Active Site Amino Acid Residues Research Articles

Pyrimidine appended heterocycles: synthesis, characterization, and biological evaluation

A new class of pyrimidine-based heterocycles has been successfully synthesized via a simple and efficient way starting from 2-keto-thiophene (1). The α,β-unsaturated ketone (chalcone) (3) was first synthesized by treating the ketone (1) with 4-methoxy benzaldehyde (2), followed by treating with amino formamidine hydrochloride to afford the intermediate pyrimidine moiety (4) which was converted to amide functionalized pyrimidines (5a–e), pyrimidine-2-imines (6a–e) and thiophene tethered pyrimidine-imidazoles (7a–e). The structures assigned to the new compounds are confirmed by IR, NMR, and MS studies. The newly synthesized compounds were subjected to a molecular docking study with the protein of Staphylococcus aureus (PDB ID: 1JIJ), for antioxidant and antibacterial activity. The compounds 5a, 6a, 6b, and 7d were found to show hydrogen bond interaction with active site amino acid residues with the binding energy of −8,08, −8.39, −8.64, and −8.02 kJ/mol, respectively, and may act as potential inhibitors of bacterial tyrosyl-tRNA synthetases. The biological study envisioned that the synthesized compound 7b has shown potent antioxidant properties with 74.07% scavenging activity at 400 µg/mL and compound 6b was found to exhibit excellent antibacterial activity with 16, 20, 21, and 22 mm zone of inhibition against S. aureus, Escherichia coli, Salmonella paratyphi-A and Bacillus subtilis bacterial strains, respectively.

Crucial Structural Understanding for Selective HDAC8 Inhibition: Common Pharmacophores, Molecular Docking, Molecular Dynamics, and Zinc Binder Analysis of selective HDAC8 inhibitors.

Overexpression of HDAC8 was observed in various cancers and inhibition of HDAC8 has emerged as a promising therapeutic approach in recent decades. This review aims to facilitate the discovery of novel selective HDAC8 inhibitors by analyzing the structural scaffolds of 66 known selective HDAC8 inhibitors, along with their IC50 values against HDAC8 and other HDACs. The inhibitors were clustered based on structural symmetry, and common pharmacophores for each cluster were identified using Phase. Molecular docking with all HDACs was performed to determine binding affinity and crucial interacting residues for HDAC8 inhibition. Representative inhibitors from each cluster were subjected to molecular dynamics simulation to analyze RMSD, RMSF, active site amino acid residues, and crucial interacting residues responsible for HDAC8 inhibition. The study reviewed the active site amino acid information, active site cavities of all HDACs, and the basic structure of Zn2+ binding groups. Common pharmacophores identified included AADHR_1, AADDR_1, ADDR_1, ADHHR_1, and AADRR_1. Molecular docking analysis revealed crucial interacting residues: HIS- 142, GLY-151, HIS-143, PHE-152, PHE-20 in the main pocket, and ARG-37, TYR-100, TYR-111, TYR-306 in the secondary pocket. The RMSD of protein and RMSF of active site amino acid residues for stable protein-ligand complexes were less than 2.4 Å and 1.0 Å, respectively, as identified from MD trajectories. The range of Molecular Mechanics Generalized Born Surface Area (MMGBSA) ΔG predicted from MD trajectories was between -15.8379 Å and -61.5017 Å kcal/mol. These findings may expedite the rapid discovery of selective HDAC8 inhibitors subject to experimental evaluation.

