Green synthesized silver nanoparticles of Terminalia bellirica leaves extract: synthesis, characterization, in-silico studies, and antimalarial activity.

Malaria is a mosquito-borne infectious disease that is caused by the Plasmodium parasite. Most of the available medication are losing their efficacy. Therefore, it is crucial to create fresh leads to combat malaria. Green silver nanoparticles (AgNPs) have recently attracted a lot of attention in biomedical research. As a result, green mediated AgNPs from leaves of Terminalia bellirica, a medicinal plant with purported antimalarial effects, were used in this investigation. Initially, cysteine-rich proteins from Plasmodium species were studied in silico as potential therapeutic targets. With docking scores between -9.93 and -11.25 kcal/mol, four leaf constituents of Terminalia bellirica were identified. The green mediated silver nanoparticles were afterward produced using leaf extract and were further examined using UV-vis spectrophotometer, DLS, Zeta potential, FTIR, XRD, and FESEM. The size of synthesized TBL-AgNPs was validated by the FESEM results; the average size of TBL-AgNPs was around 44.05 nm. The zeta potential study also supported green mediated AgNPs stability. Additionally, Plasmodium falciparum (3D7) cultures were used to assess the antimalarial efficacy, and green mediated AgNPs could effectively inhibit the parasitized red blood cells (pRBCs). In conclusion, this novel class of AgNPs may be used as a potential therapeutic replacement for the treatment of malaria.

PEX1 is essential for glycosome biogenesis and trypanosomatid parasite survival.

Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we functionally characterized a putative Trypanosoma PEX1 orthologue by bioinformatic and experimental approaches and show that it is a true PEX1 orthologue. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to the bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggest that these parasites possess a highly efficient quality control mechanism that exports the import receptors from glycosomes to the cytosol in the absence of a functional TbPEX1-TbPEX6 complex.

Targeting survivin for cancer therapy: Strategies, small molecule inhibitors and vaccine based therapeutics in development.

Survivin is a member of the family of inhibitors of apoptosis proteins (IAPs). It is involved in the normal mitotic process and acts as an anti-apoptotic molecule. While terminally differentiated normal tissues lack survivin, several human malignancies have significant protein levels. Resistance to chemotherapy and radiation in tumor cells is associated with survivin expression. Decreased tumor development, apoptosis, and increased sensitivity to chemotherapy and radiation are all effects of downregulating survivin expression or activity. As a prospective cancer treatment, small molecules targeting the transcription and translation of survivin and molecules that can directly bind with the survivin are being explored both in pre-clinical and clinics. Pre-clinical investigations have found and demonstrated the effectiveness of several small-molecule survivin inhibitors. Unfortunately, these inhibitors have also been shown to have off-target effects, which could limit their clinical utility. In addition to small molecules, several survivin peptide vaccines are currently under development. These vaccines are designed to elicit a cytotoxic T-cell response against survivin, which could lead to the destruction of tumor cells expressing survivin. Some survivin-based vaccines are advancing through Phase II clinical studies. Overall, survivin is a promising cancer drug target. However, challenges still need to be addressed before the survivin targeted therapies can be widely used in the clinics.

Malaria therapeutics: are we close enough?

Malaria is a vector-borne parasitic disease caused by the apicomplexan protozoan parasite Plasmodium. Malaria is a significant health problem and the leading cause of socioeconomic losses in developing countries. WHO approved several antimalarials in the last 2 decades, but the growing resistance against the available drugs has worsened the scenario. Drug resistance and diversity among Plasmodium strains hinder the path of eradicating malaria leading to the use of new technologies and strategies to develop effective vaccines and drugs. A timely and accurate diagnosis is crucial for any disease, including malaria. The available diagnostic methods for malaria include microscopy, RDT, PCR, and non-invasive diagnosis. Recently, there have been several developments in detecting malaria, with improvements leading to achieving an accurate, quick, cost-effective, and non-invasive diagnostic tool for malaria. Several vaccine candidates with new methods and antigens are under investigation and moving forward to be considered for clinical trials. This article concisely reviews basic malaria biology, the parasite's life cycle, approved drugs, vaccine candidates, and available diagnostic approaches. It emphasizes new avenues of therapeutics for malaria.

