In silico exploration of hypothetical proteins in Neisseria gonorrhoeae for identification of therapeutic targets.

Neisseria gonorrhoeae, a World Health Organization (WHO) declared superbug and the second-most frequent cause of bacterial sexually transmitted infections worldwide is responsible for gonorrhea. Hypothetical proteins are gene products that are predicted to be encoded by a particular gene based on the DNA sequence, but their specific functions and characteristics have not been experimentally determined or verified. In the context of this research, annotating hypothetical proteins is crucial for identifying their potential as therapeutic targets. Without proper annotation, these proteins would remain vague, hindering efforts to understand their roles in disease. The methodology used aims to bridge this gap by employing algorithm-based tools and software to annotate hypothetical proteins and assess their suitability as therapeutic targets based on factors such as essentiality, virulence, subcellular localization, and druggability. Out of 716 N. gonorrhoeae hypothetical proteins reported in UniProt, assessment of crucial pathogenic factors, including essentiality, virulence, subcellular localization, and druggability, effectively filtered and prioritized the hypothetical proteins for further therapeutic exploration and lead to 5 proteins being chosen as targets. The molecular docking studies conducted identified 10 hits targeting the five targets. Conclusively, this study aided in identification of targets and hit compounds for therapeutic targeting of gonorrhea disease. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00186-w.

Unveiling the cytotoxic and anti-proliferative potential of green-synthesized silver nanoparticles mediated by Colletotrichum gloeosporioides.

Fungal endophytes are a putative source of bioactive metabolites that have found significant applications in nanomedicine due to their metabolic versatility. In the present study, an aqueous extract of the fungal endophyte, Colletotrichum gloeosporioides associated with a medicinal plant Oroxylum indicum, has been used for the fabrication of green silver nanoparticles (CgAgNPs) and further evaluated their cytotoxic and anti-proliferative activity. Bioanalytical techniques including UV-Vis spectral analysis revealed a sharp band at 435 nm and functional molecules from the aqueous extract involved in the synthesis of CgAgNPs were evidenced through FTIR. Further, the crystalline nature of CgAgNPs was determined through XRD analysis and microscopy techniques including AFM, TEM and FESEM demonstrated the spherical shape of CgAgNPs exhibiting a crystalline hexagonal lattice and the size was found to be in the range of 9-29 nm. The significant cytotoxic potential of CgAgNPs was observed against breast cancer cells, MDA-MB-231 and MCF-7 with IC50 values of 18.398 ± 0.376 and 38.587 ± 1.828 µg mL-1, respectively. The biochemical study revealed that the treatment of MDA-MB-231 and MCF-7 cells with CgAgNPs reduces glucose uptake, suppresses cell proliferation, and enhances LDH release, indicating reduced cell viability and progression. Moreover, our research revealed differential expression of genes associated with apoptosis, cell cycle inhibition and metastasis suppression, evidencing anti-proliferative activity of CgAgNPs. The main objective of the present study is to harness anti-breast cancer activity of novel biogenic nanoparticles synthesized using the aqueous extract of O. indicum associated C. gloeosporioides and study the underlying mechanistic pathway exerted by these mycogenic nanoparticles.

Recent Advancements in the Formulation of Nanomaterials-Based Nanozymes, Their Catalytic Activity, and Biomedical Applications.

Nanozymes are nanoparticles with intrinsic enzyme-mimicking properties that have become more prevalent because of their ability to outperform conventional enzymes by overcoming their drawbacks related to stability, cost, and storage. Nanozymes have the potential to manipulate active sites of natural enzymes, which is why they are considered promising candidates to function as enzyme mimetics. Several microscopy- and spectroscopy-based techniques have been used for the characterization of nanozymes. To date, a wide range of nanozymes, including catalase, oxidase, peroxidase, and superoxide dismutase, have been designed to effectively mimic natural enzymes. The activity of nanozymes can be controlled by regulating the structural and morphological aspects of the nanozymes. Nanozymes have multifaceted benefits, which is why they are exploited on a large scale for their application in the biomedical sector. The versatility of nanozymes aids in monitoring and treating cancer, other neurodegenerative diseases, and metabolic disorders. Due to the compelling advantages of nanozymes, significant research advancements have been made in this area. Although a wide range of nanozymes act as potent mimetics of natural enzymes, their activity and specificities are suboptimal, and there is still room for their diversification for analytical purposes. Designing diverse nanozyme systems that are sensitive to one or more substrates through specialized techniques has been the subject of an in-depth study. Hence, we believe that stimuli-responsive nanozymes may open avenues for diagnosis and treatment by fusing the catalytic activity and intrinsic nanomaterial properties of nanozyme systems.

