4-(Azolyl)-Benzamidines as a Novel Chemotype for ASIC1a Inhibitors.

Acid-sensing ion channels (ASICs) play a key role in the perception and response to extracellular acidification changes. These proton-gated cation channels are critical for neuronal functions, like learning and memory, fear, mechanosensation and internal adjustments like synaptic plasticity. Moreover, they play a key role in neuronal degeneration, ischemic neuronal injury, seizure termination, pain-sensing, etc. Functional ASICs are homo or heterotrimers formed with (ASIC1-ASIC3) homologous subunits. ASIC1a, a major ASIC isoform in the central nervous system (CNS), possesses an acidic pocket in the extracellular region, which is a key regulator of channel gating. Growing data suggest that ASIC1a channels are a potential therapeutic target for treating a variety of neurological disorders, including stroke, epilepsy and pain. Many studies were aimed at identifying allosteric modulators of ASIC channels. However, the regulation of ASICs remains poorly understood. Using all available crystal structures, which correspond to different functional states of ASIC1, and a molecular dynamics simulation (MD) protocol, we analyzed the process of channel inactivation. Then we applied a molecular docking procedure to predict the protein conformation suitable for the amiloride binding. To confirm the effect of its sole active blocker against the ASIC1 state transition route we studied the complex with another MD simulation run. Further experiments evaluated various compounds in the Enamine library that emerge with a detectable ASIC inhibitory activity. We performed a detailed analysis of the structural basis of ASIC1a inhibition by amiloride, using a combination of in silico approaches to visualize its interaction with the ion pore in the open state. An artificial activation (otherwise, expansion of the central pore) causes a complex modification of the channel structure, namely its transmembrane domain. The output protein conformations were used as a set of docking models, suitable for a high-throughput virtual screening of the Enamine chemical library. The outcome of the virtual screening was confirmed by electrophysiological assays with the best results shown for three hit compounds.

Integrated workflow for the identification of new GABAA R positive allosteric modulators based on the in†silico screening with further in†vitro validation. Case study using Enamine's stock chemical space.

Numerous studies reported an association between GABAA R subunit genes and epilepsy, eating disorders, autism spectrum disorders, neurodevelopmental disorders, and bipolar disorders. This study was aimed to find some potential positive allosteric modulators and was performed by combining the in silico approach with further in vitro evaluation of its real activity. We started from the GABAA R-diazepam complexes and assembled a lipid embedded protein ensemble to refine it via molecular dynamics (MD) simulation. Then we focused on the interaction of α1ß2γ2 with some Z-drugs (non-benzodiazepine compounds) using an Induced Fit Docking (IFD) into the relaxed binding site to generate a pharmacophore model. The pharmacophore model was validated with a reference set and applied to decrease the pre-filtered Enamine database before the main docking procedure. Finally, we succeeded in identifying a set of compounds, which met all features of the docking model. The aqueous solubility and stability of these compounds in mouse plasma were assessed. Then they were tested for the biological activity using the rat Purkinje neurons and CHO cells with heterologously expressed human α1ß2γ2 GABAA receptors. Whole-cell patch clamp recordings were used to reveal the GABA induced currents. Our study represents a convenient and tunable model for the discovery of novel positive allosteric modulators of GABAA receptors. A High-throughput virtual screening of the largest available database of chemical compounds resulted in the selection of 23 compounds. Further electrophysiological tests allowed us to determine a set of 3 the most outstanding active compounds. Considering the structural features of leader compounds, the study can develop into the MedChem project soon.

In silico induced effect of N-ε-lysine acetylation on microtubule stability and subsequent interaction of microtubule-associated proteins.

Plant systems have been considered valuable models for addressing fundamental questions of microtubule (MT) organization due to their considerable practical utility. Protein acetylation is a very common protein modification, and therate of acetylation can be modulated in cells in different biological states, and these changes can be detected at a molecular level. Here, we focused on K40, K112, and K394 residues as putative acetylation sites, which were shown to exist in both plants and mammals. Such residual effect of acetylation causes critical but unclear effect on MT stability. In turn, it was shown that acetylation indirectly affects the probability of interaction with different MAPs (Microtubule-associated proteins). In a multiscale study using an all-atom force field to reproduce several lattice-forming elements found on the surface the microtubule, we assembled a fragment of a plant microtubule composed of nine tubulins and used it as a model object along with the existing human complex. Triplets of tubulins assembled in a lattice cell were then simulated for both human and plant protein complexes, using a coarse-grained force field. We then analyzed the trajectories and identified some critical deformations of the MAP interaction surface. The initial coordinates were used to investigate the structural scenario in which autophagy-related protein 8 (ATG8) was able to interact with the MT fragment.