Drug repurposing based novel anti-leishmanial drug screening using in-silico and in-vitro approaches.

Visceral leishmaniasis is a neglected tropical disease and is mainly caused by L. donovani in the Indian subcontinent. The mitochondria genome replication in Leishmania spp. is having a very specific mechanism, and it is initiated by a key enzyme called mitochondrial primase. This enzyme is essential for the onset of the replication process and growth of the parasite. Therefore, we focused on the primase protein as a potential therapeutic target for combating leishmaniasis diseases. We started our studies molecular modeling and followed by docking of the FDA-approved drug library into the binding site of the primase protein. The top 30 selected compounds were subjected for molecular dynamics studies. Also, the target protein was cloned, purified, and tested experimentally (primase activity assays and inhibition assays). Some compounds were very effective against the Leishmania cell culture. All these approaches helped us to identify few possible novel anti-leishmanial drugs such as Pioglitazone and Mupirocin. These drugs are effectively involved in inhibiting the promastigote of L. donovani, and it can be utilized in the next level of clinical trials. Communicated by Ramaswamy H. Sarma.

SARS-CoV-2 Variants of Concern and Variations within Their Genome Architecture: Does Nucleotide Distribution and Mutation Rate Alter the Functionality and Evolution of the Virus?

SARS-CoV-2 virus pathogenicity and transmissibility are correlated with the mutations acquired over time, giving rise to variants of concern (VOCs). Mutations can significantly influence the genetic make-up of the virus. Herein, we analyzed the SARS-CoV-2 genomes and sub-genomic nucleotide composition in relation to the mutation rate. Nucleotide percentage distributions of 1397 in-house-sequenced SARS-CoV-2 genomes were enumerated, and comparative analyses (i) within the VOCs and of (ii) recovered and mortality patients were performed. Fisher's test was carried out to highlight the significant mutations, followed by RNA secondary structure prediction and protein modeling for their functional impacts. Subsequently, a uniform dinucleotide composition of AT and GC was found across study cohorts. Notably, the N gene was observed to have a high GC percentage coupled with a relatively higher mutation rate. Functional analysis demonstrated the N gene mutations, C29144T and G29332T, to induce structural changes at the RNA level. Protein secondary structure prediction with N gene missense mutations revealed a differential composition of alpha helices, beta sheets, and coils, whereas the tertiary structure displayed no significant changes. Additionally, the N gene CTD region displayed no mutations. The analysis highlighted the importance of N protein in viral evolution with CTD as a possible target for antiviral drugs.

Synthesis and inhibition studies towards the discovery of benzodiazepines as potential antimalarial compounds.

The target-based discovery of therapeutics against apicoplast, an all-important organelle is an overriding perspective. MEP pathway, an accredited drug target provides an insight into the importance of apicoplast in the survival of the parasite. In this study, we present the rational design strategy employing sustainable catalysis for the synthesis of benzodiazepine (BDZ) conformers followed by their biological evaluation as prospective inhibitors against the potential target of the IPP pathway, 1-deoxy-D-xylulose-5-phosphatereductoisomerase (DXR). The study reported the inhibitory profile of 8c and 6d against the quintessential step of the only drug target in the erythrocytic stages of parasite development. The potential compounds were identified to represent a novel class of inhibitors that serve as the lead molecules to impede the pathway and further affect the survival of the parasite.

Identification of natural inhibitors to inhibit C. acnes lipase through docking and simulation studies.

Acne vulgaris is a common skin disease affecting 80-90% of teenagers worldwide. C. acnes producing lipases are the main virulence factor that catalyzes sebum lipid into free fatty acid that is used for C. acnes growth. Recently, computational biology and bioinformatics play a significant role in drug discovery programs and the identification of novel lead(s). In this study, potential inhibitors against the C. acnes lipase have been identified via cost-effective computational investigations. Molecular docking, MD simulations, and binding affinity analysis have been performed between the active site of C. acnes lipase protein and selected natural plant constituents. First, C. acnes lipase protein was downloaded from PDB and defined the catalytically active site. Next, 16 active natural plant constituents were shortlisted from the PubChem library (based on their pharmacokinetics, pharmacodynamics, and antibacterial activity). Docking studies identified the best five active compounds that showed significantly strong binding affinity interacted through hydrogen bonding, hydrophobic interactions, and π-stacking with the active site residues of the target protein. Furthermore, a 100 ns MD simulation run showed a stable RMSD and less fluctuating RMSF graph for luteolin and neryl acetate. In silico investigation suggested that luteolin, neryl acetate, and isotretinoin were involved in stable interactions which were maintained throughout the MD run with the C. acnes lipase enzyme, virtually. The results advocated that these could potentially inhibit lipase activity and be used in the clinical management of acne.

