A Computational Study of Green Tea Extracts and their Derivatives as Potential Inhibitors for Squalene Monooxygenase.

BACKGROUND: According to the World Health Organisation, cardiovascular complications have been recognized as the leading course of death between 2000 and 2019. Cardiovascular complications are caused by excess LDL cholesterol in the body or arteries that can build up to form a plaque. There are drugs currently in clinical use called statins that target HMGCoA reductase. However, these drugs result in several side effects. This work investigated using computational approaches to lower cholesterol by investigating green tea extracts as an inhibitors for squalene monooxygenase (the second-rate-controlling step in cholesterol synthesis). METHODS: Pharmacophore modeling was done to identify possible pharmacophoric sites based on the pIC50 values. The best hypothesis generated by pharmacophore modeling was further validated by atom-based 3D QSAR, where 70% of the data set was treated as the training set. Prior molecular docking ADMET studies were done to investigate the physiochemical properties of these molecules. Glide docking was performed, followed by molecular dynamics to evaluate the protein conformational changes. RESULTS: Pharmacophore results suggest that the best molecules to interact with the biological target should have at least one hydrogen acceptor (A5), two hydrogen donors (D9 and D10), and two benzene rings (R14 and R15) for green tea polyphenols and theasinensin A. ADMET result shows that all molecules in this class have low oral adsorption. Molecular docking results showed that some green tea polyphenols have good binding affinities, with most of these structures having a docking score of less than -10 kcal/mol. Molecular dynamics further illustrated that the best-docked ligands perfectly stay within the active site over a 100 ns simulation. CONCLUSION: The results obtained from this study suggest that green tea polyphenols have the potential for inhibition of squalene monooxygenase, except for theasinensin A.

Inhibitory effects of selected cannabinoids against dipeptidyl peptidase IV, an enzyme linked to type 2 diabetes.

Ethnopharmacological relevance: In recent times the decriminalisation of cannabis globally has increased its use as an alternative medication. Where it has been used in modern medicinal practises since the 1800s, there is limited scientific investigation to understand the biological activities of this plant. Aim of the study: Dipeptidyl peptidase IV (DPP-IV) plays a key role in regulating glucose homeostasis, and inhibition of this enzyme has been used as a therapeutic approach to treat type 2 diabetes. However, some of the synthetic inhibitors for this enzyme available on the market may cause undesirable side effects. Therefore, it is important to identify new inhibitors of DPP-IV and to understand their interaction with this enzyme. Methods: In this study, four cannabinoids (cannabidiol, cannabigerol, cannabinol and Δ9-tetrahydrocannabinol) were evaluated for their inhibitory effects against recombinant human DPP-IV and their potential inhibition mechanism was explored using both in vitro and in silico approaches. Results: All four cannabinoids resulted in a dose-dependent response with IC50 values of between 4.0 and 6.9 µg/mL. Kinetic analysis revealed a mixed mode of inhibition. CD spectra indicated that binding of cannabinoids results in structural and conformational changes in the secondary structure of the enzyme. These findings were supported by molecular docking studies which revealed best docking scores at both active and allosteric sites for all tested inhibitors. Furthermore, molecular dynamics simulations showed that cannabinoids formed a stable complex with DPP-IV protein via hydrogen bonds at an allosteric site, suggesting that cannabinoids act by either inducing conformational changes or blocking the active site of the enzyme. Conclusion: These results demonstrated that cannabinoids may modulate DPP-IV activity and thereby potentially assist in improving glycaemic regulation in type 2 diabetes.

AMADAR: a python-based package for large scale prediction of Diels-Alder transition state geometries and IRC path analysis.

