Amino Acid Residues Research Articles

Interaction of heparin with human cardiac troponin complex and its influence on the immunodetection of troponins in human blood samples.

Heparin is a highly charged polysaccharide used as an anticoagulant to prevent blood coagulation in patients with presumed myocardial infarction and to prepare heparin plasma samples for laboratory tests. There are conflicting data regarding the effects of heparin on the measurement of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT), which are used for the immunodiagnosis of acute myocardial infarction. In this study, we investigated the influence of heparin on the immunodetection of human cardiac troponins. Gel filtration (GF) techniques and sandwich fluoroimmunoassay were performed. The regions of сTnI and cTnT that are affected by heparin were investigated with a panel of anti-cTnI and anti-cTnT monoclonal antibodies, specific to different epitopes. Heparin was shown to bind to the human cardiac full-size ternary troponin complex (ITC-complex) and free cTnT, which increased their apparent molecular weights in GF studies. Heparin did not bind to the low molecular weight ITC-complex and to binary cTnI-troponin С complex. We did not detect any sites on cTnI in the ITC-complex that were specifically affected by heparin. In contrast, cTnT regions limited to approximately 69-99, 119-138 and 145-164 amino acid residues (aar) in the ITC-complex and a region that lies approximately between 236 and 255 aar of free cTnT were prone to heparin influence. Heparin binds to the ITC-complex via cTnT, interacting with several sites on the N-terminal and/or central parts of the cTnT molecule, which might influence the immunodetection of analytes in human blood.

Direct Interaction of Minocycline to p47phox Contributes to its Attenuation of TNF-α-Mediated Neuronal PC12 Cell Death: Experimental and Simulation Validation.

Kaempferol illustrates an example of attempting to discover new treatments for neurodegeneration by investigating the potential efficacy of natural products. Despite the identification of several molecular targets for this bio-active compound, the precise underlying pathways are not well elucidated yet. Recently, it has been shown through pulldown assay that kaemferol directly interacts with p47phox, the organizer subunit of NADPH oxidase-2 (NOX2) complex. Hence, in this study, we used homology modelling, computational docking, mutation analysis, molecular dynamics simulations and free energy calculations to determine how kaempferol interacts with p47phox. Firstly, 3D structure of p47phox was generated using x-ray structures of its domains. Then, it was docked with kaempferol, and finally 100-ns molecular dynamics (MD) simulations were performed and the global properties like root-mean square deviation (RMSD) and root-mean square fluctuations (RMSF) were calculated. Literature survey and computational analysis of key interacting amino acid residues of p47phox provided insights into a possible binding site for kaempferol, approximately around Trp193 and Cys196 located within the N-terminal SH3 domain of p47phox. Moreover, free energy calculations indicated that in silico substitution of Trp193 and Cys196 with arginine and alanine, respectively, results in less favorable interaction corroborating their importance in binding with kaempferol. Taken together, these findings suggest that kaempferol directly attaches to N-SH3 domain p47 phox, with a subsequent diminution of p47phox protein-protein interaction and possibly attenuation of NOX2 complex assembly, which reduces reactive oxygen species (ROS) generation. These observations will be beneficial for researchers exploring neuroprotection and for the development of p47phox inhibitors.

Structural and functional characterization of the role of acetylation on the interactions of the human Atg8-family proteins with the autophagy receptor TP53INP2/DOR

