In silico discovery of novel inhibitors from Northern African natural products database against main protease (Mpro) of SARS-CoV-2.

The recent outbreak of COVID-19 (Coronavirus Disease 2019), caused by a novel SARS-CoV-2 virus, has led to public health emergencies worldwide where time is as important as equipment to save lives. Antimalarial drugs such as hydroxychloroquine and chloroquine derivatives are used in emergencies but they are not suitable for patients with high blood pressure, diabetes and heart problems. Since there are no approved drugs for this disease, science is challenged to find vaccines and new drugs. Therefore, as part of our Silico drug design strategy, we identified drug-like compounds that inhibit replication of the main protease (Mpro) of SARS-CoV-2 based on receptor-based virtual database screening, molecular docking, molecular dynamics, and drug-similarity profiling from the NANPDB natural products database available at North African. The two resulting hit compounds named 5- Chloro-Omega-hydroxy-1-O-methylemodin and cystodion E showed the highest binding energy with Mpro of SARS-CoV-2 and strong inhibitory activity compared with the previously published N3 inhibitor. The complexes of these two compounds were validated by molecular dynamics analysis (RMSD, RMSF, Rg, total number of hydrogen bonds and secondary structure fractions of the protein in the complex) as the best method to evaluate the biological stability of the system. Therefore, these molecules deserve more attention in drug development compared to COVID-19. HighlightsA large database of natural compounds was screened against nCoV-2's Mpro.Molecular docking and Molecular dynamics were used as powerful methods.Two compounds were found are very attractive to inhibit Mpro of nCoV-2.ADME-Tox profiling is evaluated the active compounds are not cancerogenic.Communicated by Ramaswamy H. Sarma.

Chemical profiling, cytotoxic activities through apoptosis induction in human fibrosarcoma and carcinoma cells, and molecular docking of some 1,2,3-triazole-isoxazoline hybrids using the eugenol as a precursors.

In this research paper, we report the cytotoxic and apoptotic effects of 1,2,3-triazole derivatives in a unique 7a-g or hybrid form with isoxazoline 8a-g using the eugenol as a precursor in HT-1080 fibrosarcoma, MCF-7, and MDA-MB-231 breast carcinoma, and A-549 lung carcinoma. Data obtained on the cytotoxic effects have shown that hybrid compounds 8a-e induced a significant anticancer activity and are more important than the ones of 1,2,3-triazole derivatives 7a-g with IC50 ranging from 18 to 43 µM for the hybrids 8a-e and from 15 to 29 µM for mono-adducts 7a-g in all cell lines. Concerning the apoptotic study, compounds 7b and 8a can induce apoptosis in HT-1080 and A-549 cells as revealed by Annexin-V labeling and caspase-3/7 activity, also, the apoptotic effect was accompanied by cell cycle arrest at G2/M phase in the case of compounds 7b and 8a. Both compounds were evaluated in-silico through molecular docking and molecular dynamics and compound 8a is very active against Bcl-2 protein triggering apoptosis phenomenon by intrinsic pathway, therefore compound 8a is a potential candidate to inhibit the anti-apoptotic protein (Bcl-2).Communicated by Ramaswamy H. Sarma.

Design, synthesis, in vitro - In vivo biological evaluation of novel thiazolopyrimidine compounds as antileishmanial agent with PTR1 inhibition.

The leishmaniasis are a group of vector-borne diseases caused by a protozoan parasite from the genus Leishmania. In this study, a series of thiazolopyrimidine derivatives were designed and synthesized as novel antileishmanial agents with LmPTR1 inhibitory activity. The final compounds were evaluated for their in vitro antipromastigote activity, LmPTR1 and hDHFR enzyme inhibitory activities, and cytotoxicity on RAW264.7 and L929 cell lines. Based on the bioactivity results, three compounds, namely L24f, L24h and L25c, were selected for evaluation of their in vivo efficacy on CL and VL models in BALB/c mice. Among them, two promising compounds, L24h and L25c, showed in vitro antipromastigote activity against L. tropica with the IC50 values of 0.04 µg/ml and 6.68 µg/ml; against L. infantum with the IC50 values of 0.042 µg/ml and 6.77 µg/ml, respectively. Moreover, the title compounds were found to have low in vitro cytotoxicity on L929 and RAW264.7 cell lines with the IC50 14.08 µg/ml and 21.03 µg/ml, and IC50 15.02 µg/ml and 8.75 µg/ml, respectively. LmPTR1 enzyme inhibitory activity of these compounds was determined as 257.40 µg/ml and 59.12 µg/ml and their selectivity index (SI) over hDHFR was reported as 42.62 and 7.02, respectively. In vivo studies presented that L24h and L25c have a significant antileishmanial activity against footpad lesion development of CL and at weight measurement of VL group in comparison to the reference compound, Glucantime®. Also, docking studies were carried out with selected compounds and other potential Leishmania targets to detect the putative targets of the title compounds. Taken together, all these findings provide an important novel lead structure for the antileishmanial drug development.

