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.

Electrophilic MiniFrags Revealed Unprecedented Binding Sites for Covalent HDAC8 Inhibitors.

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

Covalent fragment approaches targeting non-cysteine residues.

Covalent fragment approaches combine advantages of covalent binders and fragment-based drug discovery (FBDD) for target identification and validation. Although early applications focused mostly on cysteine labeling, the chemistries of available warheads that target other orthosteric and allosteric protein nucleophiles has recently been extended. The range of different warheads and labeling chemistries provide unique opportunities for screening and optimizing warheads necessary for targeting non-cysteine residues. In this review, we discuss these recently developed amino-acid-specific and promiscuous warheads, as well as emerging labeling chemistries, which includes novel transition metal catalyzed, photoactive, electroactive, and noncatalytic methodologies. We also highlight recent applications of covalent fragments for the development of molecular glues and proteolysis-targeting chimeras (PROTACs), and their utility in chemical proteomics-based target identification and validation.

SH2db, an information system for the SH2 domain.

SH2 domains are key mediators of phosphotyrosine-based signalling, and therapeutic targets for diverse, mostly oncological, disease indications. They have a highly conserved structure with a central beta sheet that divides the binding surface of the protein into two main pockets, responsible for phosphotyrosine binding (pY pocket) and substrate specificity (pY + 3 pocket). In recent years, structural databases have proven to be invaluable resources for the drug discovery community, as they contain highly relevant and up-to-date information on important protein classes. Here, we present SH2db, a comprehensive structural database and webserver for SH2 domain structures. To organize these protein structures efficiently, we introduce (i) a generic residue numbering scheme to enhance the comparability of different SH2 domains, (ii) a structure-based multiple sequence alignment of all 120 human wild-type SH2 domain sequences and their PDB and AlphaFold structures. The aligned sequences and structures can be searched, browsed and downloaded from the online interface of SH2db (http://sh2db.ttk.hu), with functions to conveniently prepare multiple structures into a Pymol session, and to export simple charts on the contents of the database. Our hope is that SH2db can assist researchers in their day-to-day work by becoming a one-stop shop for SH2 domain related research.

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.

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.

Covalent inhibitors of bacterial peptidoglycan biosynthesis enzyme MurA with chloroacetamide warhead.

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the cytoplasmic part of peptidoglycan biosynthesis and is a validated target enzyme for antibacterial drug discovery; the inhibitor fosfomycin has been used clinically for decades. Like fosfomycin, most MurA inhibitors are small heterocyclic compounds that inhibit the enzyme by forming a covalent bond with the active site cysteine. The reactive chloroacetamide group was selected from a series of suitable electrophilic thiol-reactive warheads. The predominantly one-step synthesis led to the construction of the final library of 47 fragment-sized chloroacetamide compounds. Several new E. coli MurA inhibitors were identified, with the most potent compound having an IC50 value in the low micromolar range. The electrophilic reactivity of all chloroacetamide fragments in our library was evaluated by a high-throughput spectrophotometric assay using the reduced Ellman reagent as a surrogate for the cysteine thiol. LC-MS/MS experiments confirmed the covalent binding of the most potent inhibitor to Cys115 of the digested MurA enzyme. The covalent binding was further investigated by a biochemical time-dependent assay and a dilution assay, which confirmed the irreversible and time-dependent mode of action. The efficacy of chloroacetamide derivatives against MurA does not correlate with their thiol reactivity, making the active fragments valuable starting points for fragment-based development of new antibacterial agents targeting MurA.

Consensus Virtual Screening Identified [1,2,4]Triazolo[1,5-b]isoquinolines As MELK Inhibitor Chemotypes.

Maternal Embryonic Leucine-zipper Kinase (MELK) is a current oncotarget involved in a diverse range of human cancers, with the usage of MELK inhibitors being explored clinically. Here, we aimed to discover new MELK inhibitor chemotypes from our in-house compound library with a consensus-based virtual screening workflow, employing three screening concepts. After careful retrospective validation, prospective screening and in vitro enzyme inhibition testing revealed a series of [1,2,4]triazolo[1,5-b]isoquinolines as a new structural class of MELK inhibitors, with the lead compound of the series exhibiting a sub-micromolar inhibitory activity. The structure-activity relationship of the series was explored by testing further analogs based on a structure-guided selection process. Importantly, the present work marks the first disclosure of the synthesis and bioactivity of this class of compounds.

Fragment-Sized and Bidentate (Immuno)Proteasome Inhibitors Derived from Cysteine and Threonine Targeting Warheads.

Constitutive- and immunoproteasomes are part of the ubiquitin-proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the ß5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting (R)-boroleucine moieties. These compounds were designed to bind both the Thr1 and ß5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the ß5, ß5i, ß1, and ß1i subunits with submicromolar to low-micromolar IC50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either ß5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.

Discovery of a Non-Nucleoside SETD2 Methyltransferase Inhibitor against Acute Myeloid Leukemia.