Arg40 is critical for stability and activity of Adenylosuccinate lyase; a purine salvage enzyme from Leishmania donovani

Purine salvage enzymes have been of significant interest in anti-Leishmanial drug development due to the parasite's critical dependence on this pathway for the supply of nucleotides in the absence of a de novo purine synthesis pathway. Adenylosuccinate lyase (ADSL) one of the key enzymes in this pathway is a homo-tetramer, where the active site is formed by residues from three distinct subunits. Analysis of the subunit interfaces of LdADSL, revealed a conserved Arg40 forming critical inter-subunit interactions and also involved in substrate binding. We hypothesized that mutating this residue can affect both the structural stability and activity of the enzyme. In our study, we used biochemical, biophysical, and computational simulation approaches to understand the structural and functional role of Arg40 in LdADSL. We have replaced Arg40 with an Ala and Glu using site directed mutagenesis. The mutant enzymes were similar to wild-type enzyme in secondary structure and subunit association. Thermal shift assays indicated that the mutations affected the protein stability. Both mutants showed decreased specific activities in both forward and reverse directions with significantly weakened affinities towards succinyl-adenosine monophosphate (SAMP). The mutations resulted in changes in C3 loop conformation and D3 domain rotation. Consequently, the orientation of the active site amino acid residues changed resulting in compromised activity and stability. Studies so far have majorly focused on the ADSL active site for designing drugs against it. Our work indicates that an alternative inhibitory mechanism for the enzyme can be designed by targeting the inter-subunit interface.

Unveiling G-protein coupled receptor kinase-5 inhibitors for chronic degenerative diseases: Multilayered prioritization employing explainable machine learning-driven multi-class QSAR, ligand-based pharmacophore and free energy-inspired molecular simulation

GRK5 holds a pivotal role in cellular signaling pathways, with its overexpression in cardiomyocytes, neuronal cells, and tumor cells strongly associated with various chronic degenerative diseases, which highlights the urgent need for potential inhibitors. In this study, multiclass classification-based QSAR models were developed using diverse machine learning algorithms. These models were built from curated compounds with experimentally derived GRK5 inhibitory activity. Additionally, a pharmacophore model was constructed using active compounds from the dataset. Among the models, the SVM-based approach proved most effective and was initially used to screen DrugBank compounds within the applicability domain. Compounds showing significant GRK5 inhibitory potential underwent evaluation for key pharmacophoric features. Prospective compounds were subjected to molecular docking to assess binding affinity towards GRK5's key active site amino acid residues. Stability at the binding site was analyzed through 200 ns molecular dynamics simulations. MM-GBSA analysis quantified individual free energy components contributing to the total binding energy with respect to binding site residues. Metadynamics analysis, including PCA, FEL, and PDF, provided crucial insights into conformational changes of both apo and holo forms of GRK5 at defined energy states. The study identifies DB02844 (S-Adenosyl-1,8-Diamino-3-Thiooctane) and DB13155 (Esculin) as promising GRK5 inhibitors, warranting further in vitro and in vivo validation studies.

Structural and Dynamic Features of the Recognition of 8-oxoguanosine Paired with an 8-oxoG-clamp by Human 8-oxoguanine-DNA Glycosylase.

8-oxoguanine (oxoG) is formed in DNA by the action of reactive oxygen species. As a highly mutagenic and the most common oxidative DNA lesion, it is an important marker of oxidative stress. Human 8-oxoguanine-DNA glycosylase (OGG1) is responsible for its prompt removal in human cells. OGG1 is a bifunctional DNA glycosylase with N-glycosylase and AP lyase activities. Aspects of the detailed mechanism underlying the recognition of 8-oxoguanine among numerous intact bases and its subsequent interaction with the enzyme's active site amino acid residues are still debated. The main objective of our work was to determine the effect (structural and thermodynamic) of introducing an oxoG-clamp in model DNA substrates on the process of 8-oxoG excision by OGG1. Towards that end, we used DNA duplexes modeling OGG1-specific lesions: 8-oxoguanine or an apurinic/apyrimidinic site with either cytidine or the oxoG-clamp in the complementary strand opposite to the lesion. It was revealed that there was neither hydrolysis of the N-glycosidic bond at oxoG nor cleavage of the sugar-phosphate backbone during the reaction between OGG1 and oxoG-clamp-containing duplexes. Possible structural reasons for the absence of OGG1 enzymatic activity were studied via the stopped-flow kinetic approach and molecular dynamics simulations. The base opposite the damage was found to have a critical effect on the formation of the enzyme-substrate complex and the initiation of DNA cleavage. The oxoG-clamp residue prevented the eversion of the oxoG base into the OGG1 active site pocket and impeded the correct convergence of the apurinic/apyrimidinic site of DNA and the attacking nucleophilic group of the enzyme. An obtained three-dimensional model of the OGG1 complex with DNA containing the oxoG-clamp, together with kinetic data, allowed us to clarify the role of the contact of amino acid residues with DNA in the formation of (and rearrangements in) the enzyme-substrate complex.