Azo-Stilbene and Pyridine-Amine Hybrid Multifunctional Molecules to Target Metal-Mediated Neurotoxicity and Amyloid-ß Aggregation in Alzheimer's Disease.

Neurodegenerative diseases such as Alzheimer's disease (AD) are associated with progressive neuronal cell death, and they are commonly correlated with aberrant protein misfolding and aggregation of Aß peptides. Transition metal ions (Cu, Fe, and Zn) have been shown to promote aggregation and oxidative stress through formation of Aß-metal complexes. In this context, integrating molecular scaffolds rationally is used here to generate multifunctional molecules as modulators for metal-induced abnormalities. This work encompasses two azo-stilbene (AS)-derived compounds (AS-HL1 and AS-HL2), the rationale behind the design, their synthesis, characterization, and metal chelation ability [Cu(II) and Zn(II)]. The molecular frameworks of the designed compounds consist of stilbene as an Aß-interacting moiety, whereas N,N,O and N,N,N,O donor atoms are linked to generate the metal chelation moiety. Furthermore, we went on exploring their multifunctionality with respect to (w.r.t.) (i) their metal chelating capacities and (ii) their utility to modulate the aggregation pathways of both metal-free and metal-bound amyloid-ß, (iii) scavenge free radicals, and (iv) inhibit the activity of acetylcholinesterase and (v) cytotoxicity. Moreover, the compounds were able to sequester Cu2+ from the Aß-Cu complex as studied by the UV-visible spectroscopic assay. Molecular docking studies were also performed with Aß and acetylcholinesterase enzyme. Overall, the studies presented here qualify these molecules as promising candidates for further investigation in the quest for finding a treatment for Alzheimer's disease.

Overcoming vincristine resistance in cancer: Computational design and discovery of piperine-inspired P-glycoprotein inhibitors.

P-glycoprotein (P-gp)/MDR-1 plays a major role in the development of multidrug resistance (MDR) by pumping the chemotherapeutic drugs out of the cancer cells and reducing their efficacy. A number of P-gp inhibitors were reported to reverse the MDR when co-administered with chemotherapeutic drugs. Unfortunately, none has approved for clinical use due to toxicity issues. Some of the P-gp inhibitors tested in the clinics are reported to have cross-reactivity with CYP450 drug-metabolizing enzymes, resulting in unpredictable pharmacokinetics and toxicity of co-administered chemotherapeutic drugs. In this study, two piperine analogs (3 and 4) having lower cross-reactivity with CYP3A4 drug-metabolizing enzyme are identified as P-glycoprotein (P-gp) inhibitors through computational design, followed by synthesis and testing in MDR cancer cell lines over-expressing P-gp (KB ChR 8-5, SW480-VCR, and HCT-15). Both the analogs significantly increased the vincristine efficacy in MDR cancer cell lines at low micromole concentrations. Specifically, 3 caused complete reversal of vincristine resistance in KB ChR 8-5 cells and found to act as competitive inhibitor of P-gp as well as potentiated the vincristine-induced NF-KB-mediated apoptosis. Therefore, 3 ((2E,4E)-1-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-5-(4-hydroxy-3-methoxyphenyl)penta-2,4-dien-1-one) can serve as a potential P-gp inhibitor for in vivo investigations, to reverse multidrug resistance in cancer.

Design of novel ROCK inhibitors using fragment-based de novo drug design approach.