Mutations in STARD8 (DLC3) May Cause 46,XY Gonadal Dysgenesis.

INTRODUCTION: 46,XY gonadal dysgenesis is a condition that is characterised by undeveloped testes in individuals with a male karyotype. Mutations in many genes that underlie this condition have been identified; however, there are still a considerable number of patients with an unknown genetic background. Recently, a mutation in the STARD8 X-linked gene in two sisters with 46,XY gonadal dysgenesis has been reported. It was localised within the START domain, whose homologue in Drosophila is responsible for maintaining testes integrity during their development. METHODS: We analysed the potential pathogenicity of another STARD8 mutation, p.R887C, that was identified in a patient with 46,XY asymmetric gonadal dysgenesis. For this purpose, molecular dynamics simulations were performed. RESULTS: These simulations revealed the full rearrangement of the helix containing the p.R887C substitution upstream from the START domain, which may cause STARD8 protein dysfunction and contribute to 46,XY gonadal dysgenesis. A comparison of the phenotypes of the three described 46,XY gonadal dysgenesis patients that harbour STARD8 mutations indicated that alterations of this gene can result in a partial or complete gonadal dysgenesis phenotype. CONCLUSION: Based on these and previous results, it is reasonable to include STARD8 in gene panels for 46,XY gonadal dysgenesis.

In Vitro Evaluation of In Silico Screening Approaches in Search for Selective ACE2 Binding Chemical Probes.

New models for ACE2 receptor binding, based on QSAR and docking algorithms were developed, using XRD structural data and ChEMBL 26 database hits as training sets. The selectivity of the potential ACE2-binding ligands towards Neprilysin (NEP) and ACE was evaluated. The Enamine screening collection (3.2 million compounds) was virtually screened according to the above models, in order to find possible ACE2-chemical probes, useful for the study of SARS-CoV2-induced neurological disorders. An enzymology inhibition assay for ACE2 was optimized, and the combined diversified set of predicted selective ACE2-binding molecules from QSAR modeling, docking, and ultrafast docking was screened in vitro. The in vitro hits included two novel chemotypes suitable for further optimization.

Functional Effects In Silico Prediction for Androgen Receptor Ligand-Binding Domain Novel I836S Mutation.

Over 1000 mutations are described in the androgen receptor (AR) gene. Of those, about 600 were found in androgen insensitivity syndrome (AIS) patients, among which 400 mutations affect the ligand-binding domain (LBD) of the AR protein. Recently, we reported a novel missense mutation c.2507T>G I836S (ClinVarID: 974911) in a patient with complete AIS (CAIS) phenotype. In the present study, we applied a set of computational approaches for the structural analysis of the ligand-binding domains in a wild-type and mutant AR to evaluate the functional impact of the novel I836S mutation. We revealed that the novel I836S substitution leads to a shorter existence time of the ligand's gating tunnel and internal cavity, occurring only in the presence of S836 phosphorylation. Additionally, the analysis of phosphorylation of the 836 mutant residues explained the negative impact on AR homodimerization, since monomer surface changes indirectly impacted the binding site. Our analyses provide evidence that I836S causes disruptions of AR protein functionality and development of CAIS clinical features in patients.

Modelling of an autonomous Nav1.5 channel system as a part of in silico pharmacology study.

A homology model of Nav1.5, based mainly on the crystal structures of Nav1.2/1.5 was built, optimized and successfully inserted into the membrane bilayer. We applied steered and free MD simulation protocols for the visualization of the mechanism of Nav1.5 activation. We constrained dihedrals of S4 trigger to introduce a structural tension with further rearrangement and movement of secondary structure elements. From these, we observed an intracellular gate opening and movement of the Lys1419 residue caused by a gradual displacement of the distal S6 α-helix with the extended S4 3-10 helix of voltage-sensing domains (VSD). A construction containing the Lys1419 residue in P-loop also changed its position due to the extension of this helix and subsequent induction of the pore-forming helixes motion. From this point, a double membrane system was generated, implying a free of ligand Nav1.5 protein and on the opposite side its copy containing a docked bupivacaine molecule inside the pore channel. The system can be used for the design of selective inhibitors against the Nav1.7 channel, instead of mixed effect on both channels.

