Contribution of Noncovalent Recognition and Reactivity to the Optimization of Covalent Inhibitors: A Case Study on KRasG12C.

Covalent drugs might bear electrophiles to chemically modify their targets and have the potential to target previously undruggable proteins with high potency. Covalent binding of drug-size molecules includes a noncovalent recognition provided by secondary interactions and a chemical reaction leading to covalent complex formation. Optimization of their covalent mechanism of action should involve both types of interactions. Noncovalent and covalent binding steps can be characterized by an equilibrium dissociation constant (KI) and a reaction rate constant (kinact), respectively, and they are affected by both the warhead and the scaffold of the ligand. The relative contribution of these two steps was investigated on a prototypic drug target KRASG12C, an oncogenic mutant of KRAS. We used a synthetically more accessible nonchiral core derived from ARS-1620 that was equipped with four different warheads and a previously described KRAS-specific basic side chain. Combining these structural changes, we have synthesized novel covalent KRASG12C inhibitors and tested their binding and biological effect on KRASG12C by various biophysical and biochemical assays. These data allowed us to dissect the effect of scaffold and warhead on the noncovalent and covalent binding event. Our results revealed that the atropisomeric core of ARS-1620 is not indispensable for KRASG12C inhibition, the basic side chain has little effect on either binding step, and warheads affect the covalent reactivity but not the noncovalent binding. This type of analysis helps identify structural determinants of efficient covalent inhibition and may find use in the design of covalent agents.

Boronic acid inhibitors of penicillin-binding protein 1b: serine and lysine labelling agents.

Penicillin-binding proteins (PBPs) contribute to bacterial cell wall biosynthesis and are targets of antibacterial agents. Here, we investigated PBP1b inhibition by boronic acid derivatives. Chemical starting points were identified by structure-based virtual screening and aliphatic boronic acids were selected for further investigations. Structure-activity relationship studies focusing on the branching of the boron-connecting carbon and quantum mechanical/molecular mechanical simulations showed that reaction barrier free energies are compatible with fast reversible covalent binding and small or missing reaction free energies limit the inhibitory activity of the investigated boronic acid derivatives. Therefore, covalent labelling of the lysine residue of the catalytic dyad was also investigated. Compounds with a carbonyl warhead and an appropriately positioned boronic acid moiety were shown to inhibit and covalently label PBP1b. Reversible covalent labelling of the catalytic lysine by imine formation and the stabilisation of the imine by dative N-B bond is a new strategy for PBP1b inhibition.

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.

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.

Contribution of hydrophobic interactions to protein mechanical stability.

The role of hydrophobic and polar interactions in providing thermodynamic stability to folded proteins has been intensively studied, but the relative contribution of these interactions to the mechanical stability is less explored. We used steered molecular dynamics simulations with constant-velocity pulling to generate force-extension curves of selected protein domains and monitor hydrophobic surface unravelling upon extension. Hydrophobic contribution was found to vary between one fifth and one third of the total force while the rest of the contribution is attributed primarily to hydrogen bonds. Moreover, hydrophobic force peaks were shifted towards larger protein extensions with respect to the force peaks attributed to hydrogen bonds. The higher importance of hydrogen bonds compared to hydrophobic interactions in providing mechanical resistance is in contrast with the relative importance of the hydrophobic interactions in providing thermodynamic stability of proteins. The different contributions of these interactions to the mechanical stability are explained by the steeper free energy dependence of hydrogen bonds compared to hydrophobic interactions on the relative positions of interacting atoms. Comparative analyses for several protein domains revealed that the variation of hydrophobic forces is modest, while the contribution of hydrogen bonds to the force peaks becomes increasingly important for mechanically resistant protein domains.

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.

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.

Allosteric Molecular Switches in Metabotropic Glutamate Receptors.

Metabotropic glutamate receptors (mGlu) are class C G protein-coupled receptors of eight subtypes that are omnipresently expressed in the central nervous system. mGlus have relevance in several psychiatric and neurological disorders, therefore they raise considerable interest as drug targets. Allosteric modulators of mGlus offer advantages over orthosteric ligands owing to their increased potential to achieve subtype selectivity, and this has prompted discovery programs that have produced a large number of reported allosteric mGlu ligands. However, the optimization of allosteric ligands into drug candidates has proved to be challenging owing to induced-fit effects, flat or steep structure-activity relationships and unexpected changes in theirpharmacology. Subtle structural changes identified as molecular switches might modulate the functional activity of allosteric ligands. Here we review these switches discovered in the metabotropic glutamate receptor family..