ABSTRACT The Atg8-family proteins (MAP1LC3/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2) play a pivotal role in macroautophagy/autophagy through their ability to help form autophagosomes. Although autophagosomes form in the cytoplasm, nuclear levels of the Atg8-family proteins are significant. Recently, the nuclear/cytoplasmic shuttling of LC3B was shown to require deacetylation of two Lys residues (K49 and K51 in LC3B), which are conserved in Atg8-family proteins. To exit the nucleus, deacetylated LC3B must bind TP53INP2/TP53INP2 (tumor protein p53 inducible nuclear protein 2) through interaction with the LC3-interacting region (LIR) of TP53INP2 (TP53INP2LIR). To examine their selectivity for TP53INP2 and the role of the conserved Lys residues in Atg8-family proteins, we prepared the six human Atg8-family proteins and acetylated variants of LC3A and GABARAP for biophysical and structural characterization of their interactions with the TP53INP2LIR. Isothermal titration calorimetry (ITC) experiments demonstrate that this LIR binds preferentially to GABARAP subfamily proteins, and that only acetylation of the second Lys residue reduces binding to GABARAP and LC3A. Crystal structures of complexes with GABARAP and LC3A (acetylated and deacetylated) define a β-sheet in the TP53INP2LIR that determines the GABARAP selectivity and establishes the importance of acetylation at the second Lys. The in vitro results were confirmed in cells using acetyl-mimetic variants of GABARAP and LC3A to examine nuclear/cytoplasmic shuttling and colocalization with TP53INP2. Together, the results demonstrate that TP53INP2 shows selectivity to the GABARAP subfamily and acetylation at the second Lys of GABARAP and LC3A disrupts key interactions with TP53INP2 required for their nuclear/cytoplasmic shuttling.

Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa.

Cyclophilin B(CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S.globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant E.coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence,we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant cyclophilin B protein from S.globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB.

Mutational Analysis of RIP Type I Dianthin-30 Suggests a Role for Arg24 in Endocytosis

Saponin-mediated endosomal escape is a mechanism that increases the cytotoxicity of type I ribosome-inactivating proteins (type I RIPs). In order to actualize their cytotoxicity, type I RIPs must be released into the cytosol after endocytosis. Without release from the endosomes, type I RIPs are largely degraded and cannot exert their cytotoxic effects. Certain triterpene saponins are able to induce the endosomal escape of these type I RIPs, thus increasing their cytotoxicity. However, the molecular mechanism underlying the endosomal escape enhancement of type I RIPs by triterpene saponins has not been fully elucidated. In this report, we investigate the involvement of the basic amino acid residues of dianthin-30, a type I RIP isolated from the plant Dianthus caryophyllus L., in endosomal escape enhancement using alanine scanning. Therefore, we designed 19 alanine mutants of dianthin-30. Each mutant was combined with SO1861, a triterpene saponin isolated from the roots of Saponaria officinalis L., and subjected to a cytotoxicity screening in Neuro-2A cells. Cytotoxic screening revealed that dianthin-30 mutants with lysine substitutions did not impair the endosomal escape enhancement. There was one particular mutant dianthin, Arg24Ala, that exhibited significantly reduced synergistic cytotoxicity in three mammalian cell lines. However, this reduction was not based on an altered interaction with SO1861. It was, rather, due to the impaired endocytosis of dianthin Arg24Ala into the cells.

The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

Drymariamides A-J, Antiadipogenic Cyclopeptides Incorporating Noncanonical Amino Acids from Drymaria cordata.

Herein, we report an extensive phytochemical study on the whole plant of Drymaria cordata, which led to the isolation of ten new orbitides, named drymariamides A-J (1-10). Compounds 2, 3, and 5 incorporate rare residues of noncanonical amino acids of kynurenine (Kyn) or 3a-hydroxypyrroloindoline (HPI). Their structures with absolute configurations were elucidated by a combination of spectroscopic analysis, advanced Marfey's method, X-ray diffraction, and electronic circular dichroism analysis. Compounds 1-10 exhibited antiadipogenic effects in 3T3-L1 adipocytes, and the most potent compound 7 showed an EC50 value of 1.17 ± 0.19 μM.

Identification of Potential Therapeutics of Mentha Essential Oil Content as Antibacterial MDR Agents against AcrAB-TolC Multidrug Efflux Pump from Escherichia coli: An In Silico Exploration

Multidrug-resistant bacterial pathogens, such as E. coli, represent a major human health threat. Due to the critical need to overcome this dilemma, since the drug efflux pump has a vital function in the evolution of antimicrobial resistance in bacteria, we have investigated the potential of Mentha essential oil major constituents (1–19) as antimicrobial agents via their ability to inhibit pathogenic DNA gyrase and, in addition, their potential inhibition of the E. coli AcrB-TolC efflux pump, a potential target to inhibit MDR pathogens. The ligand docking approach was conducted to analyze the binding interactions of Mentha EO constituents with the target receptors. The obtained results proved their antimicrobial activity through the inhibition of DNA gyrase (1kzn) with binding affinity ΔG values between −4.94 and −6.49 kcal/mol. Moreover, Mentha EO constituents demonstrated their activity against MDR E. coli by their ability to inhibit AcrB-TolC (4dx7) with ΔG values ranging between −4.69 and −6.39 kcal/mol. The antimicrobial and MDR activity of Mentha EOs was supported via hydrogen bonding and hydrophobic interactions with the key amino acid residues at the binding site of the active pocket of the targeted receptors.