Rho-associated coiled-coil protein kinase (ROCK) is playing a vital role in the regulation of key cellular events and also responsible for causing several pathological conditions such as cancer, hypertension, Alzheimer's, cerebral vasospasm, and cardiac stroke. Therefore, it has attracted us to target ROCK protein as a potential therapeutic target for combating various diseases. Consequently, we investigated the active site of ROCK I protein and designed novel leads against the target using the de novo evolution drug design approach. Caffeic acid (an aglycone of acteoside) as a scaffold and fragments from 336 reported ROCK inhibitors were used for the design of novel leads. Multiple copy simultaneous search docking was used to identify the suitable fragments to be linked with the scaffold. Basic medicinal chemistry rules, coupled with structural insights generated by docking, led to the design of 7a, 8a, 9a, and 10a as potential ROCK I inhibitors. The designed leads showed better binding than the approved drug fasudil and also interacted with the key hinge region residue Met156 of ROCK I. Further, molecular dynamics (MD) simulation revealed that the protein-ligand complexes were stable and maintained the hydrogen bond with Met156 throughout the MD run. The promising in silico outcomes suggest that the designed compounds could be suitable anti-cancer leads that need to be synthesized and tested in various cancer cell lines. Graphical abstract.

Aryldiazoquinoline based multifunctional small molecules for modulating Aß42 aggregation and cholinesterase activity related to Alzheimer's disease.

Research continues to find a breakthrough for the treatment of Alzheimer's Disease (AD) due to its complicated pathology. Presented herein is a novel series of arydiazoquinoline molecules investigated for their multifunctional properties against the factors contributing to Alzheimer's disease (AD). The inhibitory properties of fourteen closely related aryldiazoquinoline derivatives have been evaluated for their inhibitory effect on Aß42 peptide aggregation. Most of these molecules inhibited Aß42 fibrillation by 50-80%. Selected molecules were also investigated for their binding behaviour to preformed Aß40 aggregates indicating a nanomolar affinity. In addition, these compounds were further investigated as cholinesterase inhibitors. Interestingly, some of the compounds turned out to be moderate in vitro inhibitors for AChE activity with IC50 values in low micro molar range. The highest anti-AChE activity was shown by compound labelled as 2a with an IC50 value of 6.2 µM followed by 2b with IC50 value of 7.0 µM. In order to understand the inhibitory effect, binding of selected molecules to AChE enzyme was studied using molecular docking. In addition, cell toxicity studies using Neuro2a cells were performed to assess their effect on neuronal cell viability which suggests that these molecules possess a non-toxic molecular framework. Overall, the study identifies a family of molecules that show good in vitro anti-Aß-aggregation properties and moderately inhibit cholinesterase activity.

Design of a potent anticancer lead inspired by natural products from traditional Indian medicine.

Among the plant constituents of Clerodendrum colebrookianum Walp., acteoside, martinoside, and osmanthuside ß6 interact with ROCK, a drug target for cancer. In this study, aglycone fragments of these plant constituents (caffeic acid, ferulic acid, and p-coumaric acid) along with the homopiperazine ring of fasudil (standard ROCK inhibitor) were used to design hybrid molecules. The designed molecules interact with the key hinge region residue Met156/Met157 of ROCK I/II in a stable manner according to our docking and molecular dynamics simulations. These compounds were synthesized and tested in vitro in SW480, MDA-MB-231, and A-549 cancer cell lines. The most promising compound was chemically optimized to obtain a thiourea analog, 6a (IC50 = 25 µM), which has >3-fold higher antiproliferative activity than fasudil (IC50 = 87 µM) in SW480 cells. Treatment with this molecule also inhibits the migration of colon cancer cells and induces cell apoptosis. Further, SPR experiments suggests that the binding affinity of 6a with ROCK I protein is better than that of fasudil. Hence, the drug-like natural product analog 6a constitutes a highly promising new anticancer lead.Communicated by Ramaswamy H. Sarma.

In Silico Investigations of Chemical Constituents of Clerodendrum colebrookianum in the Anti-Hypertensive Drug Targets: ROCK, ACE, and PDE5.

