Conformational analysis of the IQSEC2 protein by statistical thermodynamics.

Mutations in the IQSEC2 gene result in severe intellectual disability, epilepsy and autism. The primary function of IQSEC2 is to serve as a guanine exchange factor (GEF) controlling the activation of ARF6 which in turn mediates membrane trafficking and synaptic connections between neurons. As IQSEC2 is a large intrinsically disordered protein little is known of the structure of the protein and how this influences its function.

Molecular modeling of ARF6 dysregulation caused by mutations in IQSEC2.

IQSEC2 gene mutations are associated with epilepsy, autism, and intellectual disability. The primary function IQSEC2, mediated via its Sec 7 domain, is to act as a guanine nucleotide exchange factor for ARF6. We sought to develop a molecular model, which may explain the aberrant Sec 7 activity on ARF6 of different human IQSEC2 mutations.

Phage-Displayed Mimotopes of SARS-CoV-2 Spike Protein Targeted to Authentic and Alternative Cellular Receptors.

The evolution of the SARS-CoV-2 virus during the COVID-19 pandemic was accompanied by the emergence of new heavily mutated viral variants with increased infectivity and/or resistance to detection by the human immune system. To respond to the urgent need for advanced methods and materials to empower a better understanding of the mechanisms of virus's adaptation to human host cells and to the immuno-resistant human population, we suggested using recombinant filamentous bacteriophages, displaying on their surface foreign peptides termed "mimotopes", which mimic the structure of viral receptor-binding sites on the viral spike protein and can serve as molecular probes in the evaluation of molecular mechanisms of virus infectivity. In opposition to spike-binding antibodies that are commonly used in studying the interaction of the ACE2 receptor with SARS-CoV-2 variants in vitro, phage spike mimotopes targeted to other cellular receptors would allow discovery of their role in viral infection in vivo using cell culture, tissue, organs, or the whole organism.

Development and characterisation of SMURF2-targeting modifiers.

The C2-WW-HECT-domain E3 ubiquitin ligase SMURF2 emerges as an important regulator of diverse cellular processes. To date, SMURF2-specific modulators were not developed. Here, we generated and investigated a set of SMURF2-targeting synthetic peptides and peptidomimetics designed to stimulate SMURF2's autoubiquitination and turnover via a disruption of the inhibitory intramolecular interaction between its C2 and HECT domains.

How does the exosite of rhomboid protease affect substrate processing and inhibition?

Rhomboid proteases constitute a family of intramembrane serine proteases ubiquitous in all forms of life. They differ in many aspects from their soluble counterparts. We applied molecular dynamics (MD) computational approach to address several challenging issues regarding their catalytic mechanism: How does the exosite of GlpG rhomboid protease control the kinetics efficiency of substrate hydrolysis? What is the mechanism of inhibition by the non-competitive peptidyl aldehyde inhibitors bound to the GlpG rhomboid active site (AS)? What is the underlying mechanism that explains the hypothesis that GlpG rhomboid protease is not adopted for the hydrolysis of short peptides that do not contain a transmembrane domain (TMD)? Two fundamental features of rhomboid catalysis, the enzyme recognition and discrimination of substrates by TMD interactions in the exosite, and the concerted mechanism of non-covalent pre-catalytic complex to covalent tetrahedral complex (TC) conversion, provide answers to these mechanistic questions.

Stepwise Versus Concerted Mechanisms in General-Base Catalysis by Serine Proteases.

General-base catalysis in serine proteases still poses mechanistic challenges despite decades of research. Whether proton transfer from the catalytic Ser to His and nucleophilic attack on the substrate are concerted or stepwise is still under debate, even for the classical Asp-His-Ser catalytic triad. To address these key catalytic steps, the transformation of the Michaelis complex to tetrahedral complex in the covalent inhibition of two prototype serine proteases was studied: chymotrypsin (with the catalytic triad) inhibition by a peptidyl trifluoromethane and GlpG rhomboid (with Ser-His dyad) inhibition by an isocoumarin derivative.

A new method for filtering of reactive "warheads" of transition-state analog protease inhibitors.

In light of the major contribution of the reactive warhead to the binding energy trend in reversible covalent transition-state analog inhibitors of serine and cysteine hydrolases, would it be possible to rationally design and quickly filter such warheads, especially for large-scale screening? The previously defined W1 and W2 covalent descriptors quantitatively account for the energetic effect of the covalent bonds reorganization, accompanying enzyme-inhibitor covalent binding. The quantum mechanically calculated W1 and W2 reflect the warhead binding energy by modeling of the enzyme-inhibitor reaction core. Here, we demonstrate the use of these descriptors for warhead filtering, and examine its scope and limitations.

Application of EMBM to Structure-Based Design of Warheads for Protease Inhibitors.

Most CADD tools handle non-covalent enzyme inhibitors, despite the growing interest of the pharma industry in covalent inhibitors. We have recently introduced an enzyme mechanism-based method, EMBM, as a computational tool for binding trend analysis and prediction of chemical sites (CS) of reversible covalent enzyme inhibitors. In the current study we demonstrate the utility of EMBM to structure-based applications.

The Catalytic Machinery of Rhomboid Proteases: Combined MD and QM Simulations.

Rhomboid proteases are a ubiquitous family of intramembrane serine proteases in prokaryotic and eukaryotic organisms that cleave membrane proteins in their transmembrane region. Their catalytic activity is centered at a His-Ser catalytic dyad. We applied molecular dynamics and quantum mechanics calculations in order to clarify the protonation state of the catalytic residues of E.