Structural and Computational Insights into Dynamics and Intermediate States of Orexin 2 Receptor Signaling.

Orexin 2 receptor (OX2R) is a G protein-coupled receptor (GPCR) whose activation is crucial to regulation of the sleep-wake cycle. Recently, inactive and active state structures were determined from X-ray crystallography and cryo-electron microscopy single particle analysis, and the activation mechanisms have been discussed based on these static data. GPCRs have multiscale intermediate states during activation, and insights into these dynamics and intermediate states may aid the precise control of intracellular signaling by ligands in drug discovery. Molecular dynamics (MD) simulations are used to investigate dynamics induced in response to thermal perturbations, such as structural fluctuations of main and side chains. In this study, we proposed collective motions of the TM domain during activation by performing 30 independent microsecond-scale MD simulations for various OX2R systems and applying relaxation mode analysis. The analysis results suggested that TM3 had a vertical structural movement relative to the membrane surface during activation. In addition, we extracted three characteristic amino acid residues on TM3, i.e., Q1343.32, V1423.40, and R1523.50, which exhibited large conformational fluctuations. We quantitatively evaluated the changes in their equilibrium during activation in relation to the movement of TM3. We also discuss the regulation of ligand binding recognition and intracellular signal selectivity by changes in the equilibrium of Q1343.32 and R1523.50, respectively, according to MD simulations and GPCR database. Additionally, the OX2R-Gi signaling complex is stabilized in the conformation resembling a non-canonical (NC) state, which was previously proposed as an intermediate state during activation of neurotensin 1 receptor. Insights into the dynamics and intermediate states during activation gained from this study may be useful for developing biased agonists for OX2R.

ARIP: A Tool for Precise Interatomic Contact Area and Volume Calculation in Proteins

The interplay patterns of amino acid residues are pivotal in determining the tertiary structure and flexibility of proteins, which in turn are intricately linked to their functionality and interactions with other molecules. Here, we introduce ARIP, a novel tool designed to identify contact residues within proteins. ARIP employs a modified version of the dr_sasa algorithm and an atomic overlap weighted algorithm to directly calculate the contact area and volume between atoms based on their van der Waals radius. It also allows for the selection of solvent radii, recognizing that not every atom in proteins can interact with water molecules. The solvent parameters were derived from the analysis of approximately 5000 protein and nucleic acid structures with water molecules determined using X-ray crystallography. One advantage of the modified algorithm is its capability to analyze multiple models within a single PDB file, making it suitable for molecular dynamic capture. The contact volume is symmetrically distributed between the interacting atoms, providing more informative results than contact area for the analysis of intra- and intermolecular interactions and the development of scoring functions. Furthermore, ARIP has been applied to four distinct cases: capturing key residue–residue contacts in NMR structures of P4HB, protein–drug binding of CYP17A1, protein–DNA binding of SPI1, and molecular dynamic simulations of BRD4.

Novel WFS1 Variants in Two Moroccan Families with Wolfram Syndrome

Novel <i>WFS1</i> Variants in Two Moroccan Families with Wolfram Syndrome

A review of lactate-lactylation in malignancy: its potential in immunotherapy

Lactic acid was formerly regarded as a byproduct of metabolism. However, extensive investigations into the intricacies of cancer development have revealed its significant contributions to tumor growth, migration, and invasion. Post-translational modifications involving lactate have been widely observed in histone and non-histone proteins, and these modifications play a crucial role in regulating gene expression by covalently attaching lactoyl groups to lysine residues in proteins. This discovery has greatly enhanced our comprehension of lactic acid’s involvement in disease pathogenesis. In this article, we provide a comprehensive review of the intricate relationship between lactate and tumor immunity, the occurrence of lactylation in malignant tumors, and the exploitation of targeted lactate-lactylation in tumor immunotherapy. Additionally, we discuss future research directions, aiming to offer novel insights that could inform the investigation, diagnosis, and treatment of related diseases.