Understanding the molecular mode of action of natural product is a key step for developing drugs from them. In this regard, this study is aimed to understand the molecular-level interactions of chemical constituents of Clerodendrum colebrookianum Walp., with anti-hypertensive drug targets using computational approaches. The plant has ethno-medicinal importance for the treatment of hypertension and reported to show activity against anti-hypertensive drug targets-Rho-associated coiled-coil protein kinase (ROCK), angiotensin-converting enzyme, and phosphodiesterase 5 (PDE5). Docking studies showed that three chemical constituents (acteoside, martinoside, and osmanthuside ß6) out of 21 reported from the plant to interact with the anti-hypertensive drug targets with good glide score. In addition, they formed H-bond interactions with the key residues Met156/Met157 of ROCK I/ROCK II and Gln817 of PDE5. Further, molecular dynamics (MD) simulation of protein-ligand complexes suggest that H-bond interactions between acteoside/osmanthuside ß6 and Met156/Met157 (ROCK I/ROCK II), acteoside and Gln817 (PDE5) were stable. The present investigation suggests that the anti-hypertensive activity of the plant is due to the interaction of acteoside and osmanthuside ß6 with ROCK and PDE5 drug targets. The identified molecular mode of binding of the plant constituents could help to design new drugs to treat hypertension.

Targeting P-glycoprotein: Investigation of piperine analogs for overcoming drug resistance in cancer.

P-glycoprotein (P-gp) is a drug transporter that effluxes chemotherapeutic drugs and is implicated in the development of resistance of cancer cells to chemotherapeutic drugs. To date, no drug has been approved to inhibit P-gp and restore chemotherapy efficacy. Moreover, majority of the reported inhibitors have high molecular weight and complex structures, making it difficult to understand the basic structural requirement for P-gp inhibition. In this study, two structurally simple, low molecular weight piperine analogs Pip1 and Pip2 were designed and found to better interact with P-gp than piperine in silico. A one step, acid-amine coupling reaction between piperic acid and 6,7-dimethoxytetrahydroisoquinoline or 2-(3,4-dimethoxyphenyl)ethylamine afforded Pip1 and Pip2, respectively. In vitro testing in drug resistant P-gp overexpressing KB (cervical) and SW480 (colon) cancer cells showed that both analogs, when co-administered with vincristine, colchicine or paclitaxel were able to reverse the resistance. Moreover, accumulation of P-gp substrate (rhodamine 123) in the resistant cells, a result of alteration of the P-gp efflux, was also observed. These investigations suggest that the natural product analog - Pip1 ((2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)penta-2,4-dien-1-one) - is superior to piperine and could inhibit P-gp function. Further studies are required to explore the full potential of Pip1 in treating drug resistant cancer.

Virtual screening of traditional Chinese medicine (TCM) database: identification of fragment-like lead molecules for filariasis target asparaginyl-tRNA synthetase.

Lymphatic filariasis (LF) is a vector borne infectious disease caused by the nematode Wuchereria bancrofti, Brugia malayi, and Brugia timori. Over 120 million people are affected by LF in the world, of which two-thirds are in Asia. The infection restricts the normal flow of lymph from the infected area resulting in swelling of the extremities and causing permanent disability. As the available drugs for the treatment of LF are becoming ineffective due to the development of resistance, there is an urgent need to find new leads for drug development. In this study, asparaginyl-tRNA synthetase (AsnRS; PDB ID: 2XGT) essential for the protein bio-synthesis in the filarial nematode was used to carry out virtual screening (VS) of plant constituents from traditional Chinese medicine (TCM) database. Docking as well as E-pharmacophore based VS were carried out to identify the hits. The top scoring hits, Agri 1 (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one-3-O-beta-D-glucopyranoside) and Agri 2 (5,7-dihydroxy-2-propylchromone 7-O-beta-D-glucopyranoside), constituents of Agrimonia pilosa, were selected for molecular dynamics (MD) simulation study for 10 ns. MD simulation showed that both the glycosides Agri 1 and Agri 2 were forming stable interactions with the target protein. Moreover, docking and MD simulation of the lead A (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one; Mol. Wt.: 304.25; CLogP: 3.07) and lead B (5,7-dihydroxy-2-propylchromone; Mol. Wt.: 220.22; CLogP: 3.02), the aglycones of Agri 1 and Agri 2, respectively, were carried out with the target AsnRS. The in silico investigations of the aglycones suggest that the lead B could be a suitable fragment-like lead molecule for anti-filarial drug discovery.