Covalent inhibitors of the RAS binding domain of PI3Ka impair tumor growth driven by RAS and HER2

Joseph E Klebba, Nilotpal Roy, Steffen M Bernard, Stephanie Grabow, Melissa A. Hoffman, Hui Miao, Junko Tamiya, Jinwei Wang, Cynthia Berry, Antonio Esparza-Oros, Richard Lin, Yongsheng Liu, Marie Pariollaud, Holly Parker, Igor Mochalkin, Sareena Rana, Aaron N. Snead, Eric J. Walton, Taylor E. Wyrick, Erick Aitichson, Karl Bedke, Jacyln C. Brannon, Joel M. Chick, Kenneth Hee, Benjamin D. Horning, Mohamed Ismail, Kelsey N. Lamb, Wei Lin, Justine Metzger, Martha K. Pastuszka, Jonathan Pollock, John J. Sigler, Mona Tomaschko, Eileen Tran, Todd M. Kinsella, Miriam Molina-Arcas, Gabriel M. Simon, David S. Weinstein, Julian Downward, Matthew P. Patricelli

bioRxiv 2024.12.17.629001; 

doi: https://doi.org/10.1101/2024.12.17.629001

Genetic disruption of the RAS binding domain (RBD) of PI 3-kinase (PI3K) prevents the growth of mutant RAS driven tumors in mice and does not impact PI3Ks role in insulin mediated control of glucose homeostasis. Selectively blocking the RAS-PI3K interaction may represent an attractive strategy for treating RAS-dependent cancers as it would avoid the toxicity associated with inhibitors of PI3K lipid kinase activity such as alpelisib. Here we report compounds that bind covalently to cysteine 242 in the RBD of PI3K p110a and block the ability of RAS to activate PI3K activity. These inhibitors have a profound impact on the growth of RAS mutant and also HER2 over-expressing tumors, particularly when combined with other inhibitors of the RAS/MAPK pathway, without causing hyperglycemia.

Exploring Extended Warheads toward Developing Cysteine-Targeted Covalent Kinase Inhibitors

Zheng Zhao and Philip E. Bourne

Journal of Chemical Information and Modeling 2024

DOI: 10.1021/acs.jcim.4c00890

In designing covalent kinase inhibitors (CKIs), the inclusion of electrophiles as attacking warheads demands careful choreography, ensuring not only their presence on the scaffold moiety but also their precise interaction with nucleophiles in the binding sites. Given the limited number of known electrophiles, exploring adjacent chemical space to broaden the palette of available electrophiles capable of covalent inhibition is desirable. Here, we systematically analyze the characteristics of warheads and the corresponding adjacent fragments for use in CKI design. We first collect all the released cysteine-targeted CKIs from multiple databases and create one CKI data set containing 16,961 kinase-inhibitor data points from 12,381 unique CKIs covering 146 kinases with accessible cysteines in their binding pockets. Then, we analyze this data set, focusing on the extended warheads (i.e., warheads + adjacent fragments)─including 30 common warheads and 1344 unique adjacent fragments. In so doing, we provide structural insights and delineate chemical properties and patterns in these extended warheads. Notably, we highlight the popular patterns observed within reversible CKIs for the popular warheads cyanoacrylamide and aldehyde. This study provides medicinal chemists with novel insights into extended warheads and a comprehensive source of adjacent fragments, thus guiding the design, synthesis, and optimization of CKIs.

Structure-Based Discovery of a Series of Covalent, Orally Bioavailable, and Selective BFL1 Inhibitors

Adeline Palisse, Tony Cheung, Aileen Blokhuis, Thomas Cogswell, Bruna S. Martins, Rick Riemens, Rick Schellekens, Giovanni Battocchio, Chimed Jansen, Matthew A. Cottee, Kimberly Ornell, Claudia Sacchetto, Leonardo Leon, Maaike van Hoek- Emmelot, Mark Bostock, Brooke Leann Brauer, Kevin Beaumont, Simon C. C. Lucas, Samiyah Ahmed, J. Henry Blackwell, Ulf Börjesson, Andrea Gohlke, Iva Monique T. Gramatikov, David Hargreaves, Vera van Hoeven, Vasudev Kantae, Lea Kupcova, Alexander G. Milbradt, Uthpala Seneviratne, Nancy Su, John Vales, Haiyun Wang, Michael J. White, and Olaf Kinzel

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c01995

BFL1, a member of the antiapoptotic BCL2 family, has been relatively understudied compared to its counterparts despite evidence of its overexpression in various hematological malignancies. Across two articles, we describe the development of BFL1 in vivo tools. The first article describes the hit identification from a covalent fragment library and the subsequent evolution from the hit to compound 6.22 This work reports the structure-based optimization of compound 6 into a series of BFL1 inhibitors selective over the other BCL2 family members, with low nanomolar cellular activity when combined with AZD5991, exemplified by compound 20. Compound 20 demonstrated a cell death phenotype in SUDHL1 and OCILY10 cell lines and in the in vivo study, BFL1 stabilization and cleaved caspase 3 activation were observed in a dose-dependent manner. In addition, the enzymatic turnover studies with the BFL1 protein showed that compound 20 stabilized the protein, extending the half-life to 10.8 h.

Open-source Electrophilic Fragment Screening Platform to Identify Chemical Starting Points for UCHL1 Covalent Inhibitors

Scott B. Ficarro, Zachary H. Marto, Nicholas M. Girardi, Dingyu Deng, Isabella Jaen Maisonet, Guillaume Adelmant, Laura E. Fleming, Mona Sharafi, Isidoro Tavares, Andrew Zhao, HyoJeon Kim, Hyuk-Soo Seo, Sirano Dhe-Paganon, Sara J. Buhrlage, Jarrod A. Marto

SLAS Discovery, 2024

https://doi.org/10.1016/j.slasd.2024.100198

Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC50 <5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling.