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.

Covalent Docking in Drug Discovery: Scope and Limitations.

Drug discovery efforts for new covalent inhibitors have drastically increased in the last few years. The binding mechanism of covalent compounds entails the formation of a chemical bond between their electrophilic warhead group and the protein of interest. The use of moderately reactive warheads targeting nonconserved nucleophilic residues can improve the affinity and selectivity profiles of covalent binders as compared to their non-covalent analogs. Recent advances have also enabled their use as chemical probes to disclose novel and also less tractable targets. Increasing interest in covalent drug discovery prompted the development of new computational tools, including covalent docking methods, that are available to predict the binding mode and affinity of covalent ligands. These tools integrate conventional non-covalent docking and scoring schemes by modeling the newly formed covalent bond and the interactions occurring at the reaction site. In this review, we provide a thorough analysis of state-of-the-art covalent docking programs by highlighting their main features and current limitations. Focusing on the implemented algorithms, we show the differences in handling the formation of the new covalent bond and their relative impact on the prediction. This analysis provides a comprehensive overview of the current technology and suggests future improvements in computer-aided covalent drug design. Finally, discussing successful retrospective and prospective covalent docking-based virtual screening applications, we intend to identify best practices for the drug discovery community.

Allosteric activation of metabotropic glutamate receptor 5.

Metabotropic glutamate receptor 5 (mGluR5) is a class C G protein-coupled receptor (GPCR) with both an extracellular ligand binding site and an allosteric intrahelical chamber located similarly to the orthosteric ligand binding site of Class A GPCRs. Ligands binding to this ancestral site of mGluR5 can act as positive (PAM), negative (NAM) or silent (SAM) allosteric modulators, and their medicinal chemistry optimization is notoriously difficult, as subtle structural changes may cause significant variation in activity and switch in the functional response. Here we present all atom molecular dynamics simulations of NAM, SAM and PAM complexes formed by closely related ligands and analyse the structural differences of the complexes. Several residues involved in the activation are identified and the formation of a continuous water channel in the active complex but not in the inactive ones is recognized. Our results suggest that the mechanism of mGluR5 activation is similar to that of class A GPCRs.Communicated by Ramaswamy H. Sarma.

Thermodynamic profiling for fragment-based lead discovery and optimization.

Introduction: The enthalpic and entropic components of the ligand-protein binding free energy reflect the type and quality of the interactions and relate to the physicochemical properties of the ligands. These findings have significance in medicinal chemistry optimizations since they suggest that the thermodynamic profiling of the binding may help monitor and control the unfavorable size and hydrophobicity increase typically accompanying affinity improvements and leading to suboptimal pharmacokinetic properties.Areas covered: This review describes the ligand-protein binding event in terms of elementary steps, their associated interactions, and their enthalpic and entropic consequences. The relationships among the breaking and forming interactions, the binding thermodynamic profile, and the physicochemical properties of the ligands are also discussed.Expert opinion: Analysis of the size dependence of available affinity and favorable enthalpy highlights the limitation of the simultaneous optimization of these quantities. Indeed, moderate, rather than very high affinities can be conciliated with favorable physicochemical and pharmacokinetic profiles as it is supported by the affinity range of historical oral drugs. Although thermodynamic quantities are not suitable endpoints for medicinal chemistry optimizations owing to the complexity of the binding thermodynamics, thermodynamic profiling together with structural studies can be advantageously used to understand the details of the binding process and to optimize it.

Catalytic Mechanism and Covalent Inhibition of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA): Implications to the Design of Novel Antibacterials.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the first step in the biosynthesis of the bacterial cell wall. This pathway is essential for the growth of bacteria but missing in mammals, that nominates MurA as an attractive antibacterial target. MurA has a flexible loop whose conformational change is known to be part of the activation mechanism of the enzyme. We have shown that the loop closed conformation makes the proton transfer from Cys115 to His394 possible by a low barrier exothermic process. QM/MM MD simulations revealed that the activated thiolate is able to react with phosphoenolpyruvate (PEP), the natural substrate of MurA. The binding free energy profile of several covalent inhibitors with various warheads reacting with the activated Cys115 was calculated by QM/MM MD simulations and confirmed that reaction barrier heights tend to separate active from inactive compounds. Our results give new insight into the catalytic mechanism and covalent inhibition of MurA and suggest that QM/MM MD simulations are able to support ligand design by providing sensible relative free energy barriers for covalent inhibitors with various warheads reacting with thiolate nucleophiles.

