Covalent Inhibitors Research Articles

"The End of the Golden Weather": therapeutic strategies for mantle cell lymphoma relapsed or refractory to covalent BTK inhibitors.

Mantle cell lymphoma (MCL) is a subtype of non-Hodgkin lymphoma which is often characterised by a pattern of continued relapse after frontline chemoimmunotherapy. Although patients are usually able to regain durable disease control with covalent Bruton's tyrosine kinase inhibitors (cBTKi) at first relapse, it is now appreciated that such responses are often not sustained and the management of such patients represents a significant area of unmet need. There is an imperative to better understand resistance mechanisms and identify high-risk subsets of patients for whom cBTKi responses may be particularly short. Allogeneic stem cell transplant has an established role in appropriate candidates, however contemporary consensus is to preferentially offer chimeric antigen receptor (CAR) T-cell therapy. In this Review, we consider the available data on both existing and emerging treatment options, including non-covalent BTK inhibitors, bispecific antibodies, antibody-drug conjugates and Bcl-2 inhibitors and propose a treatment strategy prioritising clinical trials where available.

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

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

Global Profiling Lysine Reactivity and Ligandability with Oxidant‐Triggered Bioconjugation Chemistry

Due to the high abundance and diverse functions of lysine residues, both in the interior and on the surface of proteins, the development of new methods to characterize their reactivity and ligandability could significantly expand the pool of druggable targets. To date, only a limited number of aminophilic electrophiles have been assessed for interactions with the lysine proteome, resulting in a substantial fraction remaining inaccessible to current probes. Here, to the best of our knowledge, we report the first oxidant‐triggered bioconjugation platform for in‐depth profiling of lysines. We quantified over 7000 covalently modifiable lysine residues, which significantly expands the coverage of ligandable lysines in the whole proteome. Chemical proteomics enabled the mapping of more than 100 endogenous kinases, thus providing a comprehensive landscape of ligandable catalytic lysines within the kinome. Moreover, we identified a suite of new ligandable lysines such as K60 of ENO1 and K31 of PPIA, offering insights for exploring new functional and targetable residues. These findings could provide valuable clues for the development of novel targeted covalent inhibitors (TCIs).

Global Profiling Lysine Reactivity and Ligandability with Oxidant-Triggered Bioconjugation Chemistry.

Due to the high abundance and diverse functions of lysine residues, both in the interior and on the surface of proteins, the development of new methods to characterize their reactivity and ligandability could significantly expand the pool of druggable targets. To date, only a limited number of aminophilic electrophiles have been assessed for interactions with the lysine proteome, resulting in a substantial fraction remaining inaccessible to current probes. Here, to the best of our knowledge, we report the first oxidant-triggered bioconjugation platform for in-depth profiling of lysines. We quantified over 7000 covalently modifiable lysine residues, which significantly expands the coverage of ligandable lysines in the whole proteome. Chemical proteomics enabled the mapping of more than 100 endogenous kinases, thus providing a comprehensive landscape of ligandable catalytic lysines within the kinome. Moreover, we identified a suite of new ligandable lysines such as K60 of ENO1 and K31 of PPIA, offering insights for exploring new functional and targetable residues. These findings could provide valuable clues for the development of novel targeted covalent inhibitors (TCIs).

Structure-Guided Design of Affinity/Covalent-Bond Dual-Driven Inhibitors Targeting the AMP Site of FBPase.

