Covalent Fragments Research Articles

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

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.

Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53.

Over the last few years, covalent fragment-based drug discovery has gained significant importance. Thus, striving for more warhead diversity, we conceived a library consisting of 20 covalently reacting compounds. Our covalent fragment library (CovLib) contains four different warhead classes, including five α-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group (SNAr/SN). After predicting the theoretical solubility of the fragments by LogP and LogS during the selection process, we determined their experimental solubility using a turbidimetric solubility assay. The reactivities of the different compounds were measured in a high-throughput 5,5'-dithiobis-(2-nitrobenzoic acid) DTNB assay, followed by a (glutathione) GSH stability assay. We employed the CovLib in a (differential scanning fluorimetry) DSF-based screening against different targets: c-Jun N-terminal kinase 3 (JNK3), ubiquitin-specific protease 7 (USP7), and the tumor suppressor p53. Finally, the covalent binding was confirmed by intact protein mass spectrometry (MS). In general, the purchased fragments turned out to be sufficiently soluble. Additionally, they covered a broad spectrum of reactivity. All investigated α-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for α-cyanoacrylamides/acrylates). The SNAr and vinyl sulfone fragments are either highly reactive or stable. DSF measurements with the different targets JNK3, USP7, and p53 identified reactive fragment hits causing a shift in the melting temperatures of the proteins. MS confirmed the covalent binding mode of all these fragments to USP7 and p53, while additionally identifying the SNAr-type electrophile SN002 as a mildly reactive covalent hit for p53. The screening and target evaluation of the CovLib revealed first interesting hits. The highly cysteine-reactive fragments VS004, SN001, SN006, and SN007 covalently modify several target proteins and showed distinct shifts in the melting temperatures up to +5.1°C and -9.1°C.

Covalent Fragment Screening and Optimization Identifies the Chloroacetohydrazide Scaffold as Inhibitors for Ubiquitin C-terminal Hydrolase L1.

Dysregulation of the ubiquitin-proteasome systems is a hallmark of various disease states including neurodegenerative diseases and cancer. Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is expressed primarily in the central nervous system under normal physiological conditions, however, is considered an oncogene in various cancers, including melanoma, lung, breast, and lymphoma. Thus, UCHL1 inhibitors could serve as a viable treatment strategy against these aggressive cancers. Herein, we describe a covalent fragment screen that identified the chloroacetohydrazide scaffold as a covalent UCHL1 inhibitor. Subsequent optimization provided an improved fragment with single-digit micromolar potency against UCHL1 and selectivity over the closely related UCHL3. The molecule demonstrated efficacy in cellular assays of metastasis. Additionally, we report a ligand-bound crystal structure of the most potent molecule in complex with UCHL1, providing insight into the binding mode and information for future optimization.

Constructing Innovative Covalent and Noncovalent Compound Libraries: Insights from 3D Protein-Ligand Interactions.

Noncovalent interactions between small-molecule drugs and protein targets assume a pivotal role in drug design. Moreover, the design of covalent inhibitors, forming covalent bonds with amino acid residues, requires rational reactivity for their covalent warheads, presenting a key challenge as well. Understanding the intricacies of these interactions provides a more comprehensive understanding of molecular binding mechanisms, thereby guiding the rational design of potent inhibitors. In this study, we adopted the fragment-based drug design approach, introducing a novel methodology to extract noncovalent and covalent fragments according to distinct three-dimensional (3D) interaction modes from noncovalent and covalent compound libraries. Additionally, we systematically replaced existing ligands with rational fragment substitutions, based on the spatial orientation of fragments in 3D space. Furthermore, we adopted a molecular generation approach to create innovative covalent inhibitors. This process resulted in the recombination of a noncovalent compound library and several covalent compound libraries, constructed by two commonly encountered covalent amino acids: cysteine and serine. We utilized noncovalent ligands in KLIFS and covalent ligands in CovBinderInPDB as examples to recombine noncovalent and covalent libraries. These recombined compound libraries cover a substantial portion of the chemical space present in the original compound libraries and exhibit superior performance in terms of molecular scaffold diversity compared to the original compound libraries and other 11 commercial libraries. We also recombined BTK-focused libraries, and 23 compounds within our libraries have been validated by former researchers to possess potential biological activity. The establishment of these compound libraries provides valuable resources for virtual screening of covalent and noncovalent drugs targeting similar molecular targets.

Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signaling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of thephosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate theactivity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.

Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics

The nucleus controls cell growth and division through coordinated interactions between nuclear proteins and chromatin. Mutations that impair nuclear protein association with chromatin are implicated in numerous diseases. Covalent ligands are a promising strategy to pharmacologically target nuclear proteins, such as transcription factors, which lack ordered small-molecule binding pockets. To identify nuclear cysteines that are susceptible to covalent liganding, we couple proximity labeling (PL), using a histone H3.3-TurboID (His-TID) construct, with chemoproteomics. Using covalent scout fragments, KB02 and KB05, we identified ligandable cysteines on proteins involved in spindle assembly, DNA repair, and transcriptional regulation, such as Cys101 of histone acetyltransferase 1 (HAT1). Furthermore, we show that covalent fragments can affect the abundance, localization, and chromatin association of nuclear proteins. Notably, the Parkinson disease protein 7 (PARK7) showed increased nuclear localization and chromatin association upon KB02 modification at Cys106. Together, this platform provides insights into targeting nuclear cysteines with covalent ligands.

Molecular Mechanism of Labelling Functional Cysteines by Heterocyclic Thiones.

Heterocyclic thiones have recently been identified as reversible covalent warheads, consistent with their mild electrophilic nature. Little is known so far about their mechanism of action in labelling nucleophilic sidechains, especially cysteines. The vast number of tractable cysteines promotes a wide range of target proteins to examine; however, our focus was put on functional cysteines. We chose the main protease of SARS-CoV-2 harboring Cys145 at the active site that is a structurally characterized and clinically validated target of covalent inhibitors. We screened an in-house, cysteine-targeting covalent inhibitor library which resulted in several covalent fragment hits with benzoxazole, benzothiazole and benzimidazole cores. Thione derivatives and Michael acceptors were selected for further investigations with the objective of exploring the mechanism of inhibition of the thiones and using the thoroughly characterized Michael acceptors for benchmarking our studies. Classical and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were carried out that revealed a new mechanism of covalent cysteine labelling by thione derivatives, which was supported by QM and free energy calculations and by a wide range of experimental results. Our study shows that the molecular recognition step plays a crucial role in the overall binding of both sets of molecules.

Identification of covalent fragment inhibitors for Plasmodium falciparum UCHL3 with anti-malarial efficacy

Malaria continues to be a major burden on global health, responsible for 619,000 deaths in 2021. The causative agent of malaria is the eukaryotic parasite Plasmodium. Resistance to artemisinin-based combination therapies (ACTs), the current first-line treatment for malaria, has emerged in Asia, South America, and more recently Africa, where >90% of all malaria-related deaths occur. This has necessitated the identification and investigation of novel parasite proteins and pathways as antimalarial targets, including components of the ubiquitin proteasome system. Here, we investigate Plasmodium falciparum deubiquitinase ubiquitin C-terminal hydrolase L3 (PfUCHL3) as one such target. We carried out a high-throughput screen with covalent fragments and identified seven scaffolds that selectively inhibit the plasmodial UCHL3, but not human UCHL3 or the closely related human UCHL1. After assessing toxicity in human cells, we identified four promising hits and demonstrated their efficacy against asexual P. falciparum blood stages and P. berghei sporozoite stages.

Covalent Fragment Inhibits RhoA Activation by Guanine Exchange Factors.

Ras homolog gene family member (RhoA) is a GTPase and a member of the RAS superfamily of GTPases. RhoA is a master regulator of the actin cytoskeleton. It inhibits axon growth preventing repair and recovery following spinal cord and traumatic brain injuries. Despite decades of research into the biological function of Rho GTPases, there exist no small-molecule Rho inhibitors. Here, we screen a library of cysteine electrophiles to explore whether covalent bond formation at Cys-107 leads to inhibition of RhoA activation by guanine exchange factor Trio. Two fragments, propiolamide 1 (ACR-895) and acrylamide 2 (ACR-917), inhibited RhoA nucleotide exchange by Trio in a time-dependent manner. The fragments formed a covalent bond with wild-type RhoA but not Cys107Ser RhoA mutant. Time- and concentration-dependent studies led to equilibrium constants KIs and reaction rates that correspond to t1/2 values in the single-digit hour range. One fragment was selective for RhoA over Rac1 GTPase and had no effect on KRAS nucleotide exchange by SOS1. The fragments did not inhibit RhoA binding to ROCK effector protein. This work establishes Cys-107 as a suitable site for Rho GTPase inhibition and provides fragment starting points for the future development of Rho GTPase covalent inhibitors that could have profound implications in the treatment of patients with injuries of the central nervous system.

AzidoTMT Enables Direct Enrichment and Highly Multiplexed Quantitation of Proteome-Wide Functional Residues.

Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.