Identification of a covalent NEK7 inhibitor to alleviate NLRP3 inflammasome-driven metainflammation

Jin, X., Yang, Y., Liu, D. et al.

 Cell Commun Signal 22, 565 (2024). 

https://doi.org/10.1186/s12964-024-01919-w

Aberrant activation of NLRP3 inflammasome is associated with a variety of inflammatory diseases. Advances in understanding the molecular mechanisms of NLRP3 inflammasome have revealed that NEK7 is an essential component for its activation, but the development of drugs specifically targeting NEK7 remains challenging. Here we identify rociletinib (ROC), an anticancer drug in phase III clinical trial with high safety profile, as a highly potent and specific small-molecule antagonists of NEK7. Mechanistically, ROC covalent binds to the cysteine 79 of NEK7 through its reactive α, β-unsaturated carbonyl group, thereby inhibiting the interaction between NLRP3 and NEK7, and the subsequent assembly and activation of NLRP3 inflammasome. Furthermore, ROC alleviates the pathological features of metainflammation on the mouse model of type 2 diabetes (T2D). In summary, our results identify ROC as a covalent inhibitor of NEK7 and demonstrates that targeting NEK7 provides a potential and promising strategy for the treatment of NLRP3 inflammasome-driven T2D.

4-Oxo-β-lactams as Covalent Inhibitors of the Mitochondrial Intramembrane Protease PARL

Shanping Ji, Kathrin Bach, Vijay Madhav Miriyala, Jan Dohnálek, Miguel Riopedre-Fernandez, Martin Lepšík, Merel van de Plassche, Roeland Vanhoutte, Marta Barniol-Xicota, Rui Moreira, Kvido Strisovsky, and Steven H. L. Verhelst

ACS Medicinal Chemistry Letters 2024

DOI: 10.1021/acsmedchemlett.4c00384

Rhomboid proteases play a variety of physiological roles, but rhomboid protease inhibitors have been mostly developed for the E. coli model rhomboid GlpG. In this work, we screened different electrophilic scaffolds against the human mitochondrial rhomboid PARL and found 4-oxo-β-lactams as submicromolar inhibitors. Multifaceted computations suggest explanations for the activity at the molecular scale and provide models of covalently bound complexes. Together with the straightforward synthesis of the 4-oxo-β-lactam scaffold, this may pave the way toward selective, nonpeptidic PARL inhibitors.

Lysine-Targeted Covalent Inhibitors of PI3Kδ Synthesis and Screening by In Situ Interaction Upgradation

Bo Yuan, Yifan Feng, Mengyan Ma, Weiming Duan, Yujie Wu, Jiaxin Liu, Hong-Yi Zhao, Zhe Yang, San-Qi Zhang, and Minhang Xin

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c01284

Targeting the lysine residue of protein kinases to develop covalent inhibitors is an emerging hotspot. Herein, we have reported an approach to develop lysine-targeted covalent inhibitors of PI3Kδ by in situ interaction upgradation of the H-bonding to covalent bonding. Several warhead groups were introduced and screened in situ, leading to lysine-targeted covalent inhibitors bearing aromatic esters with high bioactivity and PI3Kδ selectivity. Compound A11 bearing phenolic ester was finally optimized to show a long duration of action in SU-DHL-6 cells by multiple assays. Docking simulation and further protein mass spectrometry confirmed that A11 bound to PI3Kδ by covalent-bonding interactions with Lys779. Furthermore, A11 exhibited potently antitumor efficacy without obvious toxicity in the SU-DHL-6 and Pfeiffer xenograft mouse models. This study identified A11 to be a much more effective antitumor agent in vitro and in vivo as a lysine-targeted covalent inhibitor, and it also provided a practical approach for the development of lysine-targeted covalent inhibitors.

Rapid, potent, and persistent covalent chemical probes to deconvolute PI3Kα signaling

Lukas Bissegger,  Theodora A. Constantin,  Erhan Keles,  Luka Raguž,   Isobel Barlow-Busch,  Clara Orbegozo,   Thorsten Schaefer,  Valentina Borlandelli,   Thomas Bohnacker,  Rohitha Sriramaratnam,   Alexander Schäfer,   Matthias Gstaiger,   John E. Burke,   Chiara Borsari O  and  Matthias P. Wymann 

Chem. Sci. 2024

DOI: 10.1039/D4SC05459H

Chemical probes have gained importance in the elucidation of signal transduction in biology. Insufficient selectivity and potency, lack of cellular activity and inappropriate use of chemical probes has major consequences on interpretation of biological results. The catalytic subunit of phosphoinositide 3-kinase α (PI3Kα) is one of the most frequently mutated genes in cancer, but fast-acting, high-quality probes to define PI3Kα's specific function to clearly separate it from other class I PI3K isoforms, are not available. Here, we present a series of novel covalent PI3Kα-targeting probes with optimized intracellular target access and kinetic parameters. On-target TR-FRET and off-target assays provided relevant kinetic parameters (kchem, kinact and Ki) to validate our chemical probes. Additional intracellular nanoBRET tracer displacement measurements showed rapid diffusion across the cell membrane and extremely fast target engagement, while investigations of signaling downstream of PI3Kα via protein kinase B (PKB/Akt) and forkhead box O (FOXO) revealed blunted pathway activity in cancer cell lines with constitutively activated PI3Kα lasting for several days. In contrast, persistent PI3Kα inhibition was rapidly bypassed by other class I PI3K isoforms in cells lacking functional phosphatase and tensin homolog (PTEN). Comparing the rapidly-diffusing, fast target-engaging chemical probe 9 to clinical reversible PI3Kα-selective inhibitors alpelisib, inavolisib and 9r, a reversible analogue of 9, revealed 9's superior potency to inhibit growth (up to 600-fold) associated with sustained suppression of PI3Kα signaling in breast cancer cell lines. Finally, using a simple washout protocol, the utility of the highly-selective covalent PI3Kα probe 9 was demonstrated by the quantification of the coupling of insulin, EGF and CXCL12 receptors to distinct PI3K isoforms for signal transduction in response to ligand-dependent activation. Collectively, these findings along with the novel covalent chemical probes against PI3Kα provide insights into isoform-specific functions in cancer cells and highlight opportunities to achieve improved selectivity and long-lasting efficacy. 