High-throughput selection of (new) enzymes: phage display-mediated isolation of alkyl halide hydrolases from a library of active-site mutated epoxide hydrolases.

Epoxide hydrolase StEH1, from potato, is similar in overall structural fold and catalytic mechanism to haloalkane dehalogenase DhlA from Xanthobacter autotrophicus. StEH1 displays low (promiscuous) hydrolytic activity with (2-chloro)- and (2-bromo)ethanebenzene producing 2-phenylethanol. To investigate possibilities to amplify these very low dehalogenase activities, StEH1 was subjected to targeted randomized mutagenesis at five active-site amino acid residues and the resulting protein library was challenged for reactivity towards a bait chloride substrate. Enzymes catalyzing the first half-reaction of a hydrolytic cycle were isolated following monovalent phage display of the mutated proteins. Several StEH1 derived enzymes were identified with enhanced dehalogenase activities.

Molecular docking assessment of the tocolytic potential of phytoconstituents of five medicinal plants used against preterm labour

Introduction: Preterm labour is currently being treated with a number of medications with untoward side effects, but many medicinal plants have also been found useful. This study aims to assess the tocolytic potentials of the phytoconstituents of Barteria fistulosa, Ficus capensis, Ficus exasperate, Newbouldia laevis and Zingiber officinale. Methods: Phytoconstituents present in these plants were obtained from literature sources, their 3D SDF structures were obtained from PubChem; the protein Beta-2 adrenergic receptor (7DHI) was processed using Chimera and molecular docking was done using PyRx software. Post-docking analysis was done using Bio-discovery Studio 2.0 and ADMET profiling was done using the Swiss ADME web server and ProTox-II virtual lab. Results: A binding affinity value of less than -7 kcal/mol was found for nine (9) phytoconstituents in Zingiber officinale, fourteen (14) phytoconstituents in Ficus capensis, three (3) phytoconstituents in Ficus exasperata, one (1) phytoconstituent in Barteria fistulosa and forty-four (44) phytoconstituents in Newbouldia laevis. Following post-docking analysis and ADMET profiling of specific ligands from the plants, Kaempferol, Chrysoeriol and Lapachol - all present in Newbouldia laevis - were identified as putative drug molecules based on their higher binding affinity and hydrogen bond interaction with the target proteins' active site amino acid residues. Conclusion: The tocolytic potential of Zingiber officinale, Ficus capensis, Barteria fistulosa and Newbouldia laevis as a medicinal plant for the treatment of preterm labour is validated. Kaempferol, Chrysoeriol and Lapachol, phytoconstituents in Newbouldia laevis possess the potential as a source of new drugs for the treatment of preterm labour. Keywords: Preterm labour, tocolysis, molecular docking, drug design

Single crystal X-ray, DFT, molecular dynamic simulations, and biological evaluation of 3-OH pyrrolidine derivative VA10 from alkaloid vasicine for BACE1 inhibition