Abstract 5333: Combining chemoproteomics with machine learning identifies functionally active covalent fragments for hard-to-drug cancer drivers

Abstract a) Many cancer drivers are considered “undruggable” and without targeted treatments because they lack binding sites for conventional small molecules. Here, we introduce The FRONTIER™ Platform applying machine learning (ML), chemoproteomics and covalent chemistry to identify binding sites and cell-active covalent fragments across the human proteome, including against most cancer drivers and previously “undruggable” targets. Molecules tested in functional assays are active and can serve as starting points for new drug discovery initiatives. b) We are using mass spectrometry and data analysis workflows to perform high-throughput chemoproteomic profiling experiments. These experiments identify hits across the proteome using different cancer-relevant cell backgrounds and characterize binding sites for drug discovery. Customized ML algorithms using chemoproteomic, genomic and structural data to characterize and prioritize identified binding sites for covalent drug discovery. The performance of the platform allows the profiling of thousands of compounds from a custom-built library that has been optimized by ML for covalent fragment-based drug discovery. The nature of the fragment hits and the ability to map them to and focus on preferred binding sites for covalent drugs enables accelerated lead generation. c) We show details of the platform highlighting library design concepts and the hotspot map with binding site prioritization algorithms. We show coverage and applicability across important cancer target classes and signaling pathways. Covalent fragment hits have been identified for multiple difficult cancer targets, including KRAS, p53, STAT3, KEAP1, PTPN11 and others. The platform also identifies ligands for novel allosteric binding sites in established oncology targets such as CDK4, PI3KCA and BTK. We will highlight how discovered ligands show functional activity in orthogonal assays, demonstrating their fitness for drug discovery campaigns and target validation experiments. d) Undruggable targets across a variety of cancer target classes have become druggable. Citation Format: Johannes C. Hermann, Robert Everley, Laura Marholz, Matthew Berberich, Tzu-Yi Yang, Yu-Hsin Chao, Abduselam K. Awol, Michael Shaghafi, Han Yoon, Rohan Varma, Reed Stein, Karsten Krug, Emily Lachtara, Daniel Erlanson, Chris Varma, Kevin R. Webster. Combining chemoproteomics with machine learning identifies functionally active covalent fragments for hard-to-drug cancer drivers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5333.

A Critical Study on Acylating and Covalent Reversible Fragment Inhibitors of SARS-CoV-2 Main Protease Targeting the S1 Site with Pyridine.

SARS coronavirus main proteases (3CL proteases) have been validated as pharmacological targets for the treatment of coronavirus infections. Current inhibitors of SARS main protease, including the clinically admitted drug nirmatrelvir are peptidomimetics with the downsides of this class of drugs including limited oral bioavailability, cellular permeability, and rapid metabolic degradation. Here, we investigate covalent fragment inhibitors of SARS Mpro as potential alternatives to peptidomimetic inhibitors in use today. Starting from inhibitors acylating the enzyme's active site, a set of reactive fragments was synthesized, and the inhibitory potency was correlated with the chemical stability of the inhibitors and the kinetic stability of the covalent enzyme-inhibitor complex. We found that all tested acylating carboxylates, several of them published prominently, were hydrolyzed in assay buffer and the inhibitory acyl-enzyme complexes were rapidly degraded leading to the irreversible inactivation of these drugs. Acylating carbonates were found to be more stable than acylating carboxylates, however, were inactive in infected cells. Finally, reversibly covalent fragments were investigated as chemically stable SARS CoV-2 inhibitors. Best was a pyridine-aldehyde fragment with an IC50 of 1.8 μM at a molecular weight of 211 g/mol, showing that pyridine fragments indeed are able to block the active site of SARS-CoV-2 main protease.

Covalent fragment mapping of KRasG12C revealed novel chemotypes with in vivo potency

G12C mutant KRas is considered druggable by allele-specific covalent inhibitors due to the nucleophilic character of the oncogenic mutant cysteine at position 12. Discovery of these inhibitors requires the optimization of both covalent and noncovalent interactions. Here, we report covalent fragment screening of our electrophilic fragment library of diverse non-covalent scaffolds equipped with 40 different electrophilic functionalities to identify fragments as suitable starting points targeting Cys12. Screening the library against KRasG12C using Ellman's free thiol assay, followed by protein NMR and cell viability assays, resulted in two potential inhibitor chemotypes. Characterization of these scaffolds in in vitro cellular- and in vivo xenograft models revealed them as promising starting points for covalent drug discovery programs.

Covalent Library Screening by Targeted Mass Spectrometry for Rapid Binding Site Identification.