Covalent Targeting of Histidine Residues with Aryl Fluorosulfates: Application to Mcl-1 BH3 Mimetics

Giulia Alboreggia, Parima Udompholkul, Emma L. Atienza, Kendall Muzzarelli, Zahra Assar, and Maurizio Pellecchia

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c01541

Covalent drugs provide pharmacodynamic and pharmacokinetic advantages over reversible agents. However, covalent strategies have been developed mostly to target cysteine (Cys) residues, which are rarely found in binding sites. Among other nucleophilic residues that could be in principle used for the design of covalent drugs, histidine (His) has not been given proper attention despite being in principle an attractive residue to pursue but underexplored. Aryl fluorosulfates, a mild electrophile that is very stable in biological media, have been recently identified as possible electrophiles to react with the side chains of Lys; however, limited studies are available on aryl fluorosulfates’ ability to target His residues. We demonstrate that proper incorporation of an aryl fluorosulfate juxtaposing the electrophile with a His residue can be used to afford rapid optimizations of His-covalent agents. As an application, we report on His-covalent BH3 mimetics targeting His224 of Mcl-1.

Comprehensive Exploration of Isocitrate Dehydrogenase (IDH) Mutations: Tumorigenesis, Drug Discovery, and Covalent Inhibitor Advances

Conghao Gai, Hairong Zeng ,  Haoming Xu, Xiaoyun Chai, Yan Zou, Chunlin Zhuang, Guangbo Ge, Qingjie Zhao 

European Journal of Medicinal Chemistry, 2024

https://doi.org/10.1016/j.ejmech.2024.117041

Isocitrate dehydrogenase (IDH) is an enzyme that catalyses the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (α-KG) relative to the hydroxylation of substrates. However, IDH mutants can further reduce α-KG to 2-hydroxyglutarate (2-HG) which competitively inhibits α-KG dependent enzymes, leading to the downregulation of normal hydroxylation pathways. Good IDH mutant inhibitors can effectively reduce the level of 2-HG and therefore disturb cellular malignant transformation. In this review, we introduce the biological functions of IDH, describe the tumorigenesis mechanisms of IDH variants, and review the structure-based drug discovery of clinical inhibitors during 2012-2024. We also find successful applications of covalent strategy in the development of irreversible IDH inhibitors. Biological screening methods are also collected in this paper, which may help researchers to rapidly construct workflows for drug discovery and development.

Scalable Thiol Reactivity Profiling Identifies Azetidinyl Oxadiazoles as Cysteine-Targeting Electrophiles

Fereshte Ghorbani, Shaochen You, Gennadii A. Grabovyi, Mannkyu Hong, Garrett Lindsey, Arnab K. Chatterjee, and Michael J. Bollong

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c05711

Cysteine reactive groups are a mainstay in the design of covalent drugs and probe molecules, yet only a handful of electrophiles are routinely used to target this amino acid. Here, we report the development of scalable thiol reactivity (STRP), a method which enables the facile interrogation of large chemical libraries for intrinsic reactivity with cysteine. High throughput screening using STRP identified the azetidinyl oxadiazole as a moiety that selectively reacts with cysteine through a ring opening-based mechanism, capable of covalently engaging cysteine residues broadly across the human proteome. We show the utility of this reactive group with the discovery of an azetidinyl oxadiazole containing a small molecule that augments the catalytic activity of the deubiquitinase UCHL1 in vitro and in cells by covalently modifying a cysteine distal to its enzymatic active site. This study adds a novel cysteine targeting group to the electrophilic lexicon and provides robust methodology to rapidly surveil libraries for reactivity with cysteine.

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor

Sijie Wang, Emily C. Woods, Jeyun Jo, Jiyun Zhu, Althea Hansel-Harris, Matthew Holcomb, Nichole J. Pedowitz, Tulsi Upadhyay, John Bennett, Matthias Fellner, Ki Wan Park, Anna Zhang, Tulio A. Valdez, Stefano Forli, Alix I Chan, Christian N. Cunningham, Matthew Bogyo

bioRxiv 2024.11.06.622387; 

doi: https://doi.org/10.1101/2024.11.06.622387

Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

Mutant-selective AKT inhibition through lysine targeting and neo-zinc chelation

Gregory B. Craven, Hang Chu, Jessica D. Sun, Jordan D. Carelli, Brittany Coyne, Hao Chen, Ying Chen, Xiaolei Ma, Subhamoy Das, Wayne Kong, Adam D. Zajdlik, Kin S. Yang, Solomon H. Reisberg, Peter A. Thompson, J. Russell Lipford & Jack Taunton 

Nature, 2024

https://doi.org/10.1038/s41586-024-08176-4

Somatic alterations in the oncogenic kinase AKT1 have been identified in a broad spectrum of solid tumours. The most common AKT1 alteration replaces Glu17 with Lys (E17K) in the regulatory pleckstrin homology domain1, resulting in constitutive membrane localization and activation of oncogenic signalling. In clinical studies, pan-AKT inhibitors have been found to cause dose-limiting hyperglycaemia, which has motivated the search for mutant-selective inhibitors. We exploited the E17K mutation to design allosteric, lysine-targeted salicylaldehyde inhibitors with selectivity for AKT1 (E17K) over wild-type AKT paralogues, a major challenge given the presence of three conserved lysines near the allosteric site. Crystallographic analysis of the covalent inhibitor complex unexpectedly revealed an adventitious tetrahedral zinc ion that coordinates two proximal cysteines in the kinase activation loop while simultaneously engaging the E17K–imine conjugate. The salicylaldimine complex with AKT1 (E17K), but not that with wild-type AKT1, recruits endogenous Zn2+ in cells, resulting in sustained inhibition. A salicylaldehyde-based inhibitor was efficacious in AKT1 (E17K) tumour xenograft models at doses that did not induce hyperglycaemia. Our study demonstrates the potential to achieve exquisite residence-time-based selectivity for AKT1 (E17K) by targeting the mutant lysine together with Zn2+ chelation by the resulting salicylaldimine adduct.