Recent Advances in the Discovery of SIRT1/2 Inhibitors via Computational Methods: A Perspective

Sirtuins (SIRTs) are classified as class III histone deacetylases (HDACs), a family of enzymes that catalyze the removal of acetyl groups from the ε-N-acetyl lysine residues of histone proteins, thus counteracting the activity performed by histone acetyltransferares (HATs). Based on their involvement in different biological pathways, ranging from transcription to metabolism and genome stability, SIRT dysregulation was investigated in many diseases, such as cancer, neurodegenerative disorders, diabetes, and cardiovascular and autoimmune diseases. The elucidation of a consistent number of SIRT–ligand complexes helped to steer the identification of novel and more selective modulators. Due to the high diversity and quantity of the structural data thus far available, we reviewed some of the different ligands and structure-based methods that have recently been used to identify new promising SIRT1/2 modulators. The present review is structured into two sections: the first includes a comprehensive perspective of the successful computational approaches related to the discovery of SIRT1/2 inhibitors (SIRTIs); the second section deals with the most interesting SIRTIs that have recently appeared in the literature (from 2017). The data reported here are collected from different databases (SciFinder, Web of Science, Scopus, Google Scholar, and PubMed) using “SIRT”, “sirtuin”, and “sirtuin inhibitors” as keywords.

MRGPRX4 mediates phospho-drug-associated pruritus in a humanized mouse model.

The phosphate modification of drugs is a common chemical strategy to increase solubility and allow for parenteral administration. Unfortunately, phosphate modifications often elicit treatment- or dose-limiting pruritus through an unknown mechanism. Using unbiased high-throughput drug screens, we identified the Mas-related G protein-coupled receptor X4 (MRGPRX4), a primate-specific, sensory neuron receptor previously implicated in itch, as a potential target for phosphate-modified compounds. Using both Gq-mediated calcium mobilization and G protein-independent GPCR assays, we found that phosphate-modified compounds potently activate MRGPRX4. Furthermore, a humanized mouse model expressing MRGPRX4 in sensory neurons exhibited robust phosphomonoester prodrug-evoked itch. To characterize and confirm this interaction, we further determined the structure of MRGPRX4 in complex with a phosphate-modified drug through single-particle cryo-electron microscopy (cryo-EM) and identified critical amino acid residues responsible for the binding of the phosphate group. Together, these findings explain how phosphorylated drugs can elicit treatment-limiting itch and identify MRGPRX4 as a potential therapeutic target to suppress itch and to guide future drug design.

Revealing prognostic insights of programmed cell death (PCD)-associated genes in advanced non-small cell lung cancer.

The management of patients with advanced non-small cell lung cancer (NSCLC) presents significant challenges due to cancer cells' intricate and heterogeneous nature. Programmed cell death (PCD) pathways are crucial in diverse biological processes. Nevertheless, the prognostic significance of cell death in NSCLC remains incompletely understood. Our study aims to investigate the prognostic importance of PCD genes and their ability to precisely stratify and evaluate the survival outcomes of patients with advanced NSCLC. We employed Weighted Gene Co-expression Network Analysis (WGCNA), Least Absolute Shrinkage and Selection Operator (LASSO), univariate and multivariate Cox regression analyses for prognostic gene screening. Ultimately, we identified seven PCD-related genes to establish the PCD-related risk score for the advanced NSCLC model (PRAN), effectively stratifying overall survival (OS) in patients with advanced NSCLC. Multivariate Cox regression analysis revealed that the PRAN was the independent prognostic factor than clinical baseline factors. It was positively related to specific metabolic pathways, including hexosamine biosynthesis pathways, which play crucial roles in reprogramming cancer cell metabolism. Furthermore, drug prediction for different PRAN risk groups identified several sensitive drugs explicitly targeting the cell death pathway. Molecular docking analysis suggested the potential therapeutic efficacy of navitoclax in NSCLC, as it demonstrated strong binding with the amino acid residues of C-C motif chemokine ligand 14 (CCL14), carboxypeptidase A3 (CPA3), and C-X3-C motif chemokine receptor 1 (CX3CR1) proteins. The PRAN provides a robust personalized treatment and survival assessment tool in advanced NSCLC patients. Furthermore, identifying sensitive drugs for distinct PRAN risk groups holds promise for advancing targeted therapies in NSCLC.

