Binding Free Energy Research Articles

Molecular docking and molecular dynamics simulation studies reveal repurposing I-Motif ligand as a promising protease inhibitor against SARS-CoV-2 variants

ABSTRACT The COVID-19 caused by the SARS-CoV-2 virus has escalated into a global pandemic, urgently necessitating effective treatments. Tremendous research of the SARS-CoV-2 main protease have enabled insight understanding of its mechanism and potential inhibitors. Due to its crucial role in viral replication, the protease is considered an attractive target for antiviral therapy. This study aimed to identify novel potent inhibitors of main protease protein by repurposing i-Motif (iM) binding compounds from the G4LDB database. iM ligands are known for their ability to stabilise iM structures in DNA, and their repurposing as protein inhibitors is an alternative therapeutic approach. To achieve this, we performed in silico studies with six iM binding compounds. Molecular docking, molecular dynamics (MD) simulations and MM/GBSA binding free energy analysis revealed favourable conformational stability and superior binding affinity of the three proposed iM ligands, including iML0042 (−59.91 ± 3.82 kcal/mol), iML0043 (−56.37 ± 3.87 kcal/mol), and iML0094 (−84.68 ± 3.40 kcal/mol), to SARS-CoV-2 Mpro variants compared to the reference inhibitor nirmatrelvir (−34.35 ± 3.74 kcal/mol). The results suggested that repurposing iM ligands as protease inhibitors is a promising strategy. Our findings revealed significant candidate inhibitors and are recommended for further development as potential protease inhibitors against SARS-CoV-2.

Thymus algeriensis essential oil: Phytochemical investigation, bactericidal activity, synergistic effect with colistin, molecular docking, and dynamics analysis against Gram-negative bacteria resistant to colistin

Due to the increasing resistance prevalence to the last line of antibiotics, such as colistin, and the rising threat of multi-drug resistant bacteria, it is crucial to find alternative therapeutic options. The current study focuses on evaluating antibacterial activities alone and in combination with colistin of Thymus algeriensis essential oil (TA-EO) against colistin-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli co-harboring mcr-1 gene. GC/MS was used to determine the chemical composition of TA-EO. Disc diffusion and microdilution techniques were used to evaluate the antimicrobial activities of TA-EO. Synergism between colistin and TA-EO was evaluated by checkerboard assay. The major compounds of TA-EO were docked with known enzymes involved in resistance to colistin, as well as the biosynthesis of peptidoglycan and amino acids. GC/MS revealed that TA-EO was of carvacrol chemotype (67.94 %). The TA-EO showed remarkable antibacterial activities against all Gram-negative bacterial strains, with the diameter of inhibition zones varied between 30 and 50 mm and a ratio MBC/MIC equal to 1 for the vast majority of bacterial isolates. Interestingly, the checkerboard showed synergism between TA-EO and colistin against colistin-resistant Escherichia coli co-harboring mcr-1 gene (FICI˂1) and reduced the MIC of colistin by 16- to 512-fold and those of TA-EO by 4- to 16-fold. The docking study demonstrated that carvacrol had high binding free energies against MCR-1, a phosphoethanolamine transferase extracellular domain, and its catalytic domain implicated in resistance to colistin, and undecaprenyl pyrophosphate synthase in complex with magnesium which is involved in bacterial peptidoglycan biosynthesis. The molecular dynamics study for 100-ns also revealed the stability of the MCR-1/carvacrol complex with a constant surface area over the simulation. These results support using carvacrol or TA-EO as a bactericidal agent, either alone or in combination with colistin, to treat infections caused by colistin-resistant Gram-negative bacteria.