Target Ligand Separation and Identification of Isoforsythiaside as a Histone Lysine-Specific Demethylase 1 Covalent Inhibitor Against Breast Cancer Metastasis

Mengzhen Gu, Xiaoqing Xu, Xiaoping Wang, Yun Wang, Yu Zhao, Xiaoxian Hu, Lu Zhu, Zhenzhong Deng, and Chao Han

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c02277

Histone lysine-specific demethylase 1 (LSD1) is hyperactive in breast cancer, which is associated with the metastasis of the tumor. Current irreversible LSD1 inhibitors are all synthesized by covalently binding to the flavin adenine dinucleotide cofactor, which often have side effects due to the high affinity for a variety of targets. Here, we identified isoforsythiaside (IFA), a natural phenylpropanoid glycoside isolated from Forsythia suspensa, as a novel covalent inhibitor of LSD1. The target ligand fishing technique and LC–MS/MS analysis identified that IFA could covalently bind to the Ser817 residue of LSD1 by α,β-unsaturated ketone moiety to block the amine oxidase-like domain of LSD1. Moreover, RBMS3/Twist1/MMP2, the downstream signaling pathway of LSD1, was activated after IFA treatment to inhibit the metastasis of MDA-MB-231 cells in vitro and in vivo. This study provided novel molecular templates for development of LSD1 covalence-binding inhibitor and laid a foundation for developing agents against breast carcinoma metastasis for targeting LSD1.

Covalency in PROTACs: Mechanisms and applications [@RPNowak]

Thomas M. Geiger, Radosław P. Nowak

Annual Reports in Medicinal Chemistry, 2024

https://doi.org/10.1016/bs.armc.2024.10.001

Proteolysis targeting chimeras (PROTACs) are hetero-bifunctional molecules that remove disease-causing proteins through the means of targeted protein degradation (TPD). Since their proof-of-concept over 20 years ago, PROTACs emerged as new modality in drug discovery and chemical biology. Historically, the vast majority of PROTACs use reversible-binding recruiters for both target and E3 ligase. However, in recent years more covalent PROTACs have been developed to harness the advantages of covalency such as unlocking the “undruggable” proteome to expand the repertoire of addressable targets and recruitable E3 ligases. Here, we review recent advances in covalent PROTACs, discuss their distinct mechanism of action and outline the key differences of this approach.

A Practical Guide for the Assay-Dependent Characterisation of Irreversible Inhibitors

Lavleen Mader,   Jessica Borean  and  Jeffrey W Keillor

RSC Med. Chem. 2024 

DOI 10.1039/D4MD00707G

Irreversible targeted covalent inhibitors, in the past regarded as inappropriately reactive and toxic, have seen a recent resurgence in clinical interest. This paradigm shift is attributed to the exploitation of the two-step mechanism, in which a high affinity and selectivity (i.e., low KI) scaffold binds the target and only then does a pendant low intrinsic reactivity warhead react with the target (moderate kinact). This highlights the importance of evaluating inhibitors by deriving both their KI and kinact values. The development of methods to evaluate these inhibitors by accounting for their time-dependent nature has been crucial to the discovery of promising clinical candidates. Herein, we report all the practical kinetic methods available to date to derive kinact and KI values. These methods include direct observation of covalent modification, continuous assay (Kitz & Wilson) evaluation, and discontinuous incubation and pre-incubation time-dependent IC50 assays. We also provide practical guidelines and examples for performing these assays, comparison of their utility, and perspectives for their extended applications. This review aims to provide clarity about the use of these methods for reporting complete inhibitor kinetic profiles, guiding irreversible drug development towards increased target affinity and selectivity, while modulating in-vivo stability and on-target reactivity.

Pan-Transcriptional Enhanced Associated Domain Palmitoylation Pocket Covalent Inhibitor

Jinhyuk Kim, Hadong Kim, Jongwan Kim, Seon Yeon Cho, Sungho Moon, Youngki Yoo, Hanseong Kim, Jin Kwan Kim, Hyejin Jeon, Wan Namkung, Gyoonhee Han, and Kyoung Tai No

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c01393

In the Hippo signaling pathway, the palmitoylated transcriptional enhanced associated domain (TEAD) protein interacts with the coactivator Yes-associated protein/PDZ-binding motif, leading to transcriptional upregulation of oncogenes such as Ctgf and Cyr61. Consequently, targeting the palmitoylation sites of TEAD has emerged as a promising strategy for treating TEAD-dependent cancers. Compound 1 was identified using a structure-based drug design approach, leveraging the molecular insights gained from the known TEAD palmitoylation site inhibitor, K-975. Optimization of the initial hit compound resulted in the development of compound 3, a covalent pan-TEAD inhibitor characterized by high potency and oral bioavailability.