Use of plasma induced modification of biomolecules (PLIMB) to evaluate hemin dissociation from fish and bovine methemoglobins

Hemin dissociation occurs much faster from fish methemoglobin (metHb) compared to mammalian metHb yet the mechanism remains poorly understood. This may involve enhanced solvent access to His(E7) of fish metHbs by a protonation mechanism. Plasma induced modification of biomolecules (PLIMB) produces free radicals that covalently modify solvent accessible residues of proteins, and so can provide insight regarding accessibility of hydronium ions to protonate His(E7). PLIMB-induced modifications to heme crevice sites of trout IV and bovine metHb were determined using tandem mass spectrometry after generating peptides with Trypsin/Lys-C. αHis(CE3) was more modified in trout attributable to the more dynamic nature of bovine αHis(CE3) from available crystal structures. Although His(E7) was not found to be more modified in trout, aspects of trout peptides containing His(E7) hampered modification determinations. An existing computational structure-based approach was also used to estimate protonation tendencies, suggesting His(E7) of metHbs with low hemin affinity are more protonatable.

Exploring the anti-inflammatory potential of the polyphenolic compounds in Moringa oleifera leaf: in silico molecular docking

ABSTRACT A recent study conducted an in silico model to investigate the potential of Moringa oleifera’s ethanolic leaf extract as a natural anti-inflammatory agent. The HPLC analysis identified 7 phenolic acids (benzoic acid 28%, syringic acid 23%, ellagic acid 19%, chlorogenic acid 12%, catechol 11%, gallic acid 5%, and caffeic acid 2%) and 5 flavonoids (naringenin 31%, kaempferol 30%, apigenin 14%, rutin 13%, and quercetin 12%) that are known to have anti-inflammatory activities in vitro tested on human interleukin-1β (IL-1β) and interleukin-6 (IL-6) in a previously prepared study (Hamdy, 2023) The Moringa polyphenol compounds were docked against IL-1β, IL-6, and the human interleukin-1 receptor (IL1RA) using Auto-Dock Vina. Caffeic acid, chlorogenic acid, and kaempferol interacted with amino acid residues in IL-1β by creating hydrogen bonds and hydrophobic interactions. The co-crystalized ligand attached to IL-1β displayed an affinity score of −8.96 kcal/mol. Caffeic acid, chlorogenic acid, quercetin, rutin, and syringic acid had unique interaction patterns with IL-6, forming hydrogen bonds and being hydrophobic. The interactions’ binding energies ranged from −5.97 to 5.26 kcal/mol. Moringa’s polyphenolic compounds also docked with IL1RA, where rutin and chlorogenic acid exhibited binding energies of −8.96 kcal/mol and −7.12 kcal/mol, respectively, and created hydrophobic interactions and hydrogen bonds. In conclusion, Moringa oleifera polyphenols can interact with IL-1β, IL-6, and IL-1RA to reduce inflammation, making it a potential treatment strategy for improving recovery.

Recent Developments of Hybrid Fluorescence Techniques: Advances in Amyloid Detection Methods.

Amyloid fibrils are formed from various pathological proteins. Monitoring their aggregation process is necessary for early detection and treatment. Among the available detection techniques, fluorescence is simple, intuitive, and convenient due to its sensitive and selective mode of detection. It has certain disadvantages like poor photothermal stability and detection state limitation. Research has focused on minimising the limitation by developing hybrid fluorescence techniques. This review focuses on the two ways fluorescence (intrinsic and extrinsic) has been used to monitor amyloid fibrils. In intrinsic/label free fluorescence: i) The fluorescence emission through aromatic amino acid residues like phenylalanine (F), tyrosine (Y) and tryptophan (W) is present in amyloidogenic peptides/protein sequence. And ii) The structural changes from alpha helix to cross-β-sheet structures during amyloid formation contribute to the fluorescence emission. The second method focuses on the use of extrinsic fluorophores to monitor amyloid fibrils i) organic dyes/small molecules, ii) fluorescent tagged proteins, iii) nanoparticles, iv) metal complexes and v) conjugated polymers. All these fluorophores havetheir own limitations. Developing them into hybrid fluorescence techniques and converting it into biosensors can contribute to early detection of disease.