Unraveling modulation effects on albumin synthesis and inflammation by Striatin, a bioactive protein fraction isolated from Channa striata: In silico proteomics and in vitro approaches

Hypoalbuminemia, associated with inflammation in severely ill patients, can emerge due to decreased albumin production. Transforming growth factor-beta (TGF-β) and nuclear factor-kappa B (NF-κB) are critical signaling pathways responsible for decreased albumin expression. This study explores the protein content and modulation effects of Striatin on albumin synthesis and inflammation, employing in silico proteomics and in vitro investigations. In the in silico proteomics realm, LC/MS-MS protein sequencing, 3D modeling, protein-protein docking simulations, 100 ns molecular dynamics (MD) simulations, and MM/PBSA binding free energy calculations were carried out. Complementing this, in vitro studies examined Albumin gene expression and extracellular secretion in HepG2 cells subjected to lipopolysaccharides-induced hypoalbuminemia. Furthermore, the study probed Striatin's influence on the NF-ᴋB expression, given albumin's role as a negative acute-phase protein. The results unveiled nucleoside diphosphate kinase (NdK) and parvalbumin (PV) as the prominent constituents within Striatin. Notably, NdK and PV exhibited the ability to disrupt hydrogen bonds with specific residues in both TGF-β and NF-κB complexes, thereby enhancing their flexibility, akin to the action of the FKBP12 complex (antagonist complex). In the in vitro experiments, Striatin demonstrated a dose and time-dependent inhibition of hypoalbuminemia, with peak efficacy observed at a concentration of 20 μg/mL. At this concentration, Striatin also suppressed NF-κB expression when co-incubated with lipopolysaccharides. While these findings suggest potential inhibitory effects of Striatin on TGF-β and NF-κB activities, they are preliminary and warrant further investigation. This study highlights Striatin's potential as a therapeutic agent for inflammation-related hypoalbuminemia, though additional research is needed to fully validate these results.

Bioprospection for antiviral compounds from selected medicinal plants against RNA polymerase of rotavirus A using molecular modelling and density functional theory

Rotavirus A (RVA) infection remains a significant global health challenge, especially in developing countries, causing severe dehydrating diarrhoea in children under five years of age. Despite the availability of four World Health Organization (WHO) pre-qualified vaccines, their availability, particularly in low-income countries, pose significant challenges. Currently, there are no specific anti-rotaviral medications hence, the urgency to develop novel therapeutics against rotavirus infection. Thus, this study explored the potential of secondary metabolites of Spondias mombin, Macaranga barteri and Dicerocaryum eriocarpum as novel inhibitors of the RNA-dependent RNA polymerase (VP1) of rotavirus A using computational techniques. Pharmacokinetics parameters were adopted to screen the top 20 metabolites with high affinity for the target, initially identified through a docking study. Furthermore, the ability of the resulting compounds to modulate the investigated target was assessed using molecular dynamics (MD) simulation, while density functional theory (DFT) calculations were conducted to predict the molecular properties of the top-ranked compounds. Except for ellagic acid hexoside (-33.14 kcal/mol), all the leads had higher binding free energy values relative to sofosbuvir (-36.58 kcal/mol) following a 120 ns MD simulation. Overall, the resulting complexes with the lead compounds demonstrated acceptable stability, reduced flexibility and compactness, with spiraeoside (-51.02 kcal/mol) displaying more favourable thermodynamics metrics, albeit with a lesser binding free energy relative to chrysoeriol 7-glucuronide (-58.36 kcal/mol). The binding free energy and thermodynamic parameters of the top-hit compounds could be attributed to their respective bond interactions and molecular orbital properties except chrysoeriol 7-glucuronide, with a need for additional structural adjustment to enhance its thermodynamic properties. Thus, these findings indicate the potential modulatory ability of the lead compounds against the VP1 protein of RVA, underscoring the importance of further in vitro and in vivo studies to validate the predicted activity, and ongoing efforts are being made to pursue this line of investigation.