The role of water and protein flexibility in the structure-based virtual screening of allosteric GPCR modulators: an mGlu5 receptor case study.

Stabilizing unique receptor conformations, allosteric modulators of G-protein coupled receptors (GPCRs) might open novel treatment options due to their new pharmacological action, their enhanced specificity and selectivity in both binding and signaling. Ligand binding occurs at intrahelical allosteric sites and involves significant induced fit effects that include conformational changes in the local protein environment and water networks. Based on the analysis of available crystal structures of metabotropic glutamate receptor 5 (mGlu5) we investigated these effects in the binding of mGlu5 receptor negative allosteric modulators. A large set of retrospective virtual screens revealed that the use of multiple protein structures and the inclusion of selected water molecules improves virtual screening performance compared to conventional docking strategies. The role of water molecules and protein flexibility in ligand binding can be taken into account efficiently by the proposed docking protocol that provided reasonable enrichment of true positives. This protocol is expected to be useful also for identifying intrahelical allosteric modulators for other GPCR targets.

A detailed mechanism of the oxidative half-reaction of d-amino acid oxidase: another route for flavin oxidation.

d-Amino acid oxidase (DAAO) is a flavoenzyme whose inhibition is expected to have therapeutic potential in schizophrenia. DAAO catalyses hydride transfer from the substrate to the flavin in the reductive half-reaction, and the flavin is reoxidized by O2 in the oxidative half-reaction. Quantum mechanical/molecular mechanical calculations were performed and their results together with available experimental information were used to elucidate the detailed mechanism of the oxidative half-reaction. The reaction starts with a single electron transfer from FAD to O2, followed by triplet-singlet transition. FAD oxidation is completed by a proton coupled electron transfer to the oxygen species and the reaction terminates with H2O2 formation by proton transfer from the oxidized substrate to the oxygen species via a chain of water molecules. The substrate plays a double role by facilitating the first electron transfer and by providing a proton in the last step. The mechanism differs from the oxidative half-reaction of other oxidases.

Fragment-Based Approaches for Allosteric Metabotropic Glutamate Receptor (mGluR) Modulators.

Metabotropic glutamate receptors (mGluR) are members of the class C G-Protein Coupled Receptors (GPCR-s) and have eight subtypes. These receptors are responsible for a variety of functions in the central and peripheral nervous systems and their modulation has therapeutic utility in neurological and psychiatric disorders. It was previously established that selective orthosteric modulation of these receptors is challenging, and this stimulated the search for allosteric modulators. Fragment-Based Drug Discovery (FBDD) is a viable approach to find ligands binding at allosteric sites owing to their limited size and interactions. However, it was also observed that the structure-activity relationship of allosteric modulators is often sharp and inconsistent. This can be attributed to the characteristics of the allosteric binding site of mGluRs that is a water channel where ligand binding is accompanied with induced fit and interference with the water network, both playing a role in receptor activation. In this review, we summarize fragment-based drug discovery programs on mGluR allosteric modulators and their contribution identifying of new mGluR ligands with better activity and selectivity.

Discovery of Immunoproteasome Inhibitors Using Large-Scale Covalent Virtual Screening.

Large-scale virtual screening of boronic acid derivatives was performed to identify nonpeptidic covalent inhibitors of the ß5i subunit of the immunoproteasome. A hierarchical virtual screening cascade including noncovalent and covalent docking steps was applied to a virtual library of over 104,000 compounds. Then, 32 virtual hits were selected, out of which five were experimentally confirmed. Biophysical and biochemical tests showed micromolar binding affinity and time-dependent inhibitory potency for two compounds. These results validate the computational protocol that allows the screening of large compound collections. One of the lead-like boronic acid derivatives identified as a covalent immunoproteasome inhibitor is a suitable starting point for chemical optimization.