Total Synthesis of Tagitinins, Goyazensolide and RelatedFuranoheliangolides and their Covalent Interaction withImportin-5 (IPO5)

W Liu, R Patouret, E Peev, S Barluenga, N Winssinger

Helvetica Chimica Acta, 2024 
https://doi.org/10.1002/hlca.202400122

Herein, we detail an extension of our research on the synthesis of a small library of furanoheliangolides and the characterization of the covalent interaction between goyazensolide and IPO5. Using a build‐couple‐pair strategy, we assembled a small library of germacrene‐type lactones and diversified them into eight groups of structurally different analogues. The germacrene lactones were synthesized using Sonogashira coupling and Barbier‐type macrocyclization, while the furanoheliangolides were further elaborated through gold‐catalyzed transannulation followed by esterification. This synthetic approach enabled the generation of a goyazensolide alkyne‐tagged cellular probe, which was used to identify the selective binding between goyazensolide and the oncoprotein importin‐5 (IPO5). Mass spectrometry analysis of the proteolytic digest from the reaction between the goyazensolide probe and a recombinant IPO5 indicated a covalent engagement at Cys560 of IPO5, which was confirmed by site‐directed mutagenesis.

Slow-Binding and Covalent HDAC Inhibition: A New Paradigm?

Yasir S. Raouf and Carlos Moreno-Yruela

JACS Au, 2024

DOI: 10.1021/jacsau.4c00828

The dysregulated post-translational modification of proteins is an established hallmark of human disease. Through Zn2+-dependent hydrolysis of acyl-lysine modifications, histone deacetylases (HDACs) are key regulators of disease-implicated signaling pathways and tractable drug targets in the clinic. Early targeting of this family of 11 enzymes (HDAC1–11) afforded a first generation of broadly acting inhibitors with medicinal applications in oncology, specifically in cutaneous and peripheral T-cell lymphomas and in multiple myeloma. However, first-generation HDAC inhibitors are often associated with weak-to-modest patient benefits, dose-limited efficacies, pharmacokinetic liabilities, and recurring clinical toxicities. Alternative inhibitor design to target single enzymes and avoid toxic Zn2+-binding moieties have not overcome these limitations. Instead, recent literature has seen a shift toward noncanonical mechanistic approaches focused on slow-binding and covalent inhibition. Such compounds hold the potential of improving the pharmacokinetic and pharmacodynamic profiles of HDAC inhibitors through the extension of the drug–target residence time. This perspective aims to capture this emerging paradigm and discuss its potential to improve the preclinical/clinical outlook of HDAC inhibitors in the coming years.

Discovery and development of Krazati (adagrasib/MRTX849), a potent, selective, orally bioavailable, covalent KRASG12C(OFF) inhibitor

Adrian L. Gill, Mathew A. Marx

RAS Drug Discovery Past, Present and Future 2025, 205-227

https://doi.org/10.1016/B978-0-443-21861-3.00017-6

Krazati (adagrasib/MRTX849) is a potent, selective, and covalent KRASG12C inhibitor, representing a significant breakthrough in directly targeting KRAS. Adagrasib demonstrates favorable drug-like properties, selectively modifying mutant cysteine 12 in GDP-bound KRASG12C, leading to the inhibition of KRAS-dependent signaling both in vitro and in vivo, and tumor regression in many KRASG12C-positive cell lines and patient-derived xenograft models across various tumor types. Objective responses have been observed in clinical trials, particularly in lung and colon adenocarcinoma patients with KRASG12C mutations. Comprehensive pharmacodynamic and pharmacogenomic profiling in both sensitive and partially resistant nonclinical models has shed light on the mechanisms limiting adagrasib antitumor activity. These resistance mechanisms include contributing factors related to KRAS nucleotide cycling, feedback reactivation pathways through activation of receptor tyrosine kinases, instances where tumors bypass KRAS dependence, and genetic dysregulation of the cell cycle. The ongoing characterization of adagrasib's activity, along with insights into response and resistance mechanisms, provides valuable understanding of KRAS dependence and opened a long-awaited opportunity to selectively target KRASG12C in patients. Furthermore, the identification of effective preclinical combinations, such as adagrasib with agents targeting RTKs, SHP2, mTOR, or the cell cycle, demonstrates enhanced responses and marked tumor regression. These findings contribute to the rational development of this class of agents, holding promise for improving therapeutic outcomes in KRASG12C-mutant human cancers.

Tuning isatoic anhydrides’ lysine ligation chemistry for bioconjugation and drug delivery

Tiwari, Sona, Senthil, Sathyapriya, Khanna, Shweta, Duraisamy, Santhosh, Vechalapu, Sai Kumari, Mallojjala, Sharath Chandra, Allimuthu, Dharmaraja,

Cell Reports Physical Science, 2024

DOI: https://doi.org/10.1016/j.xcrp.2024.102260

The discovery of new chemical entities for the selective modification of protein lysines is a recent interest in the development of unique covalent chemical probes. Isatoic anhydride (benzoxauracil), possessing aminophilic reactivity, was employed for the profiling of ligandable lysines in the cellular proteome. Our reactivity evaluation of benzoxauracil with proteins using mass spectral peptide mapping revealed a biased reactivity profile with nearly all the nucleophilic amino acids. The chemoselective reactivity of electrophilic tags is a key determinant of their idiosyncratic reactions. We applied the hard-soft-acid-base (HSAB) principle for tuning isatoic anhydride’s reactivity through systematic chemical modifications for lysine-dominant reactivity. We demonstrated the employability of ring-opening chemistry in isatoic anhydride as a drug delivery modality for the release of a small molecule and doxorubicin in cancer cells. Broadly, the tunable reactivity of isatoic anhydride could be leveraged for developing lysine-selective probes and drug delivery cargos.

