 from the agricultural pest Lucilia cuprina, as well as a common Gly137Asp E7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase, and low to no toxicity in human cells and mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold, and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

biochemistry↗