Abstract B010: Design of personalized neoantigen mRNA vaccines against breast cancer patients in Pakistan based on next-generation sequencing data

Abstract Breast cancer is the most common malignancy among women in Pakistan, with an outrageous increase in the incidence and mortality rate. Because of decreased effectiveness and lower survival rates, there is an urgent need for developing novel, personalized vaccines with enhanced activity. The development of a personalized mRNA vaccine illustrates a promising approach to combating the prevalence of breast cancer. We used the whole exome sequencing (WES) data of Pakistani tumor samples to predict immunogenic neoantigens. The WES data was aligned to the GRCM39 reference genome using BWA. The genome analysis toolkit (GATK) was employed for variant calling to identify somatic mutations. Using immune-bioinformatics tools, we predicted cytotoxic CD8+ T cell epitopes and helper CD4+ T cell epitopes within the identified neoantigens and designed vaccines by assembling the fusion of CTL neoepitopes, helper sequences, and adjuvants together with linkers. The Insilco ImmSim algorithm was used to examine the immune responses of the vaccine. The vaccine design was further explored by checking its simulation effect on the immune system, its physiochemical characteristics, its binding to specific immune system molecules, and cloning into an appropriate vector. In addition, molecular docking, MD simulation, and binding free energies were performed to investigate the interaction mechanism between the construct vaccine and Toll-like receptors (TLR2, TLR4, TLR7, and TLR9).By examining these factors, we aimed to ensure the vaccine's safety, stability, and ability to bind tightly to specific immune cells (class I MHC molecules), ultimately triggering a comprehensive immune response against breast cancer cells. Citation Format: Kayode Yomi Raheem, Muhammad Muddassar. Design of personalized neoantigen mRNA vaccines against breast cancer patients in Pakistan based on next-generation sequencing data [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr B010.

Virtual screening, molecular docking, and molecular dynamics simulation studies on the hypoglycemic function of oat peptides

Oat contains a large amount of polysaccharides and peptides, among which oat peptides have the function of reducing blood sugar levels in the body. This paper reviewed the peptides obtained from oat extraction and identification from literature and constructed the corresponding oat peptide database. Based on the DPP4 protein, a virtual screening of the peptide database was performed. One hundred nanosecond molecular dynamics simulations were performed for the six peptides obtained from the screening. The interaction information between different peptide molecules and DPP4 was analyzed from the stable binding conformations after the simulation, and the binding free energy between different peptide molecules and DPP4 was calculated. The results show that the peptide molecules obtained from the virtual screening can all stably bind to the DPP4 protein, among which two peptide molecules have relatively strong affinity with DPP4 and can be used as lead molecules for the subsequent design and modification of DPP4 inhibitors. The simulation results are informative for a deeper understanding of the structural characteristics of DPP4 and the molecular recognition mechanism between DPP4 and oat peptides.

In silico identification of novel CDK4 inhibitors for retinoblastoma

Retinoblastoma is a kind of cancer that mostly affects children's eyes, and this study intends to find drugs that can suppress the protein kinase cyclin-dependent kinase 4 (CDK4). CDK4 overexpression leads to the hyper-phosphorylation of RB tumor suppressor protein, which ultimately results in uncontrolled cell division. Aberration of cell cycle regulation, specifically the excessive expression of transcription factors responsible for uncontrolled cell division causes retinoblastoma progression. Hence, we screened 25,000 kinase-targeted small molecules against CDK-4 by Glide in Maestro, the top 11 were chosen based on docking scores, binding modes, chemical variety, and other parameters. We ran molecular dynamics (MD) simulations of top hits found in the docking studies and determined their free binding energy. This helped us to understand their thermodynamic and dynamic properties, as well as confirm the docking results, finally the two most promising ligands (3396 and 960) were obtained. As a result of our research, we have identified promising new compounds for treating retinoblastoma. To validate the possible therapeutic and preventative effects of this ligand, rigorous experimental validation, animal studies as well as clinical trials would be required.