Fructose-1,6-bisphosphatase (FBPase) has attracted substantial interest as a target associated with cancer and type II diabetes. FBPase inhibitors targeting the AMP allosteric site have been documented, but their limited selectivity has raised concerns about adverse effects. To address this issue, we designed the affinity/covalent-bond dual-driven inhibitors based on the pharmacophore knowledge of the AMP pocket and neighboring cysteine residue (C179) of FBPase using the cysteine-targeting reactivity warhead screen followed by a structural optimization strategy. Pull-down and Western Blotting assays confirmed FBPase as a direct target in hepatic cells. X-ray cocrystallographic structure of FBPase-11 and Cov_DOX calculation demonstrated that hydrogen bonding and π-π stacking were the predominant driving force for the inhibition of sulfonylurea-based FBPase covalent inhibitors, while covalent binding with C179 enhances the inhibitors' long-lasting hypoglycemic effects. Together, this work highlights the potential of affinity/covalent-bond dual-driven inhibitors in drug development and provides a promising approach for developing potent drugs targeting AMP-associated proteins.

KRAS inhibitors in drug resistance and potential for combination therapy.

Kirsten Rat Sarcoma (KRAS) is a potent target for cancer therapy because it acts as a signaling hub, engaging in various signaling pathways and regulating a number of cellular functions like cell differentiation, proliferation, and survival. Recently, an emergency approval from the US-FDA has been issued for KRASG12C inhibitors (sotorasib and adagrasib) for metastatic lung cancer treatment. However, clinical studies on covalent KRASG12C inhibitors have rapidly confronted resistance in patients. Many methods are being assessed to overcome this resistance, along with various combinatorial clinical studies that are in process. Moreover, because KRASG12D and KRASG12V are more common than KRASG12C, focus must be placed on the therapeutic strategies for this type of patient, along with sustained efforts in research on these targets. In the present review, we try to focus on various strategies to overcome rapid resistance through the use of combinational treatments to improve the activity of KRASG12C inhibitors.

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

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 hyperglycaemia2-6, 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.

Structure-Based Design of Covalent SARS-CoV-2 Papain-like Protease Inhibitors.

The COVID-19 pandemic is caused by SARS-CoV-2, a highly transmissible and pathogenic RNA betacoronavirus. Like other RNA viruses, SARS-CoV-2 continues to evolve with or without drug selection pressure, and many variants have emerged since the beginning of the pandemic. The papain-like protease, PLpro, is a cysteine protease that cleaves viral polyproteins as well as ubiquitin and ISG15 modifications from host proteins. Leveraging our recently discovered Val70Ub binding site in PLpro, we designed covalent PLpro inhibitors by connecting cysteine reactive warheads to the biarylphenyl PLpro inhibitors via flexible linkers. Several leads displayed potent enzymatic inhibition (IC50 = 0.1-0.3 μM) and antiviral activity (EC50 = 0.09-0.96 μM). Fumaramide inhibitors Jun13567 (15), Jun13728 (16), and Jun13714 (18) showed favorable in vivo pharmacokinetic properties with intraperitoneal injection. The X-ray crystal structure of PLpro with Jun13567 (15) validated our design strategy, revealing covalent conjugation between the catalytic Cys111 and the fumaramide warhead. The results suggest these covalent PLpro inhibitors are promising SARS-CoV-2 antiviral drug candidates.

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

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.

DNA-Encoded Noncanonical Substrate Library for Protease Profiling.

Profiling the substrate sequence preferences of proteases is important for understanding both biological functions as well as for designing protease inhibitors. Several methods are available for profiling the sequence specificity of proteases. However, there is currently no rapid and high-throughput method to profile specificity of proteases for noncanonical substrates. In this study, we described a strategy to use a DNA-encoded noncanonical substrate library to identify the protease substrates composed of both canonical and noncanonical amino acids. This approach uses a DNA-encoded peptide library and introduces a biotin molecule at the N-terminus to immobilize the library on a solid support. Upon protease hydrolysis, the released DNA tag of the substrate peptides can be sequenced to identify the substrate structures. Using this approach, we profiled trypsin and fibroblast activation protein α and discovered noncanonical substrates that were more efficiently cleaved than the commonly used substrates. The identified substrates of FAP were further used to design corresponding covalent inhibitors containing non-canonical sequences with high potency for the target protease. Overall, our approach can aid in the development of new protease substrates and inhibitors.