Binding-Induced Diversity of a Human Telomeric G-Quadruplex Stability Phase Space.

The structural polymorphism of G-quadruplex nucleic acids is an important factor in their recognition by proteins and small-molecule ligands. However, it is not clear why the binding of several ligands alters G-quadruplex topology. We addressed this question by following the (un)folding and binding of the human telomeric fragment 5'-(GGGTTA)3GGGT-3' (22GT) by calorimetry (DSC, ITC) and spectroscopy (CD). A thermodynamic analysis of the obtained data led to a detailed description of the topological phase space of stability (phase diagram) of 22GT and shows how it changes in the presence of a specific bisquinolinium ligand (360A). Various 1:1 and 2:1 ligand-quadruplex complexes were observed. With increasing temperature, the 1:1 complexes transformed into 2:1 complexes, which is attributed to the preferential binding of the ligand to the folding intermediates. Overall, the dissection of the thermodynamic parameters in combination with molecular modelling clarified the driving forces of the topological quadruplex transformations in a wide range of ligand concentrations and temperatures.

Regioselective Synthesis of 5- and 3-Hydroxy-N-Aryl-1H-Pyrazole-4-Carboxylates and Their Evaluation as Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.

In silico evaluation of various regioisomeric 5- and 3-hydroxy-substituted alkyl 1-aryl-1H-pyrazole-4-carboxylates and their acyclic precursors yielded promising results with respect to their binding in the active site of dihydroorotate dehydrogenase of Plasmodium falciparum (PfDHODH). Consequently, four ethyl 1-aryl-5-hydroxy-1H-pyrazole-4-carboxylates and their 3-hydroxy regioisomers were prepared by two-step syntheses via enaminone-type reagents or key intermediates. The synthesis of 5-hydroxy-1H-pyrazoles was carried out using the literature protocol comprising acid-catalyzed transamination of diethyl [(dimethylamino)methylene]malonate with arylhydrazines followed by base-catalyzed cyclization of the intermediate hydrazones. For the synthesis of isomeric methyl 1-aryl-3-hydroxy-1H-pyrazole-4-carboxylates, a novel two-step synthesis was developed. It comprises acylation of hydrazines with methyl malonyl chloride followed by cyclization of the hydrazines with tert-butoxy-bis(dimethylamino)methane. Testing the pyrazole derivatives for the inhibition of PfDHODH showed that 1-(naphthalene-2-yl)-5-hydroxy-1H-pyrazole-4-carboxylate and 1-(naphthalene-2-yl)-, 1-(2,4,6-trichlorophenyl)-, and 1-[4-(trifluoromethyl)phenyl]-3-hydroxy-1H-pyrazole-4-carboxylates (~30% inhibition) were slightly more potent than a known inhibitor, diethyl α-{[(1H-indazol-5-yl)amino]methylidene}malonate (19% inhibition).

Novel isoxazoline-linked 1,3,4-thiadiazole hybrids as anticancer agents: Design, synthesis, biological evaluation, molecular docking, and molecular dynamics simulation.

In the current study, natural (R)-carvone was utilized as a starting material for the efficient synthesis of two series of isoxazoline derivatives bearing the 1,3,4-thiadiazole moiety. The new compounds were obtained in good yields and were characterized by 1 H and 13 C NMR and HRMS analysis. The newly synthesized monoterpenic isoxazoline 1,3,4-thiadiazole and their thiosemicarbazone intermediate derivatives were evaluated for their anticancer activity in four cancer cell lines (HT-1080, A-549, MCF-7, and MDA-MB-231). Most of the synthesized compounds exhibited moderate to high anticancer effects. Compound 13c showed the highest anticancer activity with IC50 values ranging from 19.33 ± 1.81 to 34.81 ± 3.03 µM. Further investigation revealed that compounds 12e and 13c could inhibit the cell growth of HT-1080 and MCF-7 cells by inducing apoptosis through caspase-3/7 activation. The apoptotic effect was accompanied by an S phase and G2/M cell cycle arrest for 13c and 12e, respectively. Compounds 12e and 13c were assessed in silico using molecular docking and molecular dynamics. We found that compound 13c is moderately active against the caspase-3 protein, which triggers apoptosis via intrinsic and extrinsic routes, making compound 13c a promising candidate to activate the proapoptotic protein (caspase-3).