Structure Optimization of c-Jun N-terminal Kinase 1 Inhibitors for Treating Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease with an elusive etiology. Aberrant activation of c-Jun N-terminal kinase 1 (JNK1) has been implicated in its pathogenesis. Through a combination of structure-based drug design and structure-activity relationship (SAR) optimization, a series of pyrimidine-2,4-diamine scaffold derivatives have been developed as potent JNK1 inhibitors. Compound E1 was identified with low nanomolar JNK1 inhibitory potency (IC50 = 2.7 nM). The introduction of a dimethylamine side chain has significantly enhanced the ability of E1 to inhibit c-Jun phosphorylation, surpassing the clinical candidate CC-90001. Molecular dynamics simulations revealed a binding free energy of -50.46 kcal/mol for E1. Moreover, E1 displayed satisfactory pharmacokinetic properties, with a bioavailability of 69% in rats. Furthermore, compound E1 exerted significant antifibrotic effects in a bleomycin-induced IPF mouse model and prevented a TGF-β-induced epithelial-to-mesenchymal transition in vitro. These findings position E1 as a promising lead for further drug development targeting IPF.

A205V, D376E, W574L, S653T, and S653N Substitutions in Acetohydroxy Acid Synthase from Amaranthus retroflexus L. Show Different Functional Impacts on Herbicide Resistance

Amaranthus retroflexus L. is a troublesome dicot weed in crop fields and has developed high resistance to nicosulfuron in China. The objective of this study was to determine the effects of specific resistance mutations (A205V, D376E, W574L, S653T, and S653N) of the acetohydroxy acid synthase enzyme (AHAS) on the resistance of A. retroflexus to nicosulfuron. The resistance mutations in A. retroflexus not only conferred 17.17- to 31.70-fold resistance to nicosulfuron but also greatly decreased AHAS sensitivity and increased AHAS binding affinity to substrate pyruvate, which mechanisms were primarily responsible for the observed A. retroflexus resistance. Molecular docking results indicated that these resistance mutations altered AHAS binding free energy with nicosulfuron. All the resistance mutations showed less sensitivity to feedback inhibition by branched-chain amino acids, but the mutations did not necessarily affect biosynthesis in A. retroflexus. This report to compares the various mutations of ArAHAS in vitro and contributes to understanding herbicide resistance in this field weed.

Absolute Binding Free Energies with OneOPES.

The calculation of absolute binding free energies (ABFEs) for protein-ligand systems has long been a challenge. Recently, refined force fields and algorithms have improved the quality of the ABFE calculations. However, achieving the level of accuracy required to inform drug discovery efforts remains difficult. Here, we present a transferable enhanced sampling strategy to accurately calculate absolute binding free energies using OneOPES with simple geometric collective variables. We tested the strategy on two protein targets, BRD4 and Hsp90, complexed with a total of 17 chemically diverse ligands, including both molecular fragments and drug-like molecules. Our results show that OneOPES accurately predicts protein-ligand binding affinities with a mean unsigned error within 1 kcal mol-1 of experimentally determined free energies, without the need to tailor the collective variables to each system. Furthermore, our strategy effectively samples different ligand binding modes and consistently matches the experimentally determined structures regardless of the initial protein-ligand configuration. Our results suggest that the proposed OneOPES strategy can be used to inform lead optimization campaigns in drug discovery and to study protein-ligand binding and unbinding mechanisms.

Repurposing Antiviral Drugs as Potential Anti-EGFR Agents in NSCLC: A Structure-Based Screening and Molecular Dynamics Analysis.