Alzheimer's is a progressive neurodegenerative disease with amyloid-beta peptide deposition that impairs memory and cognitive decline. Due to lack of effective treatment for Alzheimer's disease (AD), there is an interest in finding novel compounds to treat AD. The alkaloid vasicine derivative VA10 inhibited cholinesterases and amyloid beta aggregation in Alzheimer induced rats and showed multi-target activity in treating AD. Therefore, the present study uses single-crystal X-ray and DFT studies to evaluate the properties of VA10′s physicochemical properties, reactivity, and stability. Further, the scaffold VA10 was assessed through in silico, in vitro BACE1 inhibition, and in vivo CNS toxicity experiments. The VA10 Hirshfeld surface analysis and fingerprint plots were determined to understand intra and intermolecular interactions. Furthermore, HOMO-LUMO, UV, and IR studies investigated the electrical and vibrational characteristics. The molecular docking and molecular dynamics simulation studies revealed that compound VA10 formed key interactions with active site amino acid residues and well resided at the BACE1 active site. The VA10 was found to be a potent BACE1 inhibitor and showed no sign of toxicity in rats up to 50 mg/kg. The study identifies VA10, a vasicine derivative, as a possible BACE1 inhibitor in treating Alzheimer's disease. Furthermore, comparing experimental and theoretical results may be beneficial in evaluating VA10′s molecular interactions and reactivity.

Individual Contributions of Amido Acid Residues Tyr122, Ile168, and Asp173 to the Activity and Substrate Specificity of Human DNA Dioxygenase ABH2.

Human Fe(II)/α-ketoglutarate-dependent dioxygenase ABH2 plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases, e.g., N1-methyladenine (m1A), N3-methylcytosine (m3C), and some etheno derivatives. Moreover, ABH2 is capable of a less efficient oxidation of an epigenetic DNA mark called 5-methylcytosine (m5C), which typically is a specific target of DNA dioxygenases from the TET family. In this study, to elucidate the mechanism of the substrate specificity of ABH2, we investigated the role of several active-site amino acid residues. Functional mapping of the lesion-binding pocket was performed through the analysis of the functions of Tyr122, Ile168, and Asp173 in the damaged base recognition mechanism. Interactions of wild-type ABH2, or its mutants Y122A, I168A, or D173A, with damaged DNA containing the methylated base m1A or m3C or the epigenetic marker m5C were analyzed by molecular dynamics simulations and kinetic assays. Comparative analysis of the enzymes revealed an effect of the substitutions on DNA binding and on catalytic activity. Obtained data clearly demonstrate the effect of the tested amino acid residues on the catalytic activity of the enzymes rather than the DNA-binding ability. Taken together, these data shed light on the molecular and kinetic consequences of the substitution of active-site residues for the mechanism of the substrate recognition.

Mechanism and Dynamics of Photodecarboxylation Catalyzed by Lactate Monooxygenase.

Photoenzymes are a rare class of biocatalysts that use light to facilitate chemical reactions. Many of these catalysts utilize a flavin cofactor to absorb light, suggesting that other flavoproteins might have latent photochemical functions. Lactate monooxygenase is a flavin-dependent oxidoreductase previously reported to mediate the photodecarboxylation of carboxylates to afford alkylated flavin adducts. While this reaction holds a potential synthetic value, the mechanism and synthetic utility of this process are unknown. Here, we combine femtosecond spectroscopy, site-directed mutagenesis, and a hybrid quantum-classical computational approach to reveal the active site photochemistry and the role the active site amino acid residues play in facilitating this decarboxylation. Light-induced electron transfer from histidine to flavin was revealed, which has not been reported in other proteins. These mechanistic insights enable the development of catalytic oxidative photodecarboxylation of mandelic acid to produce benzaldehyde, a previously unknown reaction for photoenzymes. Our findings suggest that a much wider range of enzymes have the potential for photoenzymatic catalysis than has been realized to date.