A Set of Experimentally Validated Decoys for the Human CC Chemokine Receptor 7 (CCR7) Obtained by Virtual Screening.

We present a state-of-the-art virtual screening workflow aiming at the identification of novel CC chemokine receptor 7 (CCR7) antagonists. Although CCR7 is associated with a variety of human diseases, such as immunological disorders, inflammatory diseases, and cancer, this target is underexplored in drug discovery and there are no potent and selective CCR7 small molecule antagonists available today. Therefore, computer-aided ligand-based, structure-based, and joint virtual screening campaigns were performed. Hits from these virtual screenings were tested in a CCL19-induced calcium signaling assay. After careful evaluation, none of the in silico hits were confirmed to have an antagonistic effect on CCR7. Hence, we report here a valuable set of 287 inactive compounds that can be used as experimentally validated decoys.

Iodine-Catalysed Dissolution of Elemental Gold in Ethanol.

Gold is a scarce element in the Earth's crust but indispensable in modern electronic devices. New, sustainable methods of gold recycling are essential to meet the growing eco-social demand of gold. Here, we describe a simple, inexpensive, and environmentally benign dissolution of gold under mild conditions. Gold dissolves quantitatively in ethanol using 2-mercaptobenzimidazole as a ligand in the presence of a catalytic amount of iodine. Mechanistically, the dissolution of gold begins when I2 oxidizes Au0 and forms a [AuI I2 ]- species, which undergoes subsequent ligand-exchange reactions and forms a stable bis-ligand AuI complex. H2 O2 oxidizes free iodide and regenerated I2 returns back to the catalytic cycle. Addition of a reductant to the reaction mixture precipitates gold quantitatively and partially regenerates the ligand. We anticipate our work will open a new pathway to more sustainable metal recycling with the utilization of just catalytic amounts of reagents and green solvents.

Inhibition of the SARS-CoV-2 3CLpro main protease by plant polyphenols.

The abundance of polyphenols in edible plants makes them an important component of human nutrition. Considering the ongoing COVID-19 pandemic, a number of studies have investigated polyphenols as bioactive constituents. We applied in-silico molecular docking as well as molecular dynamics supported by in-vitro assays to determine the inhibitory potential of various plant polyphenols against an important SARS-CoV-2 therapeutic target, the protease 3CLpro. Of the polyphenols in initial in-vitro screening, quercetin, ellagic acid, curcumin, epigallocatechin gallate and resveratrol showed IC50 values of 11.8 µM to 23.4 µM. In-silico molecular dynamics simulations indicated stable interactions with the 3CLpro active site over 100 ns production runs. Moreover, surface plasmon resonance spectroscopy was used to measure the binding of polyphenols to 3CLpro in real time. Therefore, we provide evidence for inhibition of SARS-CoV-2 3CLpro by natural plant polyphenols, and suggest further research into the development of these novel 3CLpro inhibitors or biochemical probes.

Newly synthesized (R)-carvone-derived 1,2,3-triazoles: structural, mechanistic, cytotoxic and molecular docking studies.

In the current study, natural (R)-carvone was used as starting material for the efficient synthesis of several 1,2,3-triazole derivatives. The produced products were obtained in good yields and characterized by 1H and 13C NMR and HRMS analysis. The newly synthesized monoterpenic 1,2,3-triazole 4 and derivatives were analyzed by viability tests (MTT) for their cytotoxic activity against three human cancer cells. Product 5 showed a medium antitumor activity, for which the IC50 values against selected cells HT-1080, A-549 and MCF-7 were 29.25 µM, 31.62 µM and 26.02 µM, respectively. The regioselectivity of the condensation reaction and the molecular structure of the title compounds were determined by Density Functional Theory (DFT) using B3LYP calculations at 6-311 + G(d,p) level. The orbitals HOMO and LUMO were studied to determine the electronic properties of the synthesized compounds. In addition, the global reactivity indices were used to explain the regioselectivity for the formation of compound 6, and the theoretical results agree well with the experimental results. Molecular docking and molecular dynamics confirmed the empirical test results and confirmed the stability of the complex during inhibition of the anti-apoptotic protein for killing cancer cells. Communicated by Ramaswamy H. Sarma.