Interest in covalent drug discovery has surged in recent years, following the high-profile FDA approvals of covalent inhibitors that target BTK and KRAS G12C. High-throughput screening by intact protein mass spectrometry is a popular method for identifying lead matter from covalent fragment libraries. While the technique is proven in its capacity to confirm covalent binding, it does not provide binding site information on its own. Follow-up assays to identify binding sites can be time- and resource-intensive, potentially extending the hit confirmation timeline by weeks or months. Here, we describe the development of CoMPAS, a novel, targeted mass spectrometry-based covalent screening method that provides binding site information in the initial screen. The high sensitivity of targeted detection confers additional advantages over the intact protein method, including the ability to characterize more potent binders and reduced protein reagent requirements. Interpretation of the structure-activity relationship is simplified by enabling the use of binding site-specific EC50 values. To investigate higher-throughput screening beyond what is possible with standard liquid chromatography, we acquired data in parallel on an Agilent RapidFire system and compared the screening results by statistical analysis. To demonstrate the multiplexing capabilities of CoMPAS, we determined the target selectivity of screening hits against a pool of off-target kinases.

Covalent fragment libraries in drug discovery-Design, synthesis, and screening methods.

As the development of drugs with a covalent mode of action is becoming increasingly popular, well-validated covalent fragment-based drug discovery (FBDD) methods have been comparatively slow to keep up with the demand. In this chapter the principles of covalent fragment reactivity, library design, synthesis, and screening methods are explored in depth, focussing on literature examples with direct applications to practical covalent fragment library design and screening. Further, questions about the future of the field are explored and potential useful advances are proposed.

Next-Generation Heterocyclic Electrophiles as Small-Molecule Covalent MurA Inhibitors.

Heterocyclic electrophiles as small covalent fragments showed promising inhibitory activity on the antibacterial target MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase, EC:2.5.1.7). Here, we report the second generation of heterocyclic electrophiles: the quaternized analogue of the heterocyclic covalent fragment library with improved reactivity and MurA inhibitory potency. Quantum chemical reaction barrier calculations, GSH (L-glutathione) reactivity assay, and thrombin counter screen were also used to demonstrate and explain the improved reactivity and selectivity of the N-methylated heterocycles and to compare the two generations of heterocyclic electrophiles.

Reactivity of Covalent Fragments and Their Role in Fragment Based Drug Discovery.

Fragment based drug discovery has long been used for the identification of new ligands and interest in targeted covalent inhibitors has continued to grow in recent years, with high profile drugs such as osimertinib and sotorasib gaining FDA approval. It is therefore unsurprising that covalent fragment-based approaches have become popular and have recently led to the identification of novel targets and binding sites, as well as ligands for targets previously thought to be 'undruggable'. Understanding the properties of such covalent fragments is important, and characterizing and/or predicting reactivity can be highly useful. This review aims to discuss the requirements for an electrophilic fragment library and the importance of differing warhead reactivity. Successful case studies from the world of drug discovery are then be examined.

Covalent disruptor of YAP-TEAD association suppresses defective Hippo signaling.

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

Covalent fragment-based ligand screening approaches for identification of novel ubiquitin proteasome system modulators.

Ubiquitination is a key regulatory mechanism vital for maintenance of cellular homeostasis. Protein degradation is induced by E3 ligases via attachment of ubiquitin chains to substrates. Pharmacological exploitation of this phenomenon via targeted protein degradation (TPD) can be achieved with molecular glues or bifunctional molecules facilitating the formation of ternary complexes between an E3 ligase and a given protein of interest (POI), resulting in ubiquitination of the substrate and subsequent proteolysis by the proteasome. Recently, the development of novel covalent fragment screening approaches has enabled the identification of first-in-class ligands for E3 ligases and deubiquitinases revealing so far unexplored binding sites which highlights the potential of these methods to uncover and expand druggable space for new target classes.

Covalent Fragment Screening Identifies Rgl2 RalGEF Cysteine for Targeted Covalent Inhibition of Ral GTPase Activation.

Ral GTPases belong to the RAS superfamily, and they are directly activated by K-RAS. The RalGEF pathway is one of the three major K-RAS signaling pathways. Ral GTPases do not possess a cysteine nucleophile to develop a covalent inhibitor following the strategy that led to a K-RAS G12C therapeutic agent. However, several cysteine amino acids exist on the surface of guanine exchange factors that activate Ral GTPases, such as Rgl2. Here, we screen a library of cysteine electrophile fragments to determine if covalent bond formation at one of the Rgl2 surface cysteines could inhibit Ral GTPase activation. We found several chloroacetamide and acrylamide fragments that inhibited Ral GTPase exchange by Rgl2. Site-directed mutagenesis showed that covalent bond formation at Cys-284, but not other cysteines, leads to inhibition of Ral activation by Rgl2. Follow-up time- and concentration-dependent studies of derivatives identified by substructure search of commercial libraries further confirmed Cys-284 as the reaction site and identified the indoline fragments as the most promising series for further development. Cys-284 is located outside of the Ral ⋅ Rgl2 interface on a loop that has several residues that come in direct contact with Ral GTPases. Our allosteric covalent fragment inhibitors provide a starting point for the development of small-molecule covalent inhibitors to probe Ral GTPases in animal models.