Predicting transition state geometries is one of the most challenging tasks in computational chemistry, which often requires expert-based knowledge and permanent human intervention. This short communication reports technical details and preliminary results of a python-based tool (AMADAR) designed to generate any Diels-Alder (DA) transition state geometry (TS) and analyze determined IRC paths in a (quasi-)automated fashion, given the product SMILES. Two modules of the package are devoted to performing, from IRC paths, reaction force analyses (RFA) and atomic (fragment) decompositions of the reaction force F and reaction force constant [Formula: see text]. The performance of the protocol has been assessed using a dataset of 2000 DA cycloadducts retrieved from the ZINC database. The sequential location of the corresponding TSs was achieved with a success rate of 95%. RFA plots confirmed the reaction force constant [Formula: see text] to be a good indicator of the (non)synchronicity of the associated DA reactions. Moreover, the atomic decomposition of [Formula: see text] allows for the rationalization of the (a)synchronicity of each DA reaction in terms of contributions stemming from pairs of interacting atoms. The source code of the AMADAR tool is available on GitHub [ CMCDD/AMADAR(github.com) ] and can be used directly with minor customizations, mostly regarding the local working environment of the user.

The Molecular Basis of the Effect of Temperature on the Structure and Function of SARS-CoV-2 Spike Protein.

The remarkable rise of the current COVID-19 pandemic to every part of the globe has raised key concerns for the current public healthcare system. The spike (S) protein of SARS-CoV-2 shows an important part in the cell membrane fusion and receptor recognition. It is a key target for vaccine production. Several researchers studied the nature of this protein under various environmental conditions. In this work, we applied molecular modeling and extensive molecular dynamics simulation approaches at 0°C (273.15 K), 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K) to study the detailed conformational alterations in the SARS-CoV-2 S protein. Our aim is to understand the influence of temperatures on the structure, function, and dynamics of the S protein of SARS-CoV-2. The structural deviations, and atomic and residual fluctuations were least at low (0°C) and high (60°C) temperature. Even the internal residues of the SARS-CoV-2 S protein are not accessible to solvent at high temperature. Furthermore, there was no unfolding of SARS-CoV-2 spike S reported at higher temperature. The most stable conformations of the SARS-CoV-2 S protein were reported at 20°C, but the free energy minimum region of the SARS-CoV-2 S protein was sharper at 40°C than other temperatures. Our findings revealed that higher temperatures have little or no influence on the stability and folding of the SARS-CoV-2 S protein.

Drug Resistance in the HIV-1 Subtype C Protease Enzyme: A High Throughput Virtual Screening Approach in Search of New Ligands with Activity.

BACKGROUND: HIV-1 subtype C protease is a strategic target for antiretroviral treatment. However, resistance to protease inhibitors appears after months of treatment. Chromones and 2- biscoumarin derivatives show potential for inhibition of the HIV- subtype C protease. OBJECTIVE: Different heterocyclic structures from the ZINC database were docked against Human Immunodeficiency Virus-1 (HIV) subtype C protease crystal structure 2R5Q and 2R5P. The 5 best molecules were selected to be docked against 62 homology models based on HIV-protease sequences from infants failing antiretroviral protease treatment. This experimentation was performed with two molecular docking programs: Autodock and Autodock Vina. These molecules were modified by substituting protons with different moieties, and the derivatives were docked against the same targets. Ligand-protein interactions, physical/chemical proprieties of the molecules, and dynamics simulations were analyzed. METHODS: Docking of all of the molecules was performed to find out the binding sites of HIV-1 subtype C proteases. An in-house script was made to substitute protons of molecules with different moieties. According to the Lipinski rule of five, physical and chemical properties were determined. Complexes of certain ligands-protease were compared to the protein alone in molecular dynamics simulations. RESULTS: From the first docking results, the 5 best (lowest energy) ligands (dibenz[a,h]acridine, dibenz[a, i]acridine, NSC114903, dibenz[c,h]acridine, benzo[a]acridine) were selected. The binding energy of the modified ligands increased, including the poorest-performing molecules. A correlation between nature, the position, and the resulting binding energy was observed. According to the Lipinski rules, the physico-chemical characteristics of the five best-modified ligands are ideal for oral bioavailability. Molecular dynamics simulations show that some lead-protease complexes were stable. CONCLUSION: Dibenz[a,h]acridine, dibenz[a, i]acridine, NSC114903, dibenz[c,h]acridine, benzo[ a]acridine and their derivatives might be considered as promising HIV-1 subtype C protease inhibitors. This could be confirmed through synthesis and subsequent in vitro assays.

Synthesis and anti-parasitic activity of N-benzylated phosphoramidate Mg2+-chelating ligands.