Histone methyltransferases (HMTs) have attracted considerable attention as potential targets for pharmaceutical intervention in various malignant diseases. These enzymes are known for introducing methyl marks at specific locations of histone proteins, creating a complex system that regulates epigenetic control of gene expression and cell differentiation. Here, we describe the identification of first-generation cell-permeable non-nucleoside type inhibitors of SETD2, the only mammalian HMT that is able to tri-methylate the K36 residue of histone H3. By generating the epigenetic mark H3K36me3, SETD2 is involved in the progression of acute myeloid leukemia. We developed a structure-based virtual screening protocol that was first validated in retrospective studies. Next, prospective screening was performed on a large library of commercially available compounds. Experimental validation of 22 virtual hits led to the discovery of three compounds that showed dose-dependent inhibition of the enzymatic activity of SETD2. Compound C13 effectively blocked the proliferation of two acute myeloid leukemia (AML) cell lines with MLL rearrangements and led to decreased H3K36me3 levels, prioritizing this chemotype as a viable chemical starting point for drug discovery projects.

Mechanistic and thermodynamic characterization of oxathiazolones as potent and selective covalent immunoproteasome inhibitors.

The ubiquitin-proteasome system is responsible for the degradation of proteins and plays a critical role in key cellular processes. While the constitutive proteasome (cPS) is expressed in all eukaryotic cells, the immunoproteasome (iPS) is primarily induced during disease processes, and its inhibition is beneficial in the treatment of cancer, autoimmune disorders and neurodegenerative diseases. Oxathiazolones were reported to selectively inhibit iPS over cPS, and the inhibitory activity of several oxathiazolones against iPS was experimentally determined. However, the detailed mechanism of the chemical reaction leading to irreversible iPS inhibition and the key selectivity drivers are unknown, and separate characterization of the noncovalent and covalent inhibition steps is not available for several compounds. Here, we investigate the chemical reaction between oxathiazolones and the Thr1 residue of iPS by quantum mechanics/molecular mechanics (QM/MM) simulations to establish a plausible reaction mechanism and to determine the rate-determining step of covalent complex formation. The modelled binding mode and reaction mechanism are in line with the selective inhibition of iPS versus cPS by oxathiazolones. The kinact value of several ligands was estimated by constructing the potential of mean force of the rate-determining step by QM/MM simulations coupled with umbrella sampling. The equilibrium constant Ki of the noncovalent complex formation was evaluated by classical force field-based thermodynamic integration. The calculated Ki and kinact values made it possible to analyse the contribution of the noncovalent and covalent steps to the overall inhibitory activity. Compounds with similar intrinsic reactivities exhibit varying selectivities for iPS versus cPS owing to subtle differences in the binding modes that slightly affect Ki, the noncovalent affinity, and importantly alter kinact, the covalent reactivity of the bound compounds. A detailed understanding of the inhibitory mechanism of oxathiazolones is useful in designing iPS selective inhibitors with improved drug-like properties.

Discovery of selective fragment-sized immunoproteasome inhibitors.

Proteasomes contribute to maintaining protein homeostasis and their inhibition is beneficial in certain types of cancer and in autoimmune diseases. However, the inhibition of the proteasomes in healthy cells leads to unwanted side-effects and significant effort has been made to identify inhibitors specific for the immunoproteasome, especially to treat diseases which manifest increased levels and activity of this proteasome isoform. Here, we report our efforts to discover fragment-sized inhibitors of the human immunoproteasome. The screening of an in-house library of structurally diverse fragments resulted in the identification of benzo[d]oxazole-2(3H)-thiones, benzo[d]thiazole-2(3H)-thiones, benzo[d]imidazole-2(3H)-thiones, and 1-methylbenzo[d]imidazole-2(3H)-thiones (with a general term benzoXazole-2(3H)-thiones) as inhibitors of the chymotrypsin-like (ß5i) subunit of the immunoproteasome. A subsequent structure-activity relationship study provided us with an insight regarding growing vectors. Binding to the ß5i subunit was shown and selectivity against the ß5 subunit of the constitutive proteasome was determined. Thorough characterization of these compounds suggested that they inhibit the immunoproteasome by forming a disulfide bond with the Cys48 available specifically in the ß5i active site. To obtain fragments with biologically more tractable covalent interactions, we performed a warhead scan, which yielded benzoXazole-2-carbonitriles as promising starting points for the development of selective immunoproteasome inhibitors with non-peptidic scaffolds.

Binding Mode Prediction and Virtual Screening Applications by Covalent Docking.

The mechanism of action of covalent drugs involves the formation of a bond between their electrophilic warhead group and a nucleophilic residue of the protein target. The recent advances in covalent drug discovery have accelerated the development of computational tools for the design and characterization of covalent binders. Covalent docking algorithms can predict the binding mode of covalent ligands by modeling the bonds and interactions formed at the reaction site. Their scoring functions can estimate the relative binding affinity of ligands towards the target of interest, thus allowing virtual screening of compound libraries. However, most of the scoring schemes have no specific terms for the bond formation, and therefore it prevents the direct comparison of warheads with different intrinsic reactivity. Herein, we describe a protocol for the binding mode prediction of covalent ligands, a typical virtual screening of compound sets with a single warhead chemistry, and an alternative approach to screen libraries that include various warhead types, as applied in recently validated studies.

