Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp

 Guillaume Compain, Clément Monsarrat, Julie Blagojevic, Karl Brillet, Philippe Dumas, Philippe Hammann, Lauriane Kuhn, Isabelle Martiel, Sylvain Engilberge, Vincent Oliéric, Philippe Wolff, Dominique Y. Burnouf, Jérôme Wagner, and Gilles Guichard

JACS Au 2024

DOI: 10.1021/jacsau.3c00572

Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein–protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor–SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein–protein interactions targeted to histidine.

Novel Covalent Probe Selectively Targeting Glutathione Peroxidase 4 In Vivo: Potential Applications in Pancreatic Cancer Therapy

Zifeng Tang, Jie Li, Lijie Peng, Fang Xu, Yi Tan, Xiaoqiang He, Chengjun Zhu, Zhi-Min Zhang, Zhang Zhang, Pinghua Sun, Ke Ding, and Zhengqiu Li

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.3c01608

Glutathione peroxidase 4 (GPX4) emerges as a promising target for the treatment of therapy-resistant cancer through ferroptosis. Thus, there is a broad interest in the development of GPX4 inhibitors. However, a majority of reported GPX4 inhibitors utilize chloroacetamide as a reactive electrophilic warhead, and the selectivity and pharmacokinetic properties still need to be improved. Herein, we developed a compound library based on a novel electrophilic warhead, the sulfonyl ynamide, and executed phenotypic screening against pancreatic cancer cell lines. Notably, one compound A16 exhibiting potent cell toxicity was identified. Further chemical proteomics investigations have demonstrated that A16 specifically targets GPX4 under both in situ and in vivo conditions, inducing ferroptosis. Importantly, A16 exhibited superior selectivity and potency compared to reported GPX4 inhibitors, ML210 and ML162. This provides the structural diversity of tool probes for unraveling the fundamental biology of GPX4 and exploring the therapeutic potential of pancreatic cancer via ferroptosis induction.

Peptide–Drug Conjugates: An Emerging Direction for the Next Generation of Peptide Therapeutics

 Trevor T. Dean, Juliet Jelú-Reyes, A’Lester C. Allen, and Terry W. Moore

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.3c01835

Building on recent advances in peptide science, medicinal chemists have developed a hybrid class of bioconjugates, called peptide–drug conjugates, that demonstrate improved efficacy compared to peptides and small molecules independently. In this Perspective, we discuss how the conjugation of synergistic peptides and small molecules can be used to overcome complex disease states and resistance mechanisms that have eluded contemporary therapies because of their multi-component activity. We highlight how peptide–drug conjugates display a multi-factor therapeutic mechanism similar to that of antibody–drug conjugates but also demonstrate improved therapeutic properties such as less-severe off-target effects and conjugation strategies with greater site-specificity. The many considerations that go into peptide–drug conjugate design and optimization, such as peptide/small-molecule pairing and chemo-selective chemistries, are discussed. We also examine several peptide–drug conjugate series that demonstrate notable activity toward complex disease states such as neurodegenerative disorders and inflammation, as well as viral and bacterial targets with established resistance mechanisms.

Covalent Hits and Where to Find Them

Simon C.C. Lucas, J. Henry Blackwell, Sarah H. Hewitt, Hannah Semple, Benjamin C. Whitehurst, Hua Xu,

SLAS Discovery, 2024

https://doi.org/10.1016/j.slasd.2024.01.003

Covalent hits for drug discovery campaigns are neither fantastic beasts nor mythical creatures, they can be routinely identified through electrophile-first screening campaigns using a suite of different techniques. These include biophysical and biochemical methods, cellular approaches, and DNA-encoded libraries. Employing best practice, however, is critical to success. The purpose of this review is to look at state of the art covalent hit identification, how to identify hits from a covalent library and how to select compounds for medicinal chemistry programmes.