A series of N-benzylated phosphoramidate esters, containing a 3,4-dihydroxyphenyl Mg2+-chelating group, has been synthesised in five steps as analogues of fosmidomycin, a Plasmodium falciparum 1-deoxy-1-d-xylulose-5-phosphate reductoisomerase (PfDXR) inhibitor. The 3,4-dihydroxyphenyl group effectively replaces the Mg2+-chelating hydroxamic acid group in fosmidomycin. The compounds showed very encouraging anti-parasitic activity with IC50 values of 5.6-16.4 µM against Plasmodium falciparum parasites and IC50 values of 5.2 - 10.2 µM against Trypanosoma brucei brucei (T.b.brucei). Data obtained from in silico docking of the ligands in the PfDXR receptor cavity (3AU9)5 support their potential as PfDXR inhibitors.

Synthesis and anti-parasitic activity of achiral N-benzylated phosphoramidic acid derivatives.

Synthetic pathways have been developed to access a series of N-benzylated phosphoramidic acid derivatives as novel, achiral analogues of the established Plasmodium falciparum 1-deoxy-d-xylulose-5-phosphate reductase (PfDXR) enzyme inhibitor, FR900098. Bioassays of the targeted compounds and their synthetic precursors have revealed minimal antimalarial activity but encouraging anti-trypanosomal activity - in one case with an IC50 value of 5.4 µM against Trypanosoma brucei, the parasite responsible for Nagana (African cattle sleeping sickness). The results of relevant in silico modelling and docking studies undertaken in the design and evaluation of these compounds are discussed.

Identification and evaluation of bioactive natural products as potential inhibitors of human microtubule affinity-regulating kinase 4 (MARK4).

Microtubule affinity-regulating kinase 4 (MARK4) has recently been identified as a potential drug target for several complex diseases including cancer, diabetes and neurodegenerative disorders. Inhibition of MARK4 activity is an appealing therapeutic option to treat such diseases. Here, we have performed structure-based virtual high-throughput screening of 100,000 naturally occurring compounds from ZINC database against MARK4 to find its potential inhibitors. The resulted hits were selected, based on the binding affinities, docking scores and selectivity. Further, binding energy calculation, Lipinski filtration and ADMET prediction were carried out to find safe and better hits against MARK4. Best 10 compounds bearing high specificity and binding efficiency were selected, and their binding pattern to MARK4 was analyzed in detail. Finally, 100 ns molecular dynamics simulation was performed to evaluate; the dynamics stability of MARK4-compound complex. In conclusion, these selected natural compounds from ZINC database might be potential leads against MARK4, and can further be exploited in drug design and development for associated diseases.

High throughput screening, docking, and molecular dynamics studies to identify potential inhibitors of human calcium/calmodulin-dependent protein kinase IV.

Calcium/calmodulin-dependent protein kinase IV (CAMKIV) is associated with many diseases including cancer and neurodegenerative disorders and thus being considered as a potential drug target. Here, we have employed the knowledge of three-dimensional structure of CAMKIV to identify new inhibitors for possible therapeutic intervention. We have employed virtual high throughput screening of 12,500 natural compounds of Zinc database to screen the best possible inhibitors of CAMKIV. Subsequently, 40 compounds which showed significant docking scores (-11.6 to -10.0 kcal/mol) were selected and further filtered through Lipinski rule and drug likeness parameter to get best inhibitors of CAMKIV. Docking results are indicating that ligands are binding to the hydrophobic cavity of the kinase domain of CAMKIV and forming a significant number of non-covalent interactions. Four compounds, ZINC02098378, ZINC12866674, ZINC04293413, and ZINC13403020, showing excellent binding affinity and drug likeness were subjected to molecular dynamics simulation to evaluate their mechanism of interaction and stability of protein-ligand complex. Our observations clearly suggesting that these selected ligands may be further employed for therapeutic intervention to address CAMKIV associated diseases. Communicated by Ramaswamy H. Sarma.

Seed Extract of Psoralea corylifolia and Its Constituent Bakuchiol Impairs AHL-Based Quorum Sensing and Biofilm Formation in Food- and Human-Related Pathogens.