L858R/L718Q and L858R/L792H Mutations of EGFR Inducing Resistance Against Osimertinib by Forming Additional Hydrogen Bonds.

Acquired resistance to first-line treatments in various cancers both promotes cancer recurrence as well as limits effective treatment. This is true for epidermal growth factor receptor (EGFR) mutations, for which secondary EGFR mutations are one of the principal mechanisms conferring resistance to the covalent inhibitor osimertinib. Thus, it is very important to develop a deeper understanding of the secondary mutational resistance mechanisms associated with EGFR mutations arising in tumors treated with osimertinib to expedite the development of innovative therapeutic drugs to overcome acquired resistance. This work uses all-atom molecular dynamics (MD) simulations to investigate the conformational variation of two reported EGFR mutants (L858R/L718Q and L858R/L792H) that resist osimertinib. The wild-type EGFR kinase domain and the L858R mutant are used as the reference. Our MD simulation results revealed that both the L718Q and L792H secondary mutations induce additional hydrogen bonds between the residues in the active pocket and the residues with the water molecules. These additional hydrogen bonds reduce the exposure area of C797, the covalent binding target of osimertinib. The additional hydrogen bonds also influence the binding affinity of the EGFR kinase domain by altering the secondary structure and flexibility of the amino acid residues in the domain. Our work highlights how the two reported mutations may alter both residue-residue and residue-solvent hydrogen bonds, affecting protein binding properties, which could be helpful for future drug discovery.

Pan-Transcriptional Enhanced Associated Domain Palmitoylation Pocket Covalent Inhibitor.

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.

Navigating the changing landscape of BTK-targeted therapies for B cell lymphomas and chronic lymphocytic leukaemia.

The B cell receptor (BCR) signalling pathway has an integral role in the pathogenesis of many B cell malignancies, including chronic lymphocytic leukaemia, mantle cell lymphoma, diffuse large B cell lymphoma and Waldenström macroglobulinaemia. Bruton tyrosine kinase (BTK) is a key node mediating signal transduction downstream of the BCR.The advent of BTK inhibitors has revolutionized the treatment landscape of B cell malignancies, with these agents often replacing highly intensive and toxic chemoimmunotherapy regimens as the standard of care. In this Review, we discuss the pivotal trials that have led to the approval of various covalent BTK inhibitors, the current treatment indications for these agents and mechanisms of resistance. In addition, we discuss novel BTK-targeted therapies, including covalent, as well as non-covalent, BTK inhibitors, BTK degraders and combination doublet and triplet regimens, to provide insights on the best current treatment paradigms in the frontline setting and at disease relapse.

Exploring the covalent inhibition mechanisms of inhibitors with two different warheads acting on SARS-CoV-2 Mpro by QM/MM simulations

SARS-CoV-2 Mpro had been reported to react with covalent inhibitors 14c and C7, containing a vinyl sulfanilamide and chloromethylamide as the warheads, respectively. However, the detailed reaction mechanisms and warhead effects are still unclear. In this study, the initial state of the catalytic dyad Cys145/His41 is determined as neutral during the non-covalent binding, through MM-PBSA free energy calculations. The Potential of Mean Forces (PMFs) have been computed by QM/MM MD method, obtaining the free energy changes from reactants to products. Both reactions follow asynchronous mechanism. Except the similar proton transfer (PT1), subsequently, nucleophilic addition and an additional proton transfer (PT2) for 14c, while an SN2 nucleophilic substitution for C7 are underwent, respectively. The reaction activation barrier and free energy trends both are in consistent with the experimental IC50 results. Our research enhances understanding of the inhibitory mechanism, and provides insights for designing novel and more effective inhibitors targeting SARS-CoV-2 Mpro.

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

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

Machine-Learning Approach to Identify Potential Dengue Virus Protease Inhibitors: A Computational Perspective.