Boronic modified quercetin molecules: Synthesis and biological investigations with molecular docking verification

Quercetin is an essential compound belonging to the flavanol group, which is a derivative of bioflavonoids found in fruits and vegetables. Quercetin has various pharmacological properties such as anticancer, antioxidant, anti-inflammatory, antiviral, antihistamine, and antitumor. Based on these important properties, the quercetin compound was modified with various boronic acids and new types of quercetin-based boronic (QB) compounds were synthesized. In the current study, we have synthesized quercetin-based boronic compounds to obtain more effective molecules by doping the biological activity properties of quercetin naturally in its structure. In this purpose, 6‑methoxy naphthalene boronic acid, 1,4-phenyl diboronic acid, 4‑methoxy phenylboronic acid, 6‑methoxy-3-pyridinylboronic acid, 3-formyl-4-methoxyphenyl boronic acid compounds were used. The synthesized compounds were structurally enlightened by mass, 13C NMR, 1H NMR, and FTIR spectroscopy. Then, the antioxidant, anticholinesterase, antiurease, antityrosinase and toxic-cytotoxic effects of these molecules were tested. QB compounds synthesized and examined in vitro in the study were also analyzed by molecular docking. Molecular docking, one of the structure-based drug design methods, has been applied to support experimental data. It was determined that QB compounds showed more significant efficacy than the reference substance employed in antioxidant and enzyme activity studies. As a result of molecular docking analysis, the binding energy parameter values and active binding site amino acid residues of the compounds were determined. Obtained novel boronic compounds might be associated as drug candidate due to consistent results both in experimental and molecular docking results.

In vitro and in silico inhibitory validation of Tapinanthus cordifolius leaf extract on alpha-glucosidase in the management of type 2 diabetes

The anti-diabetic properties of medicinal plants are becoming more widely recognized. To identify potential anti-diabetic agents for diabetes drug discovery, the current study used in vitro and in silico approaches to assess the alpha glucosidase inhibitory activities of Tapinanthus cordifolius (TC) leaf extracts and its bioactive components respectively. In vitro alpha glucosidase inhibitory assay was carried out on TC extract and fractions at various concentrations (50–1600 µg/mL), and the compounds with alpha glucosidase inhibitory potentials were identified using molecular docking, pharmacophore modelling, and molecular dynamics simulation. The crude extract exhibited the highest activity with an IC50 value of 248 μg/mL. Out of the 42 phytocompounds of the extract, α-Tocopherol-β-d-mannoside gave the lowest binding energy of −6.20 Kcal/mol followed by, 5-Ergosterol (−5.46 kcal/mol), Acetosyringone (−4.76 kcal/mol), and Benzaldehyde, 4-(Ethylthio)-2,5-Dimethoxy-(−4.67 kcal/mol). The selected compounds interacted with critical active site amino acid residues of alpha-glucosidase, just like the reference ligand. Molecular dynamics simulation revealed the formation of a stable complex between α-glucosidase and α-Tocopherol-β-d-mannoside, with ASP 564 sustaining two hydrogen bond connections for 99.9 and 75.0% of the simulation duration, respectively. Therefore, the selected TC compounds, especially α-Tocopherol-β-d-mannoside might be explored for future research and development as diabetic medicines. Communicated by Ramaswamy H. Sarma

Identification of 1,3,6,8-Tetrahydroxynaphthalene Synthase (ThnA) from Nocardia sp. CS682.

Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds that possess crucial biological functions and significant pharmaceutical activities. Based on our sequence analysis, we have identified a putative type III polyketide synthase from Nocardia sp. CS682 was named as ThnA. The role of ThnA, in Nocardia sp. CS682 during the biosynthesis of 1,3,6,8 tetrahydroxynaphthalene (THN), which is the key intermediate of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene (IBR-3) was characterized. ThnA utilized five molecules of malonyl-CoA as a starter substrate to generate the polyketide 1,3,6,8-tetrahydroxynaphthalene, which could spontaneously be oxidized to the red flaviolin compound 2,5,7-trihydroxy-1,4-naphthoquinone. The amino acid sequence alignment of ThnA revealed similarities with a previously identified type III PKS and identified Cys138, Phe188, His270, and Asn303 as four highly conserved active site amino acid residues, as found in other known polyketide synthases. In this study, we report the heterologous expression of the type III polyketide synthase thnA in S. lividans TK24 and the identification of THN production in a mutant strain. We also compared the transcription level of thnA in S. lividans TK24 and S. lividans pIBR25-thnA and found that thnA was only transcribed in the mutant.