The emergence of multi-drug resistance in pathogenic bacteria in clinical settings as well as food-borne infections has become a serious health concern. The problem of drug resistance necessitates the need for alternative novel therapeutic strategies to combat this menace. One such approach is targeting the quorum-sensing (QS) controlled virulence and biofilm formation. In this study, we first screened different fractions of Psoralea corylifolia (seed) for their anti-QS property in the Chromobacterium violaceum 12472 strain. The methanol fraction was found to be the most active fraction and was selected for further bioassays. At sub-inhibitory concentrations, the P. corylifolia methanol fraction (PCMF) reduced QS-regulated virulence functions in C. violaceum CVO26 (violacein); Pseudomonas aeruginosa (elastase, protease, pyocyanin, chitinase, exopolysaccharides (EPS), and swarming motility), A. hydrophila (protease, EPS), and Serratia marcescens (prodigiosin). Biofilm formation in all the test pathogens was reduced significantly (p ≤ 0.005) in a concentration-dependent manner. The ß-galactosidase assay showed that the PCMF at 1,000 µg/ml downregulated las-controlled transcription in PAO1. In vivo studies with C. elegans demonstrated increased survival of the nematodes after treatment with the PCMF. Bakuchiol, a phytoconstituent of the extract, demonstrated significant inhibition of QS-regulated violacein production in C. violaceum and impaired biofilm formation in the test pathogens. The molecular docking results suggested that bakuchiol efficiently binds to the active pockets of LasR and RhlR, and the complexes were stabilized by several hydrophobic interactions. Additionally, the molecular dynamics simulation of LasR, LasR-bakuchiol, RhlR, and RhlR-bakuchiol complexes for 50 ns revealed that the binding of bakuchiol to LasR and RhlR was fairly stable. The study highlights the anti-infective potential of the PCMF and bakuchiol instead of bactericidal or bacteriostatic action, as the extract targets QS-controlled virulence and the biofilm.

Unravelling the unfolding mechanism of human integrin linked kinase by GdmCl-induced denaturation.

Integrin-linked kinase (ILK) is a ubiquitously expressed Ser/Thr kinase which plays significant role in the cell-matrix interactions and growth factor signalling. In this study, guanidinium chloride (GdmCl)-induced unfolding of kinase domain of ILK (ILK193-446) was carried out at pH 7.5 and 25 °C. Eventually, denaturation curves of mean residue ellipticity at 222 nm ([θ]222) and fluorescence emission spectrum were analysed to estimate stability parameters. The optical properties maximum emission (λmax) and difference absorption coefficient at 292 nm (Δε292) were analysed. The denaturation curve was measured only in the GdmCl molar concentration ranging 3.0-4.2 M because protein was aggregating below 3.0 M of GdmCl concentrations. The denaturation process of ILK193-446 was found as reversible at [GdmCl] ≥ 3.0 M. Moreover, a coincidence of normalized denaturation curves of optical properties ([θ]222, Δε292 and λmax) suggesting that GdmCl-induced denaturation of ILK193-446 is a two-state process. In addition, 100 ns molecular dynamics simulations were performed to see the effects of GdmCl on the structure and stability of ILK193-446. Both the spectroscopic and molecular dynamics approaches provided clear insights into the stability and conformational properties of ILK193-446.

Exploring molecular insights into the interaction mechanism of cholesterol derivatives with the Mce4A: A combined spectroscopic and molecular dynamic simulation studies.

Mammalian cell entry protein (Mce4A) is a member of MCE-family, and is being considered as a potential drug target of Mycobacterium tuberculosis infection because it is required for invasion and latent survival of pathogen by utilizing host's cholesterol. In the present study, we performed molecular docking followed by 100 ns MD simulation studies to understand the mechanism of interaction of Mce4A to the cholesterol derivatives and probucol. The selected ligands, cholesterol, 25-hydroxycholesterol, 5-cholesten-3ß-ol-7-one and probucol bind to the predicted active site cavity of Mce4A, and complexes remain stable during entire simulation of 100 ns. In silico studies were further validated by fluorescence-binding studies to calculate actual binding affinity and number of binding site(s). The non-toxicity of all ligands was confirmed on human monocytic cell (THP1) by MTT assay. This work provides a deeper insight into the mechanism of interaction of Mce4A to cholesterol derivatives, which may be further exploited to design potential and specific inhibitors to ameliorate the Mycobacterium pathogenesis.