CovalentInDB 2.0: an updated comprehensive database for structure-based and ligand-based covalent inhibitor design and screening

Hongyan Du, Xujun Zhang, Zhenxing Wu, Odin Zhang, Shukai Gu, Mingyang Wang, Feng Zhu, Dan Li, Tingjun Hou, Peichen Pan 

Nucleic Acids Research, 2024, gkae946

 https://doi.org/10.1093/nar/gkae946

The rational design of targeted covalent inhibitors (TCIs) has emerged as a powerful strategy in drug discovery, known for its ability to achieve strong binding affinity and prolonged target engagement. However, the development of covalent drugs is often challenged by the need to optimize both covalent warhead and non-covalent interactions, alongside the limitations of existing compound libraries. To address these challenges, we present CovalentInDB 2.0, an updated online database designed to support covalent drug discovery. This updated version includes 8303 inhibitors and 368 targets, supplemented by 3445 newly added cocrystal structures, providing detailed analyses of non-covalent interactions. Furthermore, we have employed an AI-based model to profile the ligandability of 144 864 cysteines across the human proteome. CovalentInDB 2.0 also features the largest covalent virtual screening library with 2 030 192 commercially available compounds and a natural product library with 105 901 molecules, crucial for covalent drug screening and discovery. To enhance the utility of these compounds, we performed structural similarity analysis and drug-likeness predictions. Additionally, a new user data upload feature enables efficient data contribution and continuous updates. CovalentInDB 2.0 is freely accessible at http://cadd.zju.edu.cn/cidb/.

Delineating cysteine-reactive compound modulation of cellular proteostasis processes

Ashley R. Julio, Flowreen Shikwana, Cindy Truong, Nikolas R. Burton, Emil R. Dominguez III, Alexandra C. Turmon, Jian Cao & Keriann M. Backus 

Nat Chem Biol 2024

https://doi.org/10.1038/s41589-024-01760-9

Covalent modulators and covalent degrader molecules have emerged as drug modalities with tremendous therapeutic potential. Toward realizing this potential, mass spectrometry-based chemoproteomic screens have generated proteome-wide maps of potential druggable cysteine residues. However, beyond these direct cysteine-target maps, the full scope of direct and indirect activities of these molecules on cellular processes and how such activities contribute to reported modes of action, such as degrader activity, remains to be fully understood. Using chemoproteomics, we identified a cysteine-reactive small molecule degrader of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host protein disulfide isomerases. This degrader activity was further potentiated by generalized electrophile-induced global protein ubiquitylation, proteasome activation and widespread aggregation and depletion of host proteins, including the formation of stress granules. Collectively, we delineate the wide-ranging impacts of cysteine-reactive electrophilic compounds on cellular proteostasis processes.

Expanding the Library of Covalent Cysteine Cathepsin Probes Featuring Sulfoxonium Ylide Electrophiles

Bangyan Xu, Simon J. Mountford, Philip E. Thompson, and Laura E. Edgington-Mitchell

ACS Omega 2024

DOI: 10.1021/acsomega.4c07604

Covalent activity-based probes are invaluable tools to monitor protease activity in vitro and in vivo. We recently discovered that dimethyl sulfoxonium ylides (SYs) bind selectively to cysteine cathepsin proteases in a mechanism-dependent manner. Herein, we present the synthetic routes and characterization of an expanded library of SY probes with a greater diversity in recognition sequences. The probes exhibit a range of potency and selectivity for the cathepsin family members. We also investigated the impact of fluorophore positioning on probes bearing P1 lysine. When sulfonated cyanine 5 was attached via the lysine side chain, the resulting probe was selective for cathepsin S. When attached to the α-amine, with the side chain amine either free or Boc-protected, the probes reacted with both cathepsin S and X. Bulk in the P1 position is thus well tolerated by cathepsin S but not cathepsin X. We examined the impact of Cy5 sulfonation on probe properties, demonstrating that unsulfonated probes exhibit greater cellular uptake, which affects their relative selectivity. Finally, we demonstrated that SY probes exhibit minimal labeling of cathepsin S in freshly prepared lysates, but this increases during the prolonged incubation of lysates. This work extends our understanding of SY probes and informs future probe development.

Redirecting the pioneering function of FOXA1 with covalent small molecules

Sang Joon Won, Yuxiang Zhang, Christopher J. Reinhardt,Lauren M. Hargis, Nicole S. MacRae,Kristen E. DeMeester,Evert Njomen,Jarrett R. Remsberg,Bruno Melillo, Benjamin F. Cravatt, Michael A. Erb

Molecular Cell, 2024

https://www.cell.com/molecular-cell/abstract/S1097-2765(24)00780-9

Pioneer transcription factors (TFs) bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic compounds that stereoselectively and site-specifically bind the pioneer TF forkhead box protein A1 (FOXA1) at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the ligands relax the canonical DNA-binding preference of FOXA1 by strengthening interactions with suboptimal sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.

Potent Inhibition and Rapid Photoactivation of Endogenous Bruton’s Tyrosine Kinase Activity in Native Cells via Opto-Covalent Modulators

Weizhi Weng, Ping Zhang, and Zhengying Pan

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c06459

Naturally, kinases exert their activities in a highly regulated fashion. A number of ingenious approaches have been developed to artificially control kinase activity by external stimuli, such as the incorporation of unnatural amino acids or the fusion of additional protein domains; however, methods that directly modulate endogenous kinases in native cells are lacking. Herein, we present a facile and potent method that takes advantage of recent developments in targeted covalent inhibitors and rapid light-mediated uncaging chemistry. Using an important drug target, Bruton’s tyrosine kinase (BTK), as an example, these opto-covalent modulators successfully blocked the activity of endogenous BTK in native cells after simple incubation and washout steps. However, upon a few minutes of light irradiation, BTK activity was cleanly restored, and could be blocked again by conventional inhibitors. Promisingly, this photoactivation strategy easily worked in human peripheral blood mononuclear cells (hPBMCs).