GraphEGFR: Multi-task and transfer learning based on molecular graph attention mechanism and fingerprints improving inhibitor bioactivity prediction for EGFR family proteins on data scarcity.

The proteins within the human epidermal growth factor receptor (EGFR) family, members of the tyrosine kinase receptor family, play a pivotal role in the molecular mechanisms driving the development of various tumors. Tyrosine kinase inhibitors, key compounds in targeted therapy, encounter challenges in cancer treatment due to emerging drug resistance mutations. Consequently, machine learning has undergone significant evolution to address the challenges of cancer drug discovery related to EGFR family proteins. However, the application of deep learning in this area is hindered by inherent difficulties associated with small-scale data, particularly the risk of overfitting. Moreover, the design of a model architecture that facilitates learning through multi-task and transfer learning, coupled with appropriate molecular representation, poses substantial challenges. In this study, we introduce GraphEGFR, a deep learning regression model designed to enhance molecular representation and model architecture for predicting the bioactivity of inhibitors against both wild-type and mutant EGFR family proteins. GraphEGFR integrates a graph attention mechanism for molecular graphs with deep and convolutional neural networks for molecular fingerprints. We observed that GraphEGFR models employing multi-task and transfer learning strategies generally achieve predictive performance comparable to existing competitive methods. The integration of molecular graphs and fingerprints adeptly captures relationships between atoms and enables both global and local pattern recognition. We further validated potential multi-targeted inhibitors for wild-type and mutant HER1 kinases, exploring key amino acid residues through molecular dynamics simulations to understand molecular interactions. This predictive model offers a robust strategy that could significantly contribute to overcoming the challenges of developing deep learning models for drug discovery with limited data and exploring new frontiers in multi-targeted kinase drug discovery for EGFR family proteins.

MOLECULAR DOCKING DYNAMICS OF SELECTED BENZYLIDENE AMINO PHENYL ACETAMIDES AS TMK INHIBITORS USING HIGH THROUGHPUT VIRTUAL SCREENING (HTVS)

Objective: Thymidylate kinase (TMK) plays a crucial role in bacterial DNA synthesis by catalyzing the phosphorylation of deoxythymidine monophosphate (dTMP) to form deoxythymidine diphosphate (dTDP). Consequently, this enzyme emerges as a promising target for developing novel antibacterial drugs. However, no antibiotics were reported for this target, especially active against Staphylococcus aureus thymidylate kinase. Methods: Benzylidene acetamide-based ligands were examined for their potency using the in silico method. These novel ligand structures were built using ChemDraw software. The protein was retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) website. The molecular docking and binding free energy calculation by prime Molecular Mechanics in Generalized Bond Surface Area (MM-GBSA) was performed for selected ligands. A 100 ns molecular dynamic simulation was also performed to assess the stability of the potential ligand as TMK inhibitors. Results: All ten molecules have shown good glide scores and hydrophobic and hydrogen hydrophobic hydrogen bonding interactions with Arg48, Arg36, and π-π stacking Phe66 in the TMK enzyme (PDB: 4HLC). Among them, N-(2-ethylphenyl)-2-(4-((4-nitrobenzylidene) amino) phenoxy) acetamide molecule had high XP-docking scores of-3.27 kcal/mol based on extra-precision data. Prime Molecular Mechanics in Generalized Bond Surface Area study (MM-GBSA) studies also showed promising binding affinities that are ΔBind (-65.80), ΔLipo (-28.55), and ΔVdW (-55.10). Phe66 amino acid residue maintained continuous connections with the ligand during MD simulation. This ligand showed promising binding affinity with the SaTMK target. Conclusion: The N-(2-ethylphenyl)-2-(4-((4-nitrobenzylidene) amino) phenoxy) acetamide ligand at the position of the benzene ring displayed nitrogen and oxygen group, thus indicating good potential activity as the inhibitor of TMK to treat antibacterial agents.