Effect of Charge Distribution in a Modified tRNA Substrate on Pre-Reaction Protein-tRNA Complex Geometry.

An important aspect of molecular mechanics simulations of a protein structure and ligand binding often involves the generation of reliable force fields for nonstandard residues and ligands. We consider the aminoacyl-tRNA synthetase (AaRS) system that involves nucleic acid and amino acid derivatives, obtaining force field atomic charges using the restrained electrostatic potential (RESP) approach. These charges are shown to predict observed properties of the post-transfer editing reaction in this system, in contrast to simulations performed using approximate charges conceived based upon standard charges for related systems present in force field databases. In particular, the simulations predicted key properties induced by mutation. The approach taken for generating the RESP charges retains established charges for known fragments, defining new charges only for the novel chemical features present in the modified residues. This approach is of general relevance for the design of force fields for pharmacological applications, and indeed the AaRS target system is itself relevant to antibiotics development.

Peptidyl inhibition of Spt4-Spt5: Protein-protein inhibitors for targeting the transcriptional pathway related to C9orf72 expansion repeats.

Spt4/Spt5 is an useful target as it is likely a transcription factor that has implications for long non-coding RNA repeats related to frontotemporal dementia (FTD) found in the C9orf72 disease pathology. Inhibitors for Spt4/Spt5 using peptides as a starting point for assays as a means for developing small molecules, which could likely lead to therapeutic development for inhibition for Spt4/Spt5 with CNS characteristics. To elucidate the specific steps of identification and modification of key interacting residues from Spt4/Spt5 complex with further effect prediction, a set of different computational methods was applied. Newly characterized, theoretically derived peptides docked on Spt4/Spt5 models, based on X-ray crystallography sources, allowed us to complete molecular dynamics simulations and docking studies for peptide libraries that give us high confident set of peptides for use to screen for Spt4/Spt5 inhibition. Several peptides with increased specificity to the Spt4/Spt5 interface were found and can be screened in cell-based assays and enzymatic assays for peptide screens that lead to small molecule campaigns. Spt4/Spt5 comprises an attractive target for neurological diseases, and applying these peptides into a screening campaign will promote the goal of therapeutic searches for FTD drug discovery.

Stereospecificity control in aminoacyl-tRNA-synthetases: new evidence of d-amino acids activation and editing.

The homochirality of amino acids is vital for the functioning of the translation apparatus. l-Amino acids predominate in proteins and d-amino acids usually represent diverse regulatory functional physiological roles in both pro- and eukaryotes. Aminoacyl-tRNA-synthetases (aaRSs) ensure activation of proteinogenic or nonproteinogenic amino acids and attach them to cognate or noncognate tRNAs. Although many editing mechanisms by aaRSs have been described, data about the protective role of aaRSs in d-amino acids incorporation remained unknown. Tyrosyl- and alanyl-tRNA-synthetases were represented as distinct members of this enzyme family. To study the potential to bind and edit noncognate substrates, Thermus thermophilus alanyl-tRNA-synthetase (AlaRS) and tyrosyl-tRNA-synthetase were investigated in the context of d-amino acids recognition. Here, we showed that d-alanine was effectively activated by AlaRS and d-Ala-tRNAAla, formed during the erroneous aminoacylation, was edited by AlaRS. On the other hand, it turned out that d-aminoacyl-tRNA-deacylase (DTD), which usually hydrolyzes d-aminoacyl-tRNAs, was inactive against d-Ala-tRNAAla. To support the finding about DTD, computational docking and molecular dynamics simulations were run. Overall, our work illustrates the novel function of the AlaRS editing domain in stereospecificity control during translation together with trans-editing factor DTD. Thus, we propose different evolutionary strategies for the maintenance of chiral selectivity during translation.

Structural and functional features of lysine acetylation of plant and animal tubulins.