WIDOCK: a reactive docking protocol for virtual screening of covalent inhibitors.

Here we present WIDOCK, a virtual screening protocol that supports the selection of diverse electrophiles as covalent inhibitors by incorporating ligand reactivity towards cysteine residues into AutoDock4. WIDOCK applies the reactive docking method (Backus et al. in Nature 534:570-574, 2016) and extends it into a virtual screening tool by introducing facile experimental or computational parametrization and a ligand focused evaluation scheme together with a retrospective and prospective validation against various therapeutically relevant targets. Parameters accounting for ligand reactivity are derived from experimental reaction kinetic data or alternatively from computed reaction barriers. The performance of this docking protocol was first evaluated by investigating compound series with diverse warhead chemotypes against KRASG12C, MurA and cathepsin B. In addition, WIDOCK was challenged on larger electrophilic libraries screened against OTUB2 and NUDT7. These retrospective analyses showed high sensitivity in retrieving experimental actives, by also leading to superior ROC curves, AUC values and better enrichments than the standard covalent docking tool available in AutoDock4 when compound collections with diverse warheads were investigated. Finally, we applied WIDOCK for the prospective identification of covalent human MAO-A inhibitors acting via a new mechanism by binding to Cys323. The inhibitory activity of several predicted compounds was experimentally confirmed and the labelling of Cys323 was proved by subsequent MS/MS measurements. These findings demonstrate the usefulness of WIDOCK as a warhead-sensitive, covalent virtual screening protocol.

Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.

Affinity and Selectivity Assessment of Covalent Inhibitors by Free Energy Calculations.

Covalent inhibitors have been gaining increased attention in drug discovery due to their beneficial properties such as long residence time, high biochemical efficiency, and specificity. Optimization of covalent inhibitors is a complex task that involves parallel monitoring of the noncovalent recognition elements and the covalent reactivity of the molecules to avoid potential idiosyncratic side effects. This challenge calls for special design protocols, including a variety of computational chemistry methods. Covalent inhibition proceeds through multiple steps, and calculating free energy changes of the subsequent binding events along the overall binding process would help us to better control the design of drug candidates. Inspired by the recent success of free energy calculations on reversible binders, we developed a complex protocol to compute free energies related to the noncovalent and covalent binding steps with thermodynamic integration and hybrid quantum mechanical/molecular mechanical (QM/MM) potential of mean force (PMF) calculations, respectively. In optimization settings, we examined two therapeutically relevant proteins complexed with congeneric sets of irreversible cysteine targeting covalent inhibitors. In the selectivity paradigm, we studied the irreversible binding of covalent inhibitors to phylogenetically close targets by a mutational approach. The results of the calculations are in good agreement with the experimental free energy values derived from the inhibition and kinetic constants (Ki and kinact) of the enzyme-inhibitor binding. The proposed method might be a powerful tool to predict the potency, selectivity, and binding mechanism of irreversible covalent inhibitors.

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

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

An electrophilic warhead library for mapping the reactivity and accessibility of tractable cysteines in protein kinases.

Targeted covalent inhibitors represent a viable strategy to block protein kinases involved in different disease pathologies. Although a number of computational protocols have been published for identifying druggable cysteines, experimental approaches are limited for mapping the reactivity and accessibility of these residues. Here, we present a ligand based approach using a toolbox of fragment-sized molecules with identical scaffold but equipped with diverse covalent warheads. Our library represents a unique opportunity for the efficient integration of warhead-optimization and target-validation into the covalent drug development process. Screening this probe kit against multiple kinases could experimentally characterize the accessibility and reactivity of the targeted cysteines and helped to identify suitable warheads for designed covalent inhibitors. The usefulness of this approach has been confirmed retrospectively on Janus kinase 3 (JAK3). Furthermore, representing a prospective validation, we identified Maternal embryonic leucine zipper kinase (MELK), as a tractable covalent target. Covalently labelling and biochemical inhibition of MELK would suggest an alternative covalent strategy for MELK inhibitor programs.

Small molecule inhibitors of RAS proteins with oncogenic mutations.

RAS proteins control a number of essential cellular processes as molecular switches in the human body. Presumably due to their important signalling role, RAS proteins are among the most frequently mutated oncogenes in human cancers. Hence, numerous efforts were done to develop appropriate therapies for RAS-mutant cancers in the last three decades. This review aimed to collect all of the reported small molecules that affect RAS signalling. These molecules can be divided in four main branches. First, we address approaches blocking RAS membrane association. Second, we focus on the stabilization efforts of non-productive RAS complexes. Third, we examine the approach to block RAS downstream signalling through disturbance of RAS-effector complex formation. Finally, we discuss direct inhibition; particularly the most recently reported covalent inhibitors, which are already advanced to human clinical trials.

Targeting STAT3 and STAT5 in Cancer.

Insights into the mutational landscape of the human cancer genome coding regions defined about 140 distinct cancer driver genes in 2013, which approximately doubled to 300 in 2018 following advances in systems cancer biology studies [...].