One of the problems resulting from recurrent hyperactivated or mutant epidermal growth factor receptors (EGFR) in non-small cell lung cancer (NSCLC) is therapeutic resistance. Consequently, this leads to increased expressionof oncogenicproteins and reduces the efficacy of EGFR tyrosine kinase inhibitors (TKIs). This study assessed antiviral drug efficacy as potential anti-EGFR agentsfor NSCLC. We used structure-based virtual screening to evaluate 66 antiviral drugs thoroughly. The top 6 antiviral drugs exhibiting impressive binding energies (i.e. surpassing a threshold of -8.5 kcalmol-1) were identified. Subsequent bioactivity analysis and ADMET profiling were performed to select the most promising candidates, followed by a molecular dynamic simulation. Among the selected antiviral regimens, dolutegravir demonstrated the highest docking score (-9.8 kcalmol-1), followed by rilpivirine and ensitrelvir, surpassing other candidates and our reference EGFR TKI. Further molecular dynamics simulations revealed promising dynamic interactions of dolutegravir, ensitrelvir, and rilpivirine with the EGFR target as compared with afatinib. Our findings highlight the repositioning potential of antiviral drugs for anti-EGFR drug discovery, supported by their robust docking scores, ADMET profiles, dynamic interactions, and binding free energies. The results open up new avenues for advanced NSCLC therapy. Further in vitro investigations are warranted to evaluate their efficacy and safety.

Stereo-selectivity of enantiomeric inhibitors to ubiquitin-specific protease 7 (USP7) dissected by molecular docking, molecular dynamics simulations, and binding free energy calculations

Stereo-selectivity of enantiomeric inhibitors to ubiquitin-specific protease 7 (USP7) dissected by molecular docking, molecular dynamics simulations, and binding free energy calculations

Adenosyl derivatives as a potential inhibitors of NS3 protease of Japanese encephalitis virus (JEV): In silico molecular insight into therapeutic discovery

The genus Flavivirus NS3 a non-structural protein of Japanes Encephalitis Virus (JEV), a serious deadly human pathogen responsible for several deaths in South East Asia, consists of helicase/NTPase domain forming a cleft known for their role in the enzymatic activity and viral replication represented biological relevance. S-Adenosyl derivatives act as powerful inhibitors in viral replication in NS5 of the same genus as of NS3, provide a powerful base to test its effectiveness in NS3 protein due to the compatibility of both proteins. The MM-GBSA (Molecular Mechanics-Generalized Born Surface Area) simulation were performed to evaluate the binding free energy, following the 100 (ns) production MD simulation in the periodic boundary condition (PBC) for the selected docked inhibitors with NS3. The residue-wise decomposition energy determined in our study revealed the active site region made the high stability with the potential inhibitors.

Virtual Screening and Molecular Dynamics Simulation to Identify Inhibitors of the m6A-RNA Reader Protein YTHDC1

YTHDC1 (YTH domain containing 1), a crucial reader protein of N6-methyladenosine (m6A) mRNA, plays a critical role in various cellular functions and is considered a promising target for therapeutic intervention in acute myeloid leukemia and other cancers. In this study, we identified orthosteric small-molecule ligands for YTHDC1. Using a molecular docking approach, we screened the eMolecules database and recognized 15 top-ranked ligands. Subsequently, molecular dynamics simulations and MM/PBSA analysis were used to assess the stability and binding free energy of these potential hit compounds in complex with YTHDC1. Notably, five compounds with IDs of ZINC82121447, ZINC02170552, ZINC65274016, ZINC10763862, and ZINC02412146 exhibited high binding affinities and favorable binding free energies. The results also showed that these compounds formed strong hydrogen bonds with residues SER378, ASN363, and ASN367 and interacted with the aromatic cage of the YTHDC1 reader protein through TRP377, TRP428, and hydrophobic residue LEU439. To assess their viability as lead compounds, we conducted absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies to reveal promising features for these identified small molecules, shedding light on their pharmacokinetic and safety profiles.