Structural insights into the substrate recognition of serine palmitoyltransferase from Sphingobacterium multivorum

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis, which catalyzes the pyridoxal-5'-phosphate-dependent decarboxylative condensation reaction of L-serine (L-Ser) and palmitoyl-CoA (PalCoA) to form 3-ketodihydrosphingosine called long chain base (LCB). SPT is also able to metabolize L-alanine (L-Ala) and glycine (Gly), albeit with much lower efficiency. Human SPT is a membrane-bound large protein complex containing SPTLC1/SPTLC2 heterodimer as the core subunits, and it is known that mutations of the SPTLC1/SPTLC2 genes increase the formation of deoxy-type of LCBs derived from L-Ala and Gly to cause some neurodegenerative diseases. In order to study the substrate recognition of SPT, we examined the reactivity of Sphingobacterium multivorum SPT on various amino acids in the presence of PalCoA. The S. multivorum SPT could convert not only L-Ala and Gly but also L-homoserine, in addition to L-Ser, into the corresponding LCBs. Furthermore, we obtained high-quality crystals of the ligand-free form and the binary complexes with a series of amino acids, including a nonproductive amino acid, L-threonine, and determined the structures at 1.40-1.55 Å resolutions. The S. multivorum SPT accommodated various amino acid substrates through subtle rearrangements of the active-site amino acid residues and water molecules. It was also suggested that non-active-site residues mutated in the human SPT genes might indirectly influence the substrate specificity by affecting the hydrogen-bonding networks involving the bound substrate, water molecules, and amino acid residues in the active site of this enzyme. Collectively, our results highlight SPT structural features affecting substrate specificity for this stage of sphingolipid biosynthesis.

Cheminformatics identification of phenolics as modulators of penicillin-binding protein-3 of Pseudomonas aeruginosa towards interventive antibacterial therapy

Antibacterial resistance to β-lactams in microorganisms has been attributed majorly to alterations in penicillin-binding proteins (PBPs) coupled with β-lactams’ inactivation by β-lactamase. Consequently, the identification of a novel class of therapeutics with improved modulatory action on the PBPs is imperative and plant secondary metabolites, including phenolics, have found relevance in this regard. For the first time in this study, the over 10,000 phenolics currently known were computationally evaluated against PBP3 of Pseudomonas aeruginosa, a superbug implicated in several nosocomial infections. In doing this, a library of phenolics with an affinity for PBP3 of P. aeruginosa was screened using structure-activity relationship-based pharmacophore and molecular docking approaches. Subsequent thermodynamic screening of the top five phenolics with higher docking scores, more drug-likeness attributes, and feasible synthetic accessibility was achieved through a 120 ns molecular dynamic (MD) simulation. Four of the top five hits had higher binding free energy than cefotaxime (−18.72 kcal/mol), with catechin-3-rhamside having the highest affinity (−28.99 kcal/mol). All the hits were stable at the active site of the PBP3, with catechin-3-rhamside being the most stable (2.14 Å), and established important interactions with Ser294, implicated in the catalytic activity of PBP3. Also, PBP3 became more compact with less fluctuation of the active site amino acid residues following the binding of the hits. These observations are indicative of the potential of the test compounds as PBP3 inhibitors, with catechin-3-rhamside being the most prominent of the compounds that could be further improved for enhanced druggability against PBP3 in vitro and in vivo. Communicated by Ramaswamy H. Sarma

Computational assessment of chemicals from Morinda citrifolia as potential inhibitors of B-Raf kinase in hepatocellular carcinoma treatment