Mechanistic insights into the urea-induced denaturation of kinase domain of human integrin linked kinase.

Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role in the oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK (ILK193-446) was examined in the presence of increasing concentrations of urea. The stability parameters of the urea-induced denaturation were measured by monitoring changes in [θ]222 (mean residue ellipticity at 222nm), difference absorption coefficient at 292nm (Δε292) and intrinsic fluorescence emission intensity at pH7.5 and 25±0.1°C. The urea-induced denaturation was found to be reversible. The protein unfolding transition occurred in the urea concentration range 3.0-7.0M. A coincidence of normalized denaturation curves of optical properties ([θ]222, Δε292 and λmax, the wavelength of maximum emission intensity) suggested that urea-induced denaturation of kinase domain of ILK is a two-state process. We further performed molecular dynamics simulation for 100ns to see the effect of urea on structural stability of kinase domain of ILK at atomic level. Structural changes with increasing concentrations of urea were analysed, and we observed a significant increase in the root mean square deviation, root mean square fluctuations, solvent accessible surface area and radius of gyration. A correlation was observed between in vitro and in silico studies.

Investigation of molecular mechanism of recognition between citral and MARK4: A newer therapeutic approach to attenuate cancer cell progression.

Microtubule affinity regulating kinase 4 (MARK4) is a member of AMP-activated protein kinase, found to be involved in apoptosis, inflammation and many other regulatory pathways. Since, its aberrant expression is directly associated with the cell cycle and thus cancer. Therefore, MARK4 is being considered as a potential drug target for cancer therapy. Here, we investigated the mechanism of inhibition of MARK4 activity by citral. Docking studies suggested that citral effectively binds to the active site cavity, and complex is stabilized by several interactions. We further performed molecular dynamics simulation of MARK4-citral complex under explicit water condition for 100ns and observed that binding of citral to MARK4 was quite stable. Fluorescence binding studies suggested that citral strongly binds to MARK4 and thereby inhibits its enzyme activity which was measured by the kinase inhibition assay. We further performed MTT assay and observed that citral inhibits proliferation of breast cancer cell line MCF-7. This work provides a newer insight into the use of citral as novel cancer therapeutics through the MARK4 inhibition. Results may be employed to design novel therapeutic molecule using citral as a scaffold for MARK4 inhibition to fight related diseases.

Structure Based Docking and Molecular Dynamic Studies of Plasmodial Cysteine Proteases against a South African Natural Compound and its Analogs.

Identification of potential drug targets as well as development of novel antimalarial chemotherapies with unique mode of actions due to drug resistance by Plasmodium parasites are inevitable. Falcipains (falcipain-2 and falcipain-3) of Plasmodium falciparum, which catalyse the haemoglobin degradation process, are validated drug targets. Previous attempts to develop peptide based drugs against these enzymes have been futile due to the poor pharmacological profiles and susceptibility to degradation by host enzymes. This study aimed to identify potential non-peptide inhibitors against falcipains and their homologs from other Plasmodium species. Structure based virtual docking approach was used to screen a small non-peptidic library of natural compounds from South Africa against 11 proteins. A potential hit, 5α-Pregna-1,20-dien-3-one (5PGA), with inhibitory activity against plasmodial proteases and selectivity on human cathepsins was identified. A 3D similarity search on the ZINC database using 5PGA identified five potential hits based on their docking energies. The key interacting residues of proteins with compounds were identified via molecular dynamics and free binding energy calculations. Overall, this study provides a basis for further chemical design for more effective derivatives of these compounds. Interestingly, as these compounds have cholesterol-like nuclei, they and their derivatives might be well tolerated in humans.

Analysis of non-peptidic compounds as potential malarial inhibitors against Plasmodial cysteine proteases via integrated virtual screening workflow.