Elucidating the resistance mechanisms and binding pattern of novel Oxa-48-like carbapenemases covalent inhibitors: A hybrid experimental and in silico approach

Klebsiella pneumoniae is a Gram negative bacteria that can cause harm to the public's health and vulnerability has increased due to the emergence of highly virulent strains resistant to carbapenem antibiotics. The K. pneumoniae resistance mechanism involves extended-spectrum beta-lactamases, which are enzymes that can break down a broad range of antibiotics. The particular concern is Class D β-lactamases like OXA-48, which further complicates treatment options for infections caused by K. pneumoniae. The current study screened 7800 samples from three hospitals in Pakistan and found 353 positive samples of K. pneumoniae. The isolates were resistant to SXT, AMP, FEP CTX, CIP, TET, MEM, and IPM, while effective antibiotics were TGC, FOS, FOX, SCF and TZP. The sequencing and mutational analysis of the OXA-48 gene revealed 45-point mutations, including five novel missense mutations (Thr104:Ala104, Asn110:Asp110, Glu168:Gln168, Ser171:Ala171 and Arg214:Ser214). These mutations can contribute to the antibiotic affinity decrease and cause resistance. Furthermore, the study aimed to develop covalent inhibitors of OXA-48 (wild type and mutant) carbapenemases using a combination of molecular docking and molecular dynamic (MD) simulation approaches from natural products from the ZNINC20 database. 10 compounds (ZINC103533306, ZINC103531564, ZINC3861001, ZINC103533375, ZINC12411532, ZINC103533290, ZINC12376465, ZINC85875819, ZINC59586513 and ZINC13370550) showed best fit and high interactions with the OXA-48 active pocket. The binding free energies calculated from the MD showed that these compounds have high affinity with both the wild type and mutated OXA-48 protein. The selected compounds showed good drug-like properties and can be the leading inhibitors to combat the resistant K. pneumoniae strains.

Marine natural compounds as potential CBP bromodomain inhibitors for treating cancer: an in-silico approach using molecular docking, ADMET, molecular dynamics simulations and MM-PBSA binding free energy calculations.

The online version contains supplementary material available at 10.1007/s40203-024-00258-5.

Design and Synthesis of Novel Indole-Derived N-Methylcarbamoylguanidinyl Chitinase Inhibitors with Significantly Improved Insecticidal Activity.

Chitinases play an important role in the molting process of insects and are potential targets for the development of green insecticides. Based on the feature that the +1/+2 sites in OfChtI, OfChtII, and OfChi-h have tryptophan residues in mismatch-parallel position, a strategy to introduce indole scaffold into chitinase inhibitors was proposed, and multitarget chitinase inhibitors containing N-methylcarbamoylguanidinyl and indole scaffold were successfully synthesized. The inhibitory activity showed that compound 8u exhibited significant inhibitory activity against OfChtI, OfChtII, and OfChi-h, with IC50 values of 0.7, 0.79, and 0.58 μM, and Ki values of 0.05 ± 0.005, 0.065 ± 0.004, and 0.025 ± 0.006 μM, respectively. In vivo insecticidal activity showed that compounds 8a and 8g exhibited excellent insecticidal activity against Plutella xylostella and Mythimna separata, with LC50 values of 0.79 and 9.17 mg/L against P. xylostella, respectively, and 3.58 and 83.09 mg/L against M. separata, respectively, making them the most potent chitinase inhibitors with in vivo insecticidal activity discovered to date. The inhibition mechanism and binding free energy results suggested that N-methylcarbamoylguanidinyl binds to the -1 catalytic site, while additional interactions acquired by π-π stacking and hydrophobic interactions of the indole scaffold with tryptophan increase the binding affinity of the targets to chitinases. This work provides a new direction for the development of chitinase inhibitors with compounds 8a and 8g potentially serving as promising candidates for pesticide development.