Quantification of Hydroperoxides by Gas Chromatography with Flame Ionisation Detection.

Hydroperoxides are of great importance in the fields of atmospheric and biological chemistry. However, there are several analytical challenges in their analysis: unknown and usually low UV absorption coefficients, high reactivity, thermal instability, and a lack of available reference standards. To overcome these limitations, we propose a GC-FID approach involving pre-column silylation and quantification via the effective carbon number approach. Four hydroperoxides of α-pinene were synthesized in the liquid phase with singlet oxygen and identified using literature data on isomer yield distribution, MS spectra, estimated boiling temperatures of each isomer (retention time), their thermal stability and derivatisation rate. The developed procedure was used for the determination of hydroperoxides in bottled and autooxidised turpentine. We anticipate that this method could also be applied in atmospheric chemistry, where the reactivity of singlet oxygen could help explain the high formation rates of secondary organic aerosols.

Hydration and counterion binding of aqueous acetylcholine chloride and carbamoylcholine chloride.

The hydration and Cl- ion binding of the neurot†ransmitter acetylcholine (ACh+) and its synthetic analogue, carbamoylcholine (CCh+), were studied by combining dilute-solution conductivity measurements with dielectric relaxation spectroscopy and statistical mechanics calculations at 1D-RISM and 3D-RISM level. Chloride ion binding was found to be weak but not negligible. From the ∼30 water molecules coordinating ACh and CCh+ only ∼1/3 is affected in its rotational dynamics by the cation, with the majority - situated close to the hydrophobic moieties - only retarded by a factor of ∼2.5. At vanishing solute concentration cations and the ∼3-4 H2O molecules hydrogen bonding to the CO group of the solute exhibit similar rotational dynamics but increasing concentration and temperature markedly dehydrates ACh+ and CCh+.

Prioritisation of Compounds for 3CLpro Inhibitor Development on SARS-CoV-2 Variants.

COVID-19 represents a new potentially life-threatening illness caused by severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 pathogen. In 2021, new variants of the virus with multiple key mutations have emerged, such as B.1.1.7, B.1.351, P.1 and B.1.617, and are threatening to render available vaccines or potential drugs ineffective. In this regard, we highlight 3CLpro, the main viral protease, as a valuable therapeutic target that possesses no mutations in the described pandemically relevant variants. 3CLpro could therefore provide trans-variant effectiveness that is supported by structural studies and possesses readily available biological evaluation experiments. With this in mind, we performed a high throughput virtual screening experiment using CmDock and the "In-Stock" chemical library to prepare prioritisation lists of compounds for further studies. We coupled the virtual screening experiment to a machine learning-supported classification and activity regression study to bring maximal enrichment and available structural data on known 3CLpro inhibitors to the prepared focused libraries. All virtual screening hits are classified according to 3CLpro inhibitor, viral cysteine protease or remaining chemical space based on the calculated set of 208 chemical descriptors. Last but not least, we analysed if the current set of 3CLpro inhibitors could be used in activity prediction and observed that the field of 3CLpro inhibitors is drastically under-represented compared to the chemical space of viral cysteine protease inhibitors. We postulate that this methodology of 3CLpro inhibitor library preparation and compound prioritisation far surpass the selection of compounds from available commercial "corona focused libraries".

Specificity of Counterion Binding to a Conjugated Polyelectrolyte: A Combined Molecular Dynamics and NOESY Investigation.

Poly(thiophen-3-ylacetic acid) (PTAA) is a representative of conjugated polyelectrolytes which are used in many optoelectronics devices. The performance of these devices is affected by the polymer conformation, which, among others, depends on the nature of the counterion. In this study, the binding of tetrabutylammonium counterions (TBA+) on PTAA was determined using a combination of nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics (MD) simulation. It was found that TBA+ ions specifically bind on the hydrophobic main chain of PTAA, while, according to MD simulations, alkali counterions predominantly bind in the vicinity of negatively charged carboxylic groups located on side chains. The MD trajectories were used to compute the relaxation matrices and the NOESY spectra. With the help of these latter calculations, the changes of intensities in experimental NOESY spectra upon binding of TBA+ ions to PTAA were interpreted.

Insights into evolutionary interaction patterns of the 'Phosphorylation Activation Segment' in kinase.