Phenotypic screening of covalent compound libraries identifies chloromethyl ketone antibiotics and MiaA as a new target

Yizhen Jin, Sadhan Jana, Mikail E. Abbasov, Hening Lin

bioRxiv 2024.01.22.576576; 

doi: https://doi.org/10.1101/2024.01.22.576576

The emerging antibiotic resistance requires the development of new antibiotics working on novel bacterial targets. Here, we reported an antibiotic discovery workflow by combining the cysteine-reactive compound library phenotypic screening with activity-based protein profiling, which enables the rapid identification of lead compounds as well as new druggable targets in pathogens. Compounds featuring chloromethyl ketone scaffolds exhibited a notably high hit rate against both gram-negative and gram-positive bacterial strains, but not the more commonly used warheads such as acrylamide or chloroacetamide. Target identification of the lead compound, 10-F05, revealed that its primary targets in S. flexneri are FabH Cys112 and MiaA Cys273. We validated the target relevance through biochemical and genetic interactions. Mechanistic studies revealed modification of MiaA by 10-F05 impair substrate tRNA binding, leading to decreased bacterial stress resistance and virulence. Our findings underscore chloromethyl ketone as a novel antibacterial warhead in covalent antibiotic design. The study showcases that combining covalent compound library phenotypic screening with chemoproteomics is an efficient way to identify new drug targets as well as lead compounds, with the potential to open new research directions in drug discovery and chemical biology.

Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development

Guanghui Tang, Wei Wang, Chengjun Zhu, Huisi Huang, Peng Chen, Xuan Wang, Manyi Xu, Jie Sun, Chong-Jing Zhang, Qicai Xiao, Liqian Gao, Zhi-Min Zhang, Shao Q. Yao, 

Angew. Chem. Int. Ed. 2024, e202316394.

https://doi.org/10.1002/anie.202316394

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine-reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell-active TCIs are however available. We report herein lysine-reactive activity-based probes (ABPs; 2-14) based on the chemistry of aryl fluorosulfates (ArOSO2F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase-binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X-ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX-680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine-reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine-reactive ArOSO2F-based TCIs.

Silicon-Containing Thiol-Specific Bioconjugating Reagent

Zhenguo Zhang, Lanyang Li, Hailun Xu, Chi-Lik Ken Lee, Zhenhua Jia, and Teck-Peng Loh

Journal of the American Chemical Society 2024 146 (3), 1776-1782

DOI: 10.1021/jacs.3c12050

A new bioconjugation reagent containing silicon has been developed for the selective reaction with thiols. The inclusion of silicon significantly improves chemoselectivity and suppresses retro processes, thereby exceeding the capabilities of traditional reagents. The method is versatile and compatible with a broad range of thiols and unsaturated carbonyl compounds and yields moderate to high results. These reactions can be conducted under biocompatible conditions, thereby making them suitable for protein bioconjugation. The resulting conjugates display good stability in the presence of various biomolecules, which suggests their potential application for the synthesis of antibody–drug conjugates. Furthermore, the presence of a silicon moiety within the conjugated products opens up new avenues for drug release and bridging inorganics with other disciplines. This new class of silicon-containing thiol-specific bioconjugation reagents has significant implications for researchers working in bioanalytical science and medicinal chemistry and leads to innovative opportunities for advancing the field of bioconjugation research and medicinal chemistry.

Expanding the ligandable proteome by paralog hopping with covalent probes

Yuanjin Zhang, Zhonglin Liu, Marsha Hirschi, Oleg Brodsky, Eric Johnson, Sang Joon Won, Asako Nagata, Matthew D Petroski, Jaimeen D Majmudar, Sherry Niessen, Todd VanArsdale, Adam M Gilbert, Matthew M Hayward, Al E Stewart, Andrew R Nager, Bruno Melillo, Benjamin F Cravatt

bioRxiv 2024.01.18.576274; 

doi: https://doi.org/10.1101/2024.01.18.576274

More than half of the ~20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR

Alok K. Sharma,Jun Pei,Yue Yang,Marcin Dyba,Brian Smith,Dana Rabara,Erik Larsen,Felice C. Lightstone,Dominic Esposito,Andrew G. Stephen,Bin Wang,Pedro J. Beltran,Eli Wallace,Dwight V. Nissley,Frank McCormick,Anna E. Maciag

Journal of Biological Chemistry, 2024

https://www.jbc.org/article/S0021-9258(24)00026-7/fulltext

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient), and state 2 (active, effector binding enabled). Here we use 31P NMR to delineate the differences in state 1 and state 2 populations present in wild-type (WT) and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analogue GppNHp. Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in the state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RBD (RAS Binding Domain) binding and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small molecule inhibitor, BBO-8956, which is effective against both GDP and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs the state 1 - state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1 RBD is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action (MOA) for this novel, active-conformation inhibitor.