Effects of graphene reinforcement on morphology, thermophysical, and mechanical properties of polyvinyl alcohol and polyacrylamide in condensed phases

The subtle interplay of noncovalent interaction in enhancing the compatibility between the graphene-based material and the oligomers of polyvinyl alcohol (PVA) and polyacrylamide (PAM) in the solvent phase is unraveled within the framework of all-atom classical force field-based molecular dynamics (MD) simulations. The decomposition of binding free energy analysis demonstrates that the interaction between polymer segments and graphene (G)-surface crucially emanates from the van der Waals (vdW) interactions, while the adsorption of polymer chains on the graphene oxide (GO)-surface is influenced by vdW and electrostatic interactions. The influence of diverse factors including the strain rate, the size of the nanofiller, the number of oligomers, and the thermal quenching rate on controlling the morphology, mechanical, and thermophysical properties of the graphene-reinforced polymer nanocomposites are further explored. The uniaxial deformation simulations of the graphene-reinforced PVA and PAM matrices show that the interfacial mechanical strength is escalated for the G-PVA nanocomposite. The inclusion of graphene nanofiller reduces the polymer chain mobility and increases the intermolecular interaction, which in turn enhances the toughness of the graphene-polymer composites. Increasing the strain rate raises the yield strength and modulus, making the composites appear stronger and stiffer. As evidenced by the predicted glass transition temperature, the thermal stability of the PVA and PAM matrices is significantly improved by the graphene reinforcement.

Novel alloxazine analogues: design, synthesis, and antitumour efficacy enhanced by kinase screening, molecular docking, and ADME studies

This study describes the development of novel alloxazine analogues as potent antitumor agents with enhanced selectivity for tumour cells. Twenty-nine out of 45 newly compounds were investigated in vitro for their growth inhibitory activities, against two human tumour cell lines, namely, the human T-cell acute lymphoblastoid leukaemia cell line (CCRF-HSB-2) and human oral epidermoid carcinoma cell line (KB), and the antitumor agent “Ara-C” was used as a positive reference in this investigation. Compounds 9e and 10J were the highest among their analogues, against both tumour cell lines (CCRF-HSB-2 and KB). Correlation analyses demonstrated a strong relationship between the IC50 values and AutoDock binding free energy or calculated inhibition (Ki ). The study delves into structure–activity relationships (SARs) through advanced modelling tools integrated with structure-based drug design (SBDD) using GOLD 5.2.2, AutoDock 4.2, and Accelrys Discovery Studio 3.5. Physicochemical properties, pharmacokinetics, drug-likeness, and toxicity predictions of the most potent alloxazine derivatives were conducted using ProTox-II and Swiss ADME for effective antitumor agents with improved selectivity.

Binding of Inhibitors to Nuclear Localization Signal Peptide from Venezuelan Equine Encephalitis Virus Capsid Protein Explored with All-Atom Replica Exchange Molecular Dynamics.

Several small molecule inhibitors have been designed to block binding of the Venezuelan equine encephalitis virus (VEEV) nuclear localization signal (NLS) sequence to the importin-α nuclear transport protein. To probe the inhibition mechanism on a molecular level, we used all-atom explicit water replica exchange molecular dynamics to study the binding of two inhibitors, I1 and I2, to the coreNLS peptide, representing the core fragment of the VEEV NLS sequence. Our objective was to evaluate the possibility of masking wherein binding of these inhibitors to the coreNLS occurs prior to its binding to importin-α. We found that the free energy of I1 and I2 binding to the coreNLS is less favorable than that to importin-α. This outcome argues against preemptive inhibitor binding to the coreNLS prior to importin-α. Instead, both inhibitors are expected to compete with the coreNLS peptide for binding to importin-α. The two factors responsible for the low affinities of the inhibitors to the coreNLS peptide are (i) the low cooperativity of binding to the peptide and (ii) the strong hydrophobic effect associated with binding to importin-α. Our results further show that upon binding to the coreNLS peptide, the inhibitors form multiple diverse binding poses. The coreNLS peptide coincubated with I1 and I2 adopts several conformational states, including open and collapsed, which underscores the fluidity of the coreNLS conformational ensemble as a target for inhibitors. Taken together with our prior investigations, this study sheds light on the molecular mechanism by which I1 and I2 ligands inhibit binding of the VEEV capsid protein to importin-α.