The mechanism of papain inhibition by peptidyl aldehydes.

Various mechanisms for the reversible formation of a covalent tetrahedral complex (TC) between papain and peptidyl aldehyde inhibitors were simulated by DFT calculations, applying the quantum mechanical/self consistent reaction field (virtual solvent) [QM/SCRF(VS)] approach. Only one mechanism correlates with the experimental kinetic data. The His-Cys catalytic diad is in an N/SH protonation state in the noncovalent papain-aldehyde Michaelis complex.

EMBM - a new enzyme mechanism-based method for rational design of chemical sites of covalent inhibitors.

We introduce an enzyme mechanism-based method (EMBM) aimed at rational design of chemical sites (CS) of reaction coordinate analog inhibitors. The energy of valence reorganization of CS, caused by the formation of the enzyme-inhibitor covalent complex, is accounted for by new covalent descriptors W1 and W2. We considered CS fragments with a carbonyl reactivity center, like in native protease substrates.

Challenging a paradigm: theoretical calculations of the protonation state of the Cys25-His159 catalytic diad in free papain.

A central mechanistic paradigm of cysteine proteases is that the His-Cys catalytic diad forms an ion-pair NH(+)/S(-) already in the catalytically active free enzyme. Most molecular modeling studies of cysteine proteases refer to this paradigm as their starting point. Nevertheless, several recent kinetics and X-ray crystallography studies of viral and bacterial cysteine proteases depart from the ion-pair mechanism, suggesting general base catalysis.

Peptidyl epoxides extended in the P' direction as cysteine protease inhibitors: effect on affinity and mechanism of inhibition.

Endo peptidyl epoxides, in which the central epoxidic moiety replaces the scissile amide bond of a P(3)-P(3)' peptide, were designed as cysteine proteases inhibitors. The additional P'-S' interactions, relative to those of an exo peptidyl epoxide of the same P(3)-P(1) sequence, significantly improved affinity to the enzymes papain and cathepsin B, but also changed the mode of inhibition from active-site directed inactivation to reversible competitive inhibition. Computational models rationalize the binding affinity and the inhibition mechanism.

Screening of the active site from water by the incoming ligand triggers catalysis and inhibition in serine proteases.

The pKa of the catalytic His57 N(epsilon)H in the tetrahedral complex (TC) of chymotrypsin with trifluoromethyl ketone inhibitors is 4-5 units higher relative to the free enzyme (FE). Such stable TC's, formed with transition state (TS) analog inhibitors, are topologically similar to the catalytic TS. Thus, analysis of this pKa shift may shed light on the role of water solvation in the general base catalysis by histidine.

The cooperative effect between active site ionized groups and water desolvation controls the alteration of acid/base catalysis in serine proteases.

What is the driving force that alters the catalytic function of His57 in serine proteases between general base and general acid in each step along the enzymatic reaction? The stable tetrahedral complexes (TC) of chymotrypsin with trifluoromethyl ketone transition state analogue inhibitors are topologically similar to the catalytic transition state. Therefore, they can serve as a good model to study the enzyme catalytic reaction. We used DFT quantum mechanical calculations to analyze the effect of solvation and of polar factors in the active site of chymotrypsin on the pKa of the catalytic histidine in FE (the free enzyme), EI (the noncovalent enzyme inhibitor complex), and TC.

Enzyme isoselective inhibitors: application to drug design.

Common methodologies of computer-assisted drug design focus on noncovalent enzyme-ligand interactions. We introduced enzyme isoselective inhibition trend analysis as a tool for the expert analysis of covalent reversible inhibitors. The methodology is applied to predict the binding affinities of a series of transition-state analogue inhibitors of medicinally important serine and cysteine hydrolases.

Enzyme isoselective inhibitors: a tool for binding-trend analysis.

A transition-state analogue inhibitor that covalently reversibly binds to an enzyme formally consists of two parts: the chemical site, CS and the recognition site, RS. We have experimentally and theoretically demonstrated that the trend of binding affinity in a series of isoselective inhibitors (with identical RS and different CS fragments) depends mainly on their CS fragments. Isoselective inhibitors have the same affinity trend toward different enzymes of the same family with a common catalytic mechanism.

Identification of protons position in acid-base enzyme catalyzed reactions: the hepatitis C viral NS3 protease.

General acid-base catalysis is a key element of the catalytic activity of most enzymes. Therefore, any explicit molecular modeling of enzyme-catalyzed chemical reactions requires correct identification of protons location on the catalytic groups. In this work, we apply our quantum mechanical/self-consistent reaction field in virtual solvent [QM/SCRF(VS)] method for identification of the position of protons shared by the enzyme catalytic groups and the polar groups of the inhibitor in a covalent tetrahedral complex (TC) of the hepatitis C virus NS3 protease with a peptidyl alpha-ketoacid inhibitor.

Is there a weak H-bond --> LBHB transition on tetrahedral complex formation in serine proteases?

The transformation of a weak hydrogen bond in the free enzyme into a low-barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H-bond in the Asp102-His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self-consistent reaction field method in our novel QM/SCRF(VS) approach.

Nitrogen inversion in cyclic amines and the bicyclic effect.

The hitherto unsolved problem of the origin of the unusually high nitrogen inversion-rotation (NIR) barriers in 7-azabicyclo[2.2.1]heptanes (the bicyclic effect) was examined using the natural bond orbital (NBO) approach.