The study of the genome and the proteome of different species and representatives of distinct kingdoms, especially detection of proteome via wide-scaled analyses has various challenges and pitfalls. Attempts to combine all available information together and isolate some common features for determination of the pathway and their mechanism of action generally have a highly complicated nature. However, microtubule (MT) monomers are highly conserved protein structures, and microtubules are structurally conserved from Homo sapiens to Arabidopsis thaliana. The interaction of MT elements with microtubule-associated proteins and post-translational modifiers is fully dependent on protein interfaces, and almost all MT modifications are well described except acetylation. Crystallography and interactome data using different approaches were combined to identify conserved proteins important in acetylation of microtubules. Application of computational methods and comparative analysis of binding modes generated a robust predictive model of acetylation of the ϵ-amino group of Lys40 in α-tubulins. In turn, the model discarded some probable mechanisms of interaction between elements of interest. Reconstruction of unresolved protein structures was carried out with modeling by homology to the existing crystal structure (PDBID: 1Z2B) from B. taurus using Swiss-model server, followed by a molecular dynamics simulation. Docking of the human tubulin fragment with Lys40 into the active site of α-tubulin acetyltransferase, reproduces the binding mode of peptidomimetic from X-ray structure (PDBID: 4PK3).

Structural and Biological Overview of Boron-containing Amino Acids in the Medicinal Chemistry Field.

BACKGROUND: Amino acids are the basic structural units of proteins as well as the precursors of many compounds with biological activity. The addition of boron reportedly induces changes in the chemical-biological profile of amino acids. METHODS: We compiled information on the biological effect of some compounds and discussed the structure-activity relationship of the addition of boron. The specific focus presently is on borinic derivatives of α-amino acids, the specific changes in biological activity caused by the addition of a boron-containing moiety, and the identification of some attractive compounds for testing as potential new drugs. RESULTS: Borinic derivatives of α-amino acids have been widely synthesized and tested as potential new therapeutic tools. The B-N (1.65 A°) or B-C (1.61 A°) or B-O (1.50 A°) bond is often key for the stability at different pHs and temperatures and activity of these compounds. The chemical features of synthesized derivatives, such as the specific moieties and the logP, polarizability and position of the boron atom are clearly linked to their pharmacodynamic and pharmacokinetic profiles. Some mechanisms of action have been suggested or demonstrated, while those responsible for other effects remain unknown. CONCLUSION: The increasing number of synthetic borinic derivatives of α-amino acids as well as the recently reported crystal structures are providing new insights into the stability of these compounds at different pHs and temperatures, their interactions on drug targets, and the ring formation of five-membered heterocycles. Further research is required to clarify the ways to achieve specific synthesis, the mechanisms involved in the observed biological effect, and the toxicological profile of this type of boron-containing compounds (BCCs).

A molecular dynamics simulation study of amino acid selectivity of LeuRS editing domain from Thermus thermophilus.

The accuracy of protein synthesis is provided by the editing functions of aminoacyl-tRNA synthetases (aaRSs), a mechanism that eliminates misactivated amino acids or mischarged tRNAs. Despite research efforts, some molecular bases of these mechanisms are still unclear. The post-transfer editing pathway of leucyl-tRNA synthetase (LeuRS) carried out in a special insertion domain (the Connective Polypeptide 1 or CP1), as editing domain. Recently, it was shown by in vivo studies and was supported by mutagenesis, and the kinetics approaches that the CP1 domain of LeuRS has discriminatory power for different substrates. The goal of this work is to investigate the structural basis for amino acid recognition of LeuRS post-transfer editing processes with molecular dynamics (MD) simulation method. To pursue this aim, the molecular modeling studies on Thermus thermophiles LeuRS (LeuRSTT) with two post-transfer substrates (norvalyl-tRNALeu and isoleucyl-tRNALeu) was performed. Our results revealed that post-transfer substrate norvalyl-tRNALeu is more favorable. Moreover, the MD simulations show that branched side chain of Ile-A76 cannot allow water molecules to get close, which leads to a significant decrease in the rate of hydrolysis. Finally, the study showed that site mutation Asp347Ala has elucidated a number of fine structural differences in the binding mode of two post-transfer substrates to the active centre of LeuRS editing domain and two conserved threonines, namely Thr247 and Thr248, are responsible for the amino acid selection through the interaction with substrates.