 

New electrophiles targeting thiols in a reversible covalent manner

Xingyu Ma,   Manyi Xu,   Fengge Wang,  Tingting Hu,  Xinyuan Chena  and  Chong-Jing Zhang

Chem. Commun., 2024

DOI

https://doi.org/10.1039/D4CC04612A

Reversible covalent electrophiles with the advantages of both reversible and covalent interactions receive much attention in the fields of chemical biology and medicinal chemistry. Here, we report two electron-deficient olefins activated by amide and ester, amide-substituted acrylamide and methyl ester-substituted acrylamide, targeting thiols in a reversible covalent manner.

Identification of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach

Eric M. Merten  and John D. Sears  and Tina M. Leisner  and P. Brian Hardy  and Anirban Ghoshal  and Mohammad Anwar Hossain  and Kesatebrhan Haile Asressu  and Peter J. Brown  and Edwin G. Tse  and Michael A. Stashko  and Kelin Li  and Jacqueline L. Norris-Drouin  and Laura E. Herring  and Angie L. Mordant  and Thomas S. Webb  and Christine A. Mills  and Natalie K. Barker  and Zachary J. Streblow  and Sumera Perveen  and Cheryl H. Arrowsmith  and Rafael Miguez Couñago  and Jamie J. Arnold  and Craig E. Cameron  and Daniel N. Streblow  and Nathaniel J. Moorman  and Mark T. Heise  and Timothy M. Willson  and Konstantin I. Popov  and Kenneth H. Pearce 

Proc. Natl. Acad. Sci. U.S.A. 2024 121 (42) e2409166121

https://doi.org/10.1073/pnas.2409166121

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV nonstructural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose–response follow-up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC50 of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a kinact /KI of 6.4 × 103 M−1s−1. LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity in proteomic experiments or against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the identification and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic.

Reversible covalent c-Jun N-terminal kinase inhibitors targeting a specific cysteine by precision-guided Michael-acceptor warheads

Bálint, D., Póti, Á.L., Alexa, A. et al. 

Nat Commun 15, 8606 (2024). 

https://doi.org/10.1038/s41467-024-52573-2

There has been a surge of interest in covalent inhibitors for protein kinases in recent years. Despite success in oncology, the off-target reactivity of these molecules is still hampering the use of covalent warhead-based strategies. Herein, we disclose the development of precision-guided warheads to mitigate the off-target challenge. These reversible warheads have a complex and cyclic structure with optional chirality center and tailored steric and electronic properties. To validate our proof-of-concept, we modified acrylamide-based covalent inhibitors of c-Jun N-terminal kinases (JNKs). We show that the cyclic warheads have high resilience against off-target thiols. Additionally, the binding affinity, residence time, and even JNK isoform specificity can be fine-tuned by adjusting the substitution pattern or using divergent and orthogonal synthetic elaboration of the warhead. Taken together, the cyclic warheads presented in this study will be a useful tool for medicinal chemists for the deliberate design of safer and functionally fine-tuned covalent inhibitors.

Targeting a key protein-protein interaction surface on mitogen-activated protein kinases by a precision-guided warhead scaffold

Póti, Á.L., Bálint, D., Alexa, A. et al. 

Nat Commun 15, 8607 (2024). 

https://doi.org/10.1038/s41467-024-52574-1

For mitogen-activated protein kinases (MAPKs) a shallow surface—distinct from the substrate binding pocket—called the D(ocking)-groove governs partner protein binding. Screening of broad range of Michael acceptor compounds identified a double-activated, sterically crowded cyclohexenone moiety as a promising scaffold. We show that compounds bearing this structurally complex chiral warhead are able to target the conserved MAPK D-groove cysteine via reversible covalent modification and interfere with the protein-protein interactions of MAPKs. The electronic and steric properties of the Michael acceptor can be tailored via different substitution patterns. The inversion of the chiral center of the warhead can reroute chemical bond formation with the targeted cysteine towards the neighboring, but less nucleophilic histidine. Compounds bind to the shallow MAPK D-groove with low micromolar affinity in vitro and perturb MAPK signaling networks in the cell. This class of chiral, cyclic and enhanced 3D shaped Michael acceptor scaffolds offers an alternative to conventional ATP-competitive drugs modulating MAPK signaling pathways.

Development of an orally bioavailable covalent STING inhibitor

NIU, G.-H., Hsiao, W.-C., Lee, P.-H., Zheng, L.-G., Yang, Y.-S., Huang, W.-C., Hsieh, C.-C., Chiu, T.-Y., Wang, J.-Y., Chen, C.-P., Huang, C.-L., You, M.-S., Kuo, Y.-P., Wang, C.-M., Wen, Z.-H., Yu, G.-Y., Chen, C.-T., Chi, Y.-H., Tung, C.-W., Hsu, S.-C., Yeh, T.-K., Sung, P.-J., Zhang, M. M., and Tsou, L. K. 

ChemRxiv, 2024

https://doi.org/10.26434/chemrxiv-2024-62g35

Pharmacological inhibition of cGAS-STING-controlled innate immune pathway is an emerging therapeutic strategy for a myriad of inflammatory diseases, including autoimmune disease, ulcerative colitis, non-alcoholic fatty liver disease and aging-related neurodegeneration. Here we report GHN105 as an orally bioavailable covalent STING inhibitor. Late-stage diversification of the briarane-type diterpenoid excavatolide B allowed the installation of solubility-enhancing functional groups while enhancing its activity as a covalent STING inhibitor against multiple human STING variants, including the S154 variant responsible for a genetic autoimmune disease. Selectively engaging the membrane-proximal Cys91 residue of STING, GHN105 dose-dependently inhibited cGAS-STING signaling and type I interferon responses in cells and in vivo. Orally administered GHN105 exerted marked therapeutic efficacy and reversed key pathological features in a delayed-treatment acute colitis mouse model. Notably, we also showed that GHN105 covalently engaged STING in the colon tissues. Our study provided proof of concept that synthetic briarane analog GHN105 serves as a safe and orally active covalent STING inhibitor. With a growing number of chronic inflammatory diseases linked to aberrant STING activation, orally bioavailable STING inhibitors would benefit patients by lowering the infection risk from frequent injections while allowing long-term systemic administration.