We are interested in studying the phosphorylation of the kinase activation loop, distinguishing the passage from the unphosphorylated to the phosphorylated form without allostery. We performed an interaction study to trace the change of interactions between the activation segment and the kinase catalytic core, before and after phosphorylation. Results show that the structural changes are mainly due to the attraction between the phosphate group and guanidine groups of the arginine side chains of RD-pocket, which are constituted mainly of guanidine groups of the catalytic loop, the ß9, and the αC helix. This attraction causes propagation of structural variation of the activation segment, principally towards the N-terminal. The structural variations are not made on all the amino acids of the activation segment; they are conditioned by the existence of two beta sheets stabilizing the loop during phosphorylation. The first,ß6-ß9 sheet is usually present in most of the kinases; the second, ß10-ß11 is formed due to the interaction between the main chain amino acids of the activation loop and the αEF/αF loop.

Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity.

During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.

Hydration and ion association of aqueous choline chloride and chlorocholine chloride.

The choline ion (Ch+) is ubiquitous in nature and also its synthetic homologue, chlorocholine (ClCh+), is widely used. Nevertheless, surprisingly little information on the hydration and counter-ion binding of these cations can be found in the literature. In this contribution we report effective hydration numbers, determined by dielectric relaxation spectroscopy, and ion-pair association constants with Cl-, determined by dilute-solution conductivity measurements. In combination with RISM calculations the obtained data suggest that for Ch+ water is bound to the hydroxy group via hydrogen bonds whereas for ClCh+ a rather stiff clathrate-like shell around the chlorine atom seems to be formed. With Cl- both cations form contact ion pairs with association constants of only ∼2 to 3 M-1.

Design and synthesis of constrained bicyclic molecules as candidate inhibitors of influenza A neuraminidase.

The rise of drug-resistant influenza A virus strains motivates the development of new antiviral drugs, with different structural motifs and substitution. Recently, we explored the use of a bicyclic (bicyclo[3.1.0]hexane) analogue of sialic acid that was designed to mimic the conformation adopted during enzymatic cleavage within the neuraminidase (NA; sialidase) active site. Given that our first series of compounds were at least four orders of magnitude less active than available drugs, we hypothesized that the new carbon skeleton did not elicit the same interactions as the cyclohexene frameworks used previously. Herein, we tried to address this critical point with the aid of molecular modeling and we proposed new structures with different functionalization, such as the introduction of free ammonium and guanidinium groups and ether side chains other than the 3-pentyl side chain, the characteristic side chain in Oseltamivir. A highly simplified synthetic route was developed, starting from the cyclopropanation of cyclopentenone and followed by an aziridination and further functionalization of the five-member ring. This allowed the efficient preparation of a small library of new bicyclic ligands that were characterized by enzyme inhibition assays against influenza A neuraminidases N1, its H274Y mutant, and N2. The results show that none of the new structural variants synthesized, including those containing guanidinium groups rather than free ammonium ions, displayed activity against influenza A neuraminidases at concentrations less than 2 mM. We conclude that the choice and positioning of functional groups on the bicyclo[3.1.0]hexyl system still need to be properly tuned for producing complementary interactions within the catalytic site.

DNA-Polyelectrolyte Complexation Study: The Effect of Polyion Charge Density and Chemical Nature of the Counterions.

Complexes of polycations and DNA, also known as polyplexes, have been extensively studied in the past decade, as potential gene delivery systems. Their stability depends strongly on the characteristics of the polycations, as well as the nature of the added salt. We present here a study of the DNA ionene complexation in which we used fluorescence, UV, and CD spectroscopy, combined with molecular dynamics computer simuations, to systematically examine the influence of the polycation charge density, as well as the influence of the nature of the counterion, on the stability of these systems. Ionenes as polycations, depending on their structural characteristics, have previously been found to possess low cytotoxicity, and are therefore particularly interesting as potential gene delivery agents. The results show that the DNA solutions in the presence of the polycation are more stable in the case of very large or very small ionene charge density, suggesting different mechanism of complexation. The computer simulations show that the ionenes with high charge density bind to the minor groove of the DNA molecules, while the ionenes with lower charge density bind to the major groove of the DNA. The nature of the counterions play only a minor role: precipitation of the DNA molecules occurs at slightly lower ionene concentration when fluoride counterion are present, compared to the bromide counterions.

Modelling the Correlation Between Molecular Electrostatic Potential and pKa on Sets of Carboxylic Acids, Phenols and Anilines.

Calculations of molecular electrostatic potential were correlated with experimental pKa values for different sets of acidic molecules (carboxylic acids, phenols, and anilines) to obtain linear relationships of variable quality. A single tri-parameter model function was constructed to describe the pKa dependence on MEP maxima together with two automatically generated molecular descriptors, namely the counts of carboxylic acid and amine functional groups.