Falcipain-2 (FP-2) and falcipain-3 (FP-3), haemoglobin-degrading enzymes in Plasmodium falciparum, are validated drug targets for the development of effective inhibitors against malaria. However, no commercial drug-targeting falcipains has been developed despite their central role in the life cycle of the parasites. In this work, in silico approaches are used to identify key structural elements that control the binding and selectivity of a diverse set of non-peptidic compounds onto FP-2, FP-3 and homologues from other Plasmodium species as well as human cathepsins. Hotspot residues and the underlying non-covalent interactions, important for the binding of ligands, are identified by interaction fingerprint analysis between the proteases and 2-cyanopyridine derivatives (best hits). It is observed that the size and chemical type of substituent groups within 2-cyanopyridine derivatives determine the strength of protein-ligand interactions. This research presents novel results that can further be exploited in the structure-based molecular-guided design of more potent antimalarial drugs.

SANCDB: a South African natural compound database.

BACKGROUND: Natural products (NPs) are important to the drug discovery process. NP research efforts are expanding world-wide and South Africa is no exception to this. While freely-accessible small molecule databases, containing compounds isolated from indigenous sources, have been established in a number of other countries, there is currently no such online database in South Africa. DESCRIPTION: The current research presents a South African natural compound database, named SANCDB. This is a curated and fully-referenced database containing compound information for 600 natural products extracted directly from journal articles, book chapters and theses. There is a web interface to the database, which is simple and easy to use, while allowing for compounds to be searched by a number of different criteria. Being fully referenced, each compound page contains links to the original referenced work from which the information was obtained. Further, the website provides a submission pipeline, allowing researchers to deposit compounds from their own research into the database. CONCLUSIONS: SANCDB is currently the only web-based NP database in Africa. It aims to provide a useful resource for the in silico screening of South African NPs for drug discovery purposes. The database is supported by a submission pipeline to allow growth by entries from researchers. As such, we currently present SANCDB the starting point of a platform for a community-driven, curated database to further natural products research in South Africa. SANCDB is freely available at https://sancdb.rubi.ru.ac.za/.

Synthetic analogues of the marine bisindole deoxytopsentin: potent selective inhibitors of MRSA pyruvate kinase.

As part of an ongoing study to elucidate the SAR of bisindole alkaloid inhibitors against the evolutionary conserved MRSA pyruvate kinase (PK), we present here the synthesis and biological activity of six dihalogenated analogues of the naturally occurring sponge metabolite deoxytopsentin, including the naturally occurring dibromodeoxytopsentin. The most active compounds displayed potent low nanomolar inhibitory activity against MRSA PK with concomitant significant selectivity for MRSA PK over human PK orthologues. Computational studies suggest that these potent MRSA PK inhibitors occupy a region of the small interface of the enzyme tetramer where amino acid sequence divergence from common human PK orthologues may contribute to the observed selectivity.

Exploring DOXP-reductoisomerase binding limits using phosphonated N-aryl and N-heteroarylcarboxamides as DXR inhibitors.

DOXP-reductoisomerase (DXR) is a validated target for the development of antimalarial drugs to address the increase in resistant strains of Plasmodium falciparum. Series of aryl- and heteroarylcarbamoylphosphonic acids, their diethyl esters and disodium salts have been prepared as analogues of the potent DXR inhibitor fosmidomycin. The effects of the carboxamide N-substituents and the length of the methylene linker have been explored using in silico docking studies, saturation transfer difference NMR spectroscopy and enzyme inhibition assays using both EcDXR and PfDXR. These studies indicate an optimal linker length of two methylene units and have confirmed the importance of an additional binding pocket in the PfDXR active site. Insights into the constraints of the PfDXR binding site provide additional scope for the rational design of DXR inhibitors with increased ligand-receptor interactions.

Synthesis and evaluation of phosphonated N-heteroarylcarboxamides as DOXP-reductoisomerase (DXR) inhibitors.

The diethyl esters and disodium salts of a range of heteroarylcarbamoylphosphonic acids have been prepared and evaluated as analogues of the highly active DOXP-reductoisomerase (DXR) inhibitor, fosmidomycin. Computer-simulated docking studies, Saturation Transfer Difference (STD) NMR analysis and enzyme inhibition assays have been used to explore enzyme-binding and -inhibition potential, while in silico analysis of the DXR active site has highlighted the importance of including a well-parameterised metal co-factor in docking studies and has revealed the availability of an additional binding pocket to guide future drug design.