The global prevalence of dengue virus (DENV), a widespread flavivirus, has led to varied epidemiological impacts, economic burdens, and health consequences. The alarming increase in infections is exacerbated by the absence of approved antiviral agents against the DENV. Within flaviviruses, the NS3/NS2B serine protease plays a pivotal role in processing the viral polyprotein into distinct components, making it an attractive target for antiviral drug development. In this study, machine-learning (ML) techniques were employed to build predictive models for the screening of a library containing 32,000 protease inhibitors. Utilizing GNINA for structure-based virtual screening, the top potential candidates underwent a subsequent evaluation of their absorption, distribution, metabolism, excretion, and toxicity properties. Selected compounds were subjected to molecular dynamics simulations and binding free energy calculations via MM/GBSA. The results suggest that comp530 possesses binding potential to DENV protease as a noncovalent inhibitor with multiple positions for chemical substitutions, presenting opportunities for optimizing their selectivity and specificity. However, other compounds predicted via ML models may still provide a promising start for covalent inhibitors.

Structure-guided design and photochemical synthesis of new carbamo(dithioperoxo)thioates with improved potencies to SARS-CoV-2 3CLpro

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has triggered a protracted global pandemic from 2019 to 2022, and posed a significant threat to human health. One of the non-structural proteins 3CLpro of SARS-CoV-2 is considered as a validated target for the development of inhibitors against the virus. Disulfiram has been reported as a covalent inhibitor of 3CLpro; however, its structure lacks bonding site with active pockets of 3CLpro and its highly symmetric structure doesn’t match well with the irregular cavity of the active center, limiting its therapeutic applications. To enhance their affinity for the 3CLpro target, in this study, two kinds of disulfiram derivatives, designed based on the reevaluation and optimization of disulfiram, have been synthesized through photoredox chemistry, and the novel carbamo(dithioperoxo)thioates 4g-m were found to display 5–17 folds potency against SARS-CoV-2 3CLpro compared to the parent disulfiram, with resulting half-maximal inhibitory concentration (IC50) values ranging from 0.14–0.47 μM. Carbamo(dithioperoxo)thioates 4i containing a 4-hydroxy piperidine and a 4-trifluoromethyl phenyl ring, was identified as the most potent inhibitor (IC50 = 0.14 μM). Furthermore, molecular dynamics simulations, binding free energy analysis and mass analysis were performed and provided insights on the stability, conformational behavior, and interactions of 4g with 3CLpro. The green synthetic methodology, the privileged carbamo(dithioperoxo)thioate scaffold, and the molecular mechanisms presented might serve as a useful system for the further discovery of highly potent inhibitors targeting SARS-CoV-2 3CLpro.

TOWARDS THE DESIGN OF LIGANDS OF THE INTERNAL POCKET TEADS C-TERMINAL DOMAIN

The Hippo pathway controls in organ size and tissue homeostasis through regulating cell growth, proliferation and apoptosis. Phosphorylation of the transcription co-activator YAP (Yes associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif) regulates their nuclear import and therefore their interaction with TEAD (Transcriptional Enhanced Associated Domain). YAP, TAZ and TEADs are dysregulated in several solid cancers making YAP/TAZ-TEAD interaction a new anti-cancer target. We identified by screening a small in-house library, 5-benzyloxindole which binds to hTEAD2 at its internal/palmitate pocket. Its optimization led to covalent inhibitors bearing different warhead. Soaking with hTEAD2 gave seven new crystal structures where the ligands occupied palmitate pocket. 5-Benzyloxyindoles armed with vinylsulfamide moiety inhibit YAP/TAZ-TEAD target genes expression and breast cancer cell proliferation at micromolar concentration.

A practical guide for the assay-dependent characterisation of irreversible inhibitors.

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 K I) scaffold binds the target and only then does a pendant low intrinsic reactivity warhead react with the target (moderate k inact). This highlights the importance of evaluating inhibitors by deriving both their K I and k inact 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 k inact and K I 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.

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

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