Covalent Targeting of Splicing in T Cells

Kevin A. Scott, Hiroyuki Kojima, Nathalie Ropek, Charles D. Warren, Tiffany L. Zhang, Simon J. Hogg, Caroline Webster, Xiaoyu Zhang, Jahan Rahman, Bruno Melillo, Benjamin F. Cravatt, Jiankun Lyu, Omar Abdel-Wahab, Ekaterina V. Vinogradova

bioRxiv 2023.12.18.572199; doi: https://doi.org/10.1101/2023.12.18.572199

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

Biomimetic Synthesis and Chemical Proteomics Reveal the Mechanism of Action and Functional Targets of Phloroglucinol Meroterpenoids

Amy K. Bracken, Colby E. Gekko, Nina O. Suss, Emma E. Lueders, Qi Cui, Qin Fu, Andy C. W. Lui, Elizabeth T. Anderson, Sheng Zhang, and Mikail E. Abbasov

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.3c10741

Natural products perennially serve as prolific sources of drug leads and chemical probes, fueling the development of numerous therapeutics. Despite their scarcity, natural products that modulate protein function through covalent interactions with lysine residues hold immense potential to unlock new therapeutic interventions and advance our understanding of the biological processes governed by these modifications. Phloroglucinol meroterpenoids constitute one of the most expansive classes of natural products, displaying a plethora of biological activities. However, their mechanism of action and cellular targets have, until now, remained elusive. In this study, we detail the concise biomimetic synthesis, computational mechanistic insights, physicochemical attributes, kinetic parameters, molecular mechanism of action, and functional cellular targets of several phloroglucinol meroterpenoids. We harness synthetic clickable analogues of natural products to probe their disparate proteome-wide reactivity and subcellular localization through in-gel fluorescence scanning and cell imaging. By implementing sample multiplexing and a redesigned lysine-targeting probe, we streamline a quantitative activity-based protein profiling, enabling the direct mapping of global reactivity and ligandability of proteinaceous lysines in human cells. Leveraging this framework, we identify numerous lysine–meroterpenoid interactions in breast cancer cells at tractable protein sites across diverse structural and functional classes, including those historically deemed undruggable. We validate that phloroglucinol meroterpenoids perturb biochemical functions through stereoselective and site-specific modification of lysines in proteins vital for breast cancer metabolism, including lipid signaling, mitochondrial respiration, and glycolysis. These findings underscore the broad potential of phloroglucinol meroterpenoids for targeting functional lysines in the human proteome.

Diazepam-based covalent modifiers of GPX4 induce ferroptosis in liver cancer cells

D. Yadav, S. Tiwari, S. Senthil, S. K. Vechalapu, S. Duraisamy, V. Rawat, M. I. Rahman, S. Khanna and D. Allimuthu, 

Chem. Commun., 2024

DOI: 10.1039/D3CC06215E

Developing new chemotherapeutics that are structurally and mechanistically unique is a need due to the rapid rise of cancer incidences across the globe. Here we report the identification of irreversible, thiol-reactive diazepam derivatives as GPX4 modifiers and nanomolar inducers of ferroptosis in liver cancer cells.

Design, Synthesis, X-ray Crystallography, and Biological Activities of Covalent, Non-Peptidic Inhibitors of SARS-CoV-2 Main Protease

Md Ashraf-Uz-Zaman, Teck Khiang Chua, Xin Li, Yuan Yao, Bala Krishna Moku, Chandra Bhushan Mishra, Vasanthi Avadhanula, Pedro A. Piedra, and Yongcheng Song

ACS Infectious Diseases 2023

DOI: 10.1021/acsinfecdis.3c00565

Highly contagious SARS-CoV-2 coronavirus has infected billions of people worldwide with flu-like symptoms since its emergence in 2019. It has caused deaths of several million people. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and therefore a drug target. Several series of covalent inhibitors of Mpro were designed and synthesized. Structure–activity relationship studies show that (1) several chloroacetamide- and epoxide-based compounds targeting Cys145 are potent inhibitors with IC50 values as low as 0.49 μM and (2) Cys44 of Mpro is not nucleophilic for covalent inhibitor design. High-resolution X-ray studies revealed the protein–inhibitor interactions and mechanisms of inhibition. It is of interest that Cys145 preferably attacks the more hindered Cα atom of several epoxide inhibitors. Chloroacetamide inhibitor 13 and epoxide inhibitor 30 were found to inhibit cellular SARS-CoV-2 replication with an EC68 (half-log reduction of virus titer) of 3 and 5 μM. These compounds represent new pharmacological leads for anti-SARS-CoV-2 drug development. 

Catalytic Protein Inhibitors

Prof. Dr. Thomas Kodadek

Angewandte Chemie International Edition 2024 e202316726

https://onlinelibrary.wiley.com/doi/10.1002/anie.202316726

Many of the highest priority targets in a wide range of disease states are difficult-to-drug proteins. The development of reversible small molecule inhibitors for the active sites of these proteins with sufficient affinity and residence time on-target is an enormous challenge. This has engendered interest in strategies to increase the potency of a given protein inhibitor by routes other than further improvement in gross affinity. Amongst these, the development of catalytic protein inhibitors has garnered the most attention and investment, particularly with respect to protein degraders, which catalyze the destruction of the target protein. This article discusses the genesis of the burgeoning field of catalytic inhibitors, the current state of the art, and exciting future directions.

Electrophilic MiniFrags Revealed Unprecedented Binding Sites for Covalent HDAC8 Inhibitors

Aaron B. Keeley, Aleksandra Kopranovic, Vincenzo Di Lorenzo, Péter Ábrányi-Balogh, Niklas Jänsch, Linh N. Lai, László Petri, Zoltán Orgován, Daniel Pölöske, Anna Orlova, András György Németh, Charlotte Desczyk, Tímea Imre, Dávid Bajusz, Richard Moriggl, Franz-Josef Meyer-Almes, and György M. Keserü

Journal of Medicinal Chemistry 2024 67 (1), 572-585

 https://doi.org/10.1021/acs.jmedchem.3c01779

Screening of ultra-low-molecular weight ligands (MiniFrags) successfully identified viable chemical starting points for a variety of drug targets. Here we report the electrophilic analogues of MiniFrags that allow the mapping of potential binding sites for covalent inhibitors by biochemical screening and mass spectrometry. Small electrophilic heterocycles and their N-quaternized analogues were first characterized in the glutathione assay to analyze their electrophilic reactivity. Next, the library was used for systematic mapping of potential covalent binding sites available in human histone deacetylase 8 (HDAC8). The covalent labeling of HDAC8 cysteines has been proven by tandem mass spectrometry measurements, and the observations were explained by mutating HDAC8 cysteines. As a result, screening of electrophilic MiniFrags identified three potential binding sites suitable for the development of allosteric covalent HDAC8 inhibitors. One of the hit fragments was merged with a known HDAC8 inhibitor fragment using different linkers, and the linker length was optimized to result in a lead-like covalent inhibitor.

High-Affinity Fluorogenic Substrate for Tissue Transglutaminase Reveals Enzymatic Hysteresis [@theKeillors]

Eric W. J. Gates, Adrien Prince-Hallée, Yasaman Heidari, Abootaleb Sedighi, and Jeffrey W. Keillor

Biochemistry 2023 62 (21), 3085-3095

DOI: 10.1021/acs.biochem.3c00337

Transglutaminases (TGases) are a family of calcium-dependent enzymes primarily known for their ability to cross-link proteins. Transglutaminase 2 (TG2) is one isozyme in this family whose role is multifaceted. TG2 can act not only as a typical transamidase through its catalytic core but also as a G-protein via its GTP binding site. These two discrete activities are tightly regulated by both environmental stimuli and redox reactions. Ubiquitously expressed in humans, TG2 has been implicated in numerous disease pathologies that require extensive investigation. The catalytic activity of TG2 can be monitored through various mechanisms, including hydrolysis, transamidation, or cleavage of isopeptide bonds. Activity assays are required to monitor the activity of this isozyme not only for studying its transamidation reaction but also for validation of therapeutics designed to abolish this activity. Herein, we present the design, synthesis, and evaluation of a new TG2 activity substrate based on a previously optimized inhibitor scaffold. The substrate APH7 exhibits excellent affinity, selectivity, and reactivity with TG2 (KM = 3.0 μM). Furthermore, its application also allowed the discovery of unique hysteresis at play within the catalytic activity and inhibition reactivity of TG2.