Hepatocellular carcinoma (HCC) is a tumour pathology that lacks specific treatment and is predominantly resistant to chemotherapy. The inhibitory activity of Morinda citrifolia, an evergreen tree commonly called Noni, against various carcinomas especially HCC is widely acclaimed. This study was to assess the phytochemical constituents of the plant for inhibitory activity against B-Raf kinase (3C4C) in order to design drugs for HCC treatment. Molecular docking, pharmacophore modelling, induced-fit docking, molecular dynamics (MD) simulations and ADMET predictions were the computational techniques employed in this study to detect potential inhibitors of B-Raf kinase from 135 compounds of Morinda citrifolia. Soranjidiol, Thiamine, Lucidin, 2-Methyl-1,3,5-Trihydroxyanthraquinone and Rubiadin were the five top-scoring compounds ranging from −8.39 to −8.22 kcal/mol, however, the standard ligand, PLX4720, scored −11.26 kcal/mol. The five compounds, like PLX4720 demonstrated hydrogen bond interactions with active site amino acid residues such as GLN 530, CYS 532 and ASP 594. The main energy contributor to the interactions between the compounds and B-Raf kinase were pi-stacking, hydrogen bond, van der Waals and covalent energy. Better docking scores obtained in the induced-fit docking further validates the inhibitory potential of the Soranjidiol against the flexible protein. In MD simulations, Soranjidiol revealed good stability in the active site of the protein since significant conformational changes were not evident. These five compounds, unlike the standard compound, demonstrated adequate druglike properties and good safety profiles. Therefore, further studies should be undertaken so as to develop them into drugs against HCC. Communicated by Ramaswamy H. Sarma

Structure-based pharmacophore modeling, virtual screening, and molecular dynamics simulation studies for identification of Plasmodium falciparum 5-aminolevulinate synthase inhibitors.

Plasmodium falciparum (Pf) 5-aminolevulinic acid synthase (5-ALAS) is an essential enzyme with high selectivity during liver stage development, signifying its potential as a prophylactic antimalarial drug target. The aim of this study was to identify important potential lead compounds which can serve as inhibitors of Pf 5-ALAS using pharmacophore modeling, virtual screening, qualitative structural assessment, in silico ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) evaluation and molecular dynamics simulation. The best model of the tertiary structure of Pf 5-ALAS was obtained using MolProbity, while the following databases were explored for the pharmacophore-based virtual screening: CHEMBL, ChemDiv, ChemSpace, MCULE, MCULE-ULTIMATE, MolPort, NCI Open Chemical Repository, LabNetwork and ZINC databases. 2,621 compounds were screened against the modeled Pf 5-ALAS using AutoDock vina. The post-screening analysis was carried out using Discovery Studio while molecular dynamics simulation was performed on the best hits using NAMD-VMD and Galaxy Europe platform. Compound CSMS00081585868 was observed as the best hit with a binding affinity of -9.9 kcal/mol and predicted Ki of 52.10 nM, engaging in seven hydrogen bonds with the target's active site amino acid residues. The in silico ADMET prediction showed that all ten best hits possessed relatively good pharmacokinetic properties. The qualitative structural assessment of the best hit, CSMS00081585868, revealed that the presence of two pyridine scaffolds bearing hydroxy and fluorine groups linked by a pyrrolidine scaffold contributed significantly to its ability to have a strong binding affinity with the receptor. The best hit also showed stability in the active site of Pf 5-ALAS as confirmed from the RMSD obtained during the MD simulation.

Bioprospection of Selected Plant Secondary Metabolites as Modulators of the Proteolytic Activity of Plasmodium falciparum Plasmepsin V.