Repositioning the existing drugs for neuroinflammation: a fusion of computational approach and biological validation to counter the Parkinson's disease progression.

Parkinson's disease is caused by the deficiency of striatal dopamine and the accumulation of aggregated α-synuclein in the substantia nigra pars compacta (SNpc). Neuroinflammation associated with oxidative stress is a key factor contributing to the death of dopaminergic neurons in SNpc and advancement of Parkinson's disease. Two molecular targets, i.e., nuclear factor kappa-light-chain-enhancer (NF-kB) and α-synuclein play a substantial role in neuroinflammation progression. Therefore, the compounds targeting these neuroinflammatory targets hold a great potential to combat Parkinson's disease. Thereby, in this study, molecular docking and Connectivity Map (CMap) based gene expression profiling was utilized to reposition the approved drugs as neuroprotective agents for Parkinson's disease. With in silico screening, two drugs namely theophylline and propylthiouracil were selected for anti-neuroinflammatory activity evaluation in in vivo models of chronic neuroinflammation. The neuroinflammatory effect of the identified compounds was confirmed by quantifying the expression of three important neuroinflammatory mediators, i.e. IL-6, TNF-alpha, and IL-1 beta on brain tissue using ELISA assay. The ELISA experiment demonstrated that both compounds significantly decreased the expression of neuroinflammatory mediators, highlighting the compounds' potential in neuroinflammation management. Furthermore, the drug and disease interaction network of the two identified drugs and diseases (neuroinflammation and Parkinson's disease) suggested that the two drugs might interact with various targets namely adenosine receptors, Poly [ADP-ribose] polymerase-1, myeloperoxidase (MPO) and thyroid peroxidase through multiple pathways associated with neuroinflammation and Parkinson's disease. Computational studies suggest that a particular drug may be effective in managing Parkinson's disease associated with neuroinflammation. However, further research is needed to confirm this in biological experiments.

Recent Advances in Nanomaterials-Based Targeted Drug Delivery for Preclinical Cancer Diagnosis and Therapeutics.

Nano-oncology is a branch of biomedical research and engineering that focuses on using nanotechnology in cancer diagnosis and treatment. Nanomaterials are extensively employed in the field of oncology because of their minute size and ultra-specificity. A wide range of nanocarriers, such as dendrimers, micelles, PEGylated liposomes, and polymeric nanoparticles are used to facilitate the efficient transport of anti-cancer drugs at the target tumor site. Real-time labeling and monitoring of cancer cells using quantum dots is essential for determining the level of therapy needed for treatment. The drug is targeted to the tumor site either by passive or active means. Passive targeting makes use of the tumor microenvironment and enhanced permeability and retention effect, while active targeting involves the use of ligand-coated nanoparticles. Nanotechnology is being used to diagnose the early stage of cancer by detecting cancer-specific biomarkers using tumor imaging. The implication of nanotechnology in cancer therapy employs photoinduced nanosensitizers, reverse multidrug resistance, and enabling efficient delivery of CRISPR/Cas9 and RNA molecules for therapeutic applications. However, despite recent advancements in nano-oncology, there is a need to delve deeper into the domain of designing and applying nanoparticles for improved cancer diagnostics.

Exploration of in vitro cytotoxic and in ovo antiangiogenic activity of ethyl acetate extract of Penicillium oxalicum.

Fungal endophytes have established new paradigms in the area of biomedicine due to their ability to produce metabolites of pharmacological importance. The present study reports the in vitro cytotoxic and in ovo antiangiogenic activity of the ethyl acetate (EA) extract of Penicillium oxalicum and their chemical profiling through Gas Chromatography-Mass Spectrometry analysis. Treatment of the EA extract of P. oxalicum to the selected human breast cancer cell lines (MDA-MB-231 and MCF-7) leads to the reduced glucose uptake and increased nitric oxide production suggesting the cytotoxic activity of EA extract of P. oxalicum. Our results further show that treatment of EA extract of P. oxalicum attenuates the colony number, cell migration ability and alters nuclear morphology in both the human breast cancer cell lines. Furthermore, the treatment of EA extract of P. oxalicum mediates apoptosis by increasing the expression of BAX, P21, FADD, and CASPASE-8 genes, with increased Caspase-3 activity. Additionally, in ovo chorioallantoic membrane (CAM) assay showed that the treatment of EA extract of P. oxalicum leads to antiangiogenic activity with perturbed formation of blood vessels. Overall, our findings suggest that the EA extract of P. oxalicum show in vitro cytotoxic and antiproliferative activity against human breast cancer cell lines, and in ovo antiangiogenic activity in CAM model.