Design and Evaluation of PROTACs Targeting Acyl Protein Thioesterase 1

Dr. Luís A. R. Carvalho,  Bárbara B. Sousa,  Daniel Zaidman,  Hannah Kiely-Collins,  Prof. Gonçalo J. L. Bernardes

Nat Commun 10, 5811 (2019).

https://doi.org/10.1002/cbic.202300736

PROTAC linker design remains mostly an empirical task. We employed the PRosettaC computational software in the design of sulfonyl-fluoride-based PROTACs targeting acyl protein thioesterase 1 (APT1). The software efficiently generated ternary complex models from empirically-designed PROTACs and suggested alkyl linkers to be the preferred type of linker to target APT1. Western blotting analysis revealed efficient degradation of APT1 and activity-based protein profiling showed remarkable selectivity of an alkyl linker-based PROTAC amongst serine hydrolases. Collectively, our data suggests that combining PRosettaC and chemoproteomics can effectively assist in triaging PROTACs for synthesis and providing early data on their potency and selectivity.

Modulating Liquid–Liquid Phase Separation of Nck Adaptor Protein against Enteropathogenic Escherichia coli Infection

Min Liu, Chunjian Wu, Rui Wang, Jiaming Qiu, Zhentao She, Jianan Qu, and Jiang Xia

ACS Central Science 2023 9 (12), 2358-2368

DOI: 10.1021/acscentsci.3c01068

Signaling proteins often form biomolecular condensates through liquid–liquid phase separation (LLPS) during intracellular signal transduction. Modulating the LLPS property of intracellular protein condensates will redirect intracellular signals and provide a potential way to regulate cellular physiology. Phosphorylation of multiple tyrosine residues of the transmembrane receptor nephrin is known to drive the LLPS of the adaptor protein Nck and neuronal Wiskott–Aldrich Syndrome protein (N-WASP) and form the Nck signaling complex. Phosphorylation of the translocated intimin receptor (Tir) in the host cell may recruit this enteropathogenic Escherichia coli (EPEC) virulence factor to the Nck signaling complex and lead to the entry of EPEC into the intestine cell. In this work, we first identified a phosphotyrosine (pY)-containing peptide 3pY based on the sequence similarity of nephrin and Tir; 3pY promoted the LLPS of Nck and N-WASP, mimicking the role of phosphorylated nephrin. Next, we designed a covalent blocker of Nck, peptide p1 based on the selected pY peptides, which site-selectively reacted with the SH2 domain of Nck (Nck-SH2) at Lys331 through a proximity-induced reaction. The covalent reaction of p1 with Nck blocked the protein binding site of Nck-SH2 and disintegrated the 3pY/Nck/N-WASP condensates. In the presence of membrane-translocating peptide L17E, p1 entered Caco-2 cells in the cytosol, reduced the number of Nck puncta, and rendered Caco-2 cells resistant to EPEC infection. Site-selective covalent blockage of Nck thereby disintegrates intracellular Nck condensates, inhibits actin reorganization, and shuts down the entrance pathway of EPEC. This work showcases the promotion or inhibition of protein phase separation by synthetic peptides and the use of reactive peptides as LLPS disruptors and signal modulators.

Two-Step Covalent Docking with Attracting Cavities

Mathilde Goullieux, Vincent Zoete, and Ute F. Röhrig

Journal of Chemical Information and Modeling 2023 63 (24), 7847-7859

DOI: 10.1021/acs.jcim.3c01055

Due to their various advantages, interest in the development of covalent drugs has been renewed in the past few years. It is therefore important to accurately describe and predict their interactions with biological targets by computer-aided drug design tools such as docking algorithms. Here, we report a covalent docking procedure for our in-house docking code Attracting Cavities (AC), which mimics the two-step mechanism of covalent ligand binding. Ligand binding to the protein cavity is driven by nonbonded interactions, followed by the formation of a covalent bond between the ligand and the protein through a chemical reaction. To test the performance of this method, we developed a diverse, high-quality, openly accessible re-docking benchmark set of 95 covalent complexes bound by 8 chemical reactions to 5 different reactive amino acids. Combination with structures from previous studies resulted in a set of 304 complexes, on which AC obtained a success rate (rmsd ≤ 2 Å) of 78%, outperforming two state-of-the-art covalent docking codes, genetic optimization for ligand docking (GOLD (66%)) and AutoDock (AD (35%)). Using a more stringent success criterion (rmsd ≤ 1.5 Å), AC reached a success rate of 71 vs 55% for GOLD and 26% for AD. We additionally assessed the cross-docking performance of AC on a set of 76 covalent complexes of the SARS-CoV-2 main protease. On this challenging test set of mainly small and highly solvent-exposed ligands, AC yielded success rates of 58 and 28% for re-docking and cross-docking, respectively, compared to 45 and 17% for GOLD.