Malaria is a devastating disease, and its management is only achieved through chemotherapy. However, resistance to available medication is still a challenge; therefore, there is an urgent need for the discovery and development of therapeutics with a novel mechanism of action to counter the resistance scourge consistent with the currently available antimalarials. Recently, plasmepsin V was validated as a therapeutic target for the treatment of malaria. The pepsin-like aspartic protease anchored in the endoplasmic reticulum is responsible for the trafficking of parasite-derived proteins to the erythrocytic surface of the host cells. In this study, a small library of compounds was preliminarily screened in vitro to identify novel modulators of Plasmodium falciparum plasmepsin V (PfPMV). The results obtained revealed kaempferol, quercetin, and shikonin as possible PfPMV inhibitors, and these compounds were subsequently probed for their inhibitory potentials using in vitro and in silico methods. Kaempferol and shikonin noncompetitively and competitively inhibited the specific activity of PfPMV in vitro with IC50 values of 22.4 and 43.34 μM, respectively, relative to 62.6 μM obtained for pepstatin, a known aspartic protease inhibitor. Further insight into the structure-activity relationship of the compounds through a 100 ns molecular dynamic (MD) simulation showed that all the test compounds had a significant affinity for PfPMV, with quercetin (-36.56 kcal/mol) being the most prominent metabolite displaying comparable activity to pepstatin (-35.72 kcal/mol). This observation was further supported by the compactness and flexibility of the resulting complexes where the compounds do not compromise the structural integrity of PfPMV but rather stabilized and interacted with the active site amino acid residues critical to PfPMV modulation. Considering the findings in this study, quercetin, kaempferol, and shikonin could be proposed as novel aspartic protease inhibitors worthy of further investigation in the treatment of malaria.

In vitro and in-silico inhibitory validation of Tapinanthus cordifolius leaf extract on alpha-amylase in the management of type 2 diabetes

Tapinanthus cordifolius an African mistletoe is an important medicinal plant that has been shown to lower postprandial hyperglycemia. It has been proposed that mistletoe's hypoglycemic effects are related to its ability to lower blood glucose levels and that the anti-diabetic activity of some of these parasitic plants might be due to their ability to block the action of alpha-amylase. To identify prospective alpha-amylase inhibitors for anti-diabetic drug discovery, the current study used in vitro experiments and computational methods such as molecular docking, pharmacophore modelling, and ADMET profiling to evaluate the alpha-amylase inhibitory ability of Tapinanthus cordifolius leaf extracts and its bioactive components. The crude extract of the plant showed the highest inhibiting activity with an IC50 value of 26.88 μg/ml. Gas chromatography-mass spectroscopic analysis of the extract gave 43 phytochemicals, including sesquiterpenes, diterpenes, triterpenes and their derivatives, phytosterols, and tocopherols. The molecular docking analysis of these compounds with alpha-amylase identified benzaldehyde, 4-(Ethylthio)-2,5-dimethoxy, alpha-Tocopherol-Beta-D-Mannoside, 5-ergosterol, 3,4,5-trimethoxybenzoic acid, and acetosyringone as the five top-scoring compounds. Their binding energies which ranged from −4.944 to −4.365 kcal/mol, were close to that of the reference compound, which was −5.67 kcal/mol. Like the reference ligand, these compounds interacted with crucial active site amino acid residues of alpha-amylase. They also possess favorable drug-like properties as well as a low-risk profile. Hence, these compounds could be subjected to lead optimization and experimental studies for further development into novel drugs for managing type 2 diabetes mellitus.

MSALigMap-A Tool for Mapping Active-Site Amino Acids in PDB Structures onto Known and Novel Unannotated Homologous Sequences with Similar Function.

MSALigMap (Multiple Sequence Alignment Ligand Mapping) is a tool for mapping active-site amino-acid residues that bind selected ligands on to target protein sequences of interest. Users can also provide novel sequences (unavailable in public databases) for analysis. MSALigMap is written in Python. There are several tools and servers available for comparing and mapping active-site amino-acid residues among protein structures. However, there has not previously been a tool for mapping ligand binding amino-acid residues onto protein sequences of interest. Using MSALigMap, users can compare multiple protein sequences, such as those from different organisms or clinical strains, with sequences of proteins with crystal structures in PDB that are bound with the ligand/drug and DNA of interest. This allows users to easily map the binding residues and to predict the consequences of different mutations observed in the binding site. The MSALigMap server can be accessed at https://albiorix.bioenv.gu.se/MSALigMap/HomePage.py.