Role and Recent Advancements of Ionic Liquids in Drug Delivery Systems.

Advancements in the fields of ionic liquids (ILs) broaden its applications not only in traditional use but also in different pharmaceutical and biomedical fields. Ionic liquids "Solutions for Your Success" have received a lot of interest from scientists due to a myriad of applications in the pharmaceutical industry for drug delivery systems as well as targeting different diseases. Solubility is a critical physicochemical property that determines the drug's fate at the target site. Many promising drug candidates fail in various phases of drug research due to poor solubility. In this context, ionic liquids are regarded as effective drug delivery systems for poorly soluble medicines. ILs are also able to combine different anions/cations with other cations/anions to produce salts that satisfy the concept behind the ILs. The important characteristics of ionic liquids are the modularity of their physicochemical properties depending on the application. The review highlights the recent advancement and further applications of ionic liquids to deliver drugs in the pharmaceutical and biomedical fields.

Setomimycin as a potential molecule for COVID­19 target: in silico approach and in vitro validation.

COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a worldwide crisis. In view of emerging variants time to time, there is a pressing need of effective COVID-19 therapeutics. Setomimycin, a rare tetrahydroanthracene antibiotic, remained unexplored for its therapeutic uses. Herein, we report our investigations on the potential of setomimycin as COVID-19 therapeutic. Pure setomimycin was isolated from Streptomyces sp. strain RA-WS2 from NW Himalayan region followed by establishing in silico as well as in vitro anti-SARS-CoV-2 property of the compound against SARS-CoV-2 main protease (Mpro). It was found that the compound targets Mpro enzyme with an IC50 value of 12.02 ± 0.046 µM. The molecular docking study revealed that the compound targets Glu166 residue of Mpro enzyme, hence preventing dimerization of SARS-CoV-2 Mpro monomer. Additionally, the compound also exhibited anti-inflammatory and anti-oxidant property, suggesting that setomimycin may be a viable option for application against COVID-19 infections.

Identification of novel MurA inhibitors using in silico approach, their validation and elucidation of mode of inhibition.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an important enzyme involved in the first cytosolic step of bacterial cell wall synthesis. In this study a combination of ligand based and structure based in silico virtual screening methods were utilised for screening of more than 50,000 drug-like compounds from CSIR-IIIM in-house compound library in order to identify potent inhibitors of MurA. The identified hits were validated in vitro under various incubation conditions using Malachite green phosphate assay, and two potent hits viz 3772-9534 and D396-0012 were identified. Among these hits, compound 3772-9534 showed significant changes in the activity values in different assay conditions. The MD simulation study of 3772-9534 suggested a novel binding site in MurA enzyme, independent of the two-substrate binding sites. Binding of inhibitors at the allosteric site induces conformational changes in the enzyme, which leads to inhibition of enzymatic activity. Overall, the study offers new insight for targeting MurA, which may promote the discovery of novel MurA allosteric site inhibitors.

Epigenetic manipulation for secondary metabolite activation in endophytic fungi: current progress and future directions.

Fungal endophytes have emerged as a promising source of secondary metabolites with significant potential for various applications in the field of biomedicine. The biosynthetic gene clusters of endophytic fungi are responsible for encoding several enzymes and transcriptional factors that are involved in the biosynthesis of secondary metabolites. The investigation of fungal metabolic potential at genetic level faces certain challenges, including the synthesis of appropriate amounts of chemicals, and loss of the ability of fungal endophytes to produce secondary metabolites in an artificial culture medium. Therefore, there is a need to delve deeper into the field of fungal genomics and transcriptomics to explore the potential of fungal endophytes in generating secondary metabolites governed by biosynthetic gene clusters. The silent biosynthetic gene clusters can be activated by modulating the chromatin structure using chemical compounds. Epigenetic modification plays a significant role by inducing cryptic gene responsible for the production of secondary metabolites using DNA methyl transferase and histone deacetylase. CRISPR-Cas9-based genome editing emerges an effective tool to enhance the production of desired metabolites by modulating gene expression. This review primarily focuses on the significance of epigenetic elicitors and their capacity to boost the production of secondary metabolites from endophytes. This article holds the potential to rejuvenate the drug discovery pipeline by introducing new chemical compounds.

Study of potent CDK7 inhibitor secondary metabolites from Tecomella undulata.