Discovery of electrophilic degraders that exploit SNAr chemistry

Zhe Zhuang, Woong Sub Byun, Zuzanna Kozicka, Brendan G. Dwyer, Katherine A. Donovan, Zixuan Jiang, Hannah M. Jones, Dinah M. Abeja, Meredith N. Nix, Jianing Zhong, Mikołaj Słabicki, Eric S. Fischer, Benjamin L. Ebert, Nathanael S. Gray

bioRxiv 2024.09.25.615094; 

doi: https://doi.org/10.1101/2024.09.25.615094

Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly ‘undruggable’ targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (SNAr) reactions in the rational design of electrophilic protein degraders. Appending an SNAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The SNAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an SNAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.

Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells

Njomen, E., Hayward, R.E., DeMeester, K.E. et al. Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells. Nat. Chem. (2024). 

https://doi.org/10.1038/s41557-024-01601-1

Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. However, the extent to which such residue-directed platforms fully assess the protein targets of electrophilic compounds in cells remains unclear. Here we evaluate a complementary protein-directed ABPP method that identifies proteins showing stereoselective reactivity with alkynylated, chiral electrophilic compounds—termed stereoprobes. Integration of protein- and cysteine-directed data from cancer cells treated with tryptoline acrylamide stereoprobes revealed generally well-correlated ligandability maps and highlighted features, such as protein size and the proteotypicity of cysteine-containing peptides, that explain gaps in each ABPP platform. In total, we identified stereoprobe binding events for >300 structurally and functionally diverse proteins, including compounds that stereoselectively and site-specifically disrupt MAD2L1BP interactions with the spindle assembly checkpoint complex leading to delayed mitotic exit in cancer cells.

Activity-Based Acylome Profiling with N-(Cyanomethyl)-N-(phenylsulfonyl)amides for Targeted Lysine Acylation and Post-Translational Control of Protein Function in Cells

Elizabeth M. Ryan, Michael A. Norinskiy, Amy K. Bracken, Emma E. Lueders, Xueer Chen, Qin Fu, Elizabeth T. Anderson, Sheng Zhang, and Mikail E. Abbasov

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c09073

Lysine acylations are ubiquitous and structurally diverse post-translational modifications that vastly expand the functional heterogeneity of the human proteome. Hence, the targeted acylation of lysine residues has emerged as a strategic approach to exert biomimetic control over the protein function. However, existing strategies for targeted lysine acylation in cells often rely on genetic intervention, recruitment of endogenous acylation machinery, or nonspecific acylating agents and lack methods to quantify the magnitude of specific acylations on a global level. In this study, we develop activity-based acylome profiling (ABAP), a chemoproteomic strategy that exploits elaborate N-(cyanomethyl)-N-(phenylsulfonyl)amides and lysine-centric probes for site-specific introduction and proteome-wide mapping of posttranslational lysine acylations in human cells. Harnessing this framework, we quantify various artificial acylations and rediscover numerous endogenous lysine acylations. We validate site-specific acetylation of target lysines and establish a structure–activity relationship for N-(cyanomethyl)-N-(phenylsulfonyl)amides in proteins from diverse structural and functional classes. We identify paralog-selective chemical probes that acetylate conserved lysines within interferon-stimulated antiviral RNA-binding proteins, generating de novo proteoforms with obstructed RNA interactions. We further demonstrate that targeted acetylation of a key enzyme in retinoid metabolism engenders a proteoform with a conformational change in the protein structure, leading to a gain-of-function phenotype and reduced drug potency. These findings underscore the versatility of our strategy in biomimetic control over protein function through targeted delivery and global profiling of endogenous and artificial lysine acylations, potentially advancing therapeutic modalities and our understanding of biological processes orchestrated by these post-translational modifications.

Isocyanides inhibit bacterial pathogens by covalent targeting of essential metabolic enzymes

Alexandra Geißler, Howard Junca , Andreas M. Kany , Lena J. Daumann, Anna K. H. Hirsch  Dietmar H. Pieper b and Stephan A. Sieber 

Chem. Sci., 2024, 15, 11946-11955

 https://doi.org/10.1039/D4SC01940G

Isonitrile natural products, also known as isocyanides, demonstrate potent antimicrobial activities, yet our understanding of their molecular targets remains limited. Here, we focus on the so far neglected group of monoisonitriles to gain further insights into their antimicrobial mode of action (MoA). Screening a focused monoisonitrile library revealed a potent S. aureus growth inhibitor with a different MoA compared to previously described isonitrile antibiotics. Chemical proteomics via competitive cysteine reactivity profiling, uncovered covalent modifications of two essential metabolic enzymes involved in the fatty acid biosynthetic process (FabF) and the hexosamine pathway (GlmS) at their active site cysteines. In-depth studies with the recombinant enzymes demonstrated concentration-dependent labeling, covalent binding to the catalytic site and corresponding functional inhibition by the isocyanide. Thermal proteome profiling and full proteome studies of compound-treated S. aureus further highlighted the destabilization and dysregulation of proteins related to the targeted pathways. Cytotoxicity and the inhibition of cytochrome P450 enzymes require optimization of the hit molecule prior to therapeutic application. The here described novel, covalent isocyanide MoA highlights the versatility of the functional group, making it a useful tool and out-of-the-box starting point for the development of innovative antibiotics.