Chemical investigation of the petroleum ether extract of heartwood of Tecomella undulata led to the isolation of tectonaquinone B (1), 2-methylquinizarin (2) along with tecomaquinone I (3), lapachol (4), 2-isopropenylnaphtho[2,3-b]-furan-4,9-quinone (5), dehydro-α-lapachone (6), α-lapachone (7), and ß-lapachone (8). This is the first report of isolation of tectonaquinone B and 2-methylquinizarin from this plant. The structures of compounds were elucidated by advanced spectroscopic methods. Molecular docking study for potential inhibitory action toward CDK7 (cyclin-dependent kinase 7) were performed, which proved that these compounds have high binding affinities with the receptor protein (CDK7). 2-Methylquinizarin exhibited best docking score (-7.70 kcal/mol) among all the tested compounds. The present study showed that 2-methylquinizarin may exhibit potent anticancer activity through inhibiting CDK7 via interaction with Met94.

Isolation and anticancer activity evaluation of rare Bisaryl anthraquinone antibiotics from novel Streptomyces sp. strain of NW Himalayan region.

Biosynthesis of bisaryl preanthraquinone antibiotics by various microorganisms differs in monomeric subunits as well as their dimerization positions leading to different configurations. The present study relates to the production of rare bisaryl anthraquinone antibiotics by a new Streptomyces strain isolated from Shivalik region of NW Himalayas. In vitro anticancer and anti-migratory effects of Setomimycin (9,9' bisanthraquinone antibiotic) was seen with a significant reduction in the expression of both MEK as well as ERK pathways in a dose dependent manner at 6.5 µM & 8 µM concentration in HCT-116 and 5.5 µM & 7 µM concentration in MCF-7 cells. In vivo studies in aggressive orthotopic mouse mammary carcinoma model (4T1) demonstrated about 76% reduction of primary tumor weight and 90.5% reduction in the tumor volume within two weeks. In vivo pharmacokinetics study of setomimycin revealed that it can be rapidly absorbed with an adequate plasma exposure and half-life which can be linked to its in vivo efficacy.

Boswellic acids, as novel inhibitor targeting peptidoglycan biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) in Escherichia coli.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an essential cytosolic enzyme in the biosynthesis of peptidoglycan. It becomes a potential bacterial target for screening promising antibacterial compounds as it is associated with the early phases of peptidoglycan production. MurA enzyme is conserved and necessary for bacterial viability with no mammalian homolog, which is a well-proven therapeutic research target. The present study reports the natural compounds from Boswellia serrata targeting the MurA enzyme. The identified inhibitors against MurA Escherichia coli (E. coli): ß-boswellic acid (IC50 33.65 µM), Acetyl-ß-boswellic acid (IC50 30.17 µM), and Acetyl-11-keto-ß-boswellic acid (IC50 37.67 µM). Inhibitors showed a fourfold decrease in IC50 values on pre-incubation with substrate-UDP-N-acetyl-glucosamine (UDP-GlcNAc). Mode-of-inhibition studies revealed their uncompetitive nature with both the substrates. Although these boswellic acids have been explored for their pharmacological potential, this is the first study reporting these compounds' E. coli MurA inhibiting potential.

Synthesis of 3-N-/O-/S-methyl-imidazo[1,2-a] pyridine derivatives for caspase-3 mediated apoptosis induced anticancer activity.

A library of 49 analogs of imidazo[1,2-a]pyridine with 2-halo, aryl, styryl and phenylethynyl-substitution at C-2 position and N-/O-/S-methyl linkage at C-3 position, have been synthesized and evaluated for their anti-proliferative activity against breast (MCF-7, MDA-MB-231), pancreatic (MiaPaca-2), lung (A549), prostate (PC-3) and colon (HCT-116) cancer cell lines and normal cells (HEK-293). Among the screened compounds, 5b exhibited best anticancer potential in all tested cancer cells with IC50 ranging from 3.5 to 61.1 µM and no toxicity in normal cells. Further, mechanistic study of 5b revealed concentration dependent increased generation of ROS, reduced mitochondrial membrane potential (MMP), surface and nuclear morphological alterations and inhibition of colony formation in HCT-116 cells. Western blot results had shown that the cell death in HCT-116 colon cancer cells was achieved through the induction of apoptosis via upregulation of the PTEN gene and downregulation of AKT pathway. Similarly, 5b treatment induced caspase-3 cleavage which is a hallmark of apoptosis. Molecular docking and binding energy (ΔG) studies of hit 5b with respect to three important cancer targets (EGFR, mTOR and PI3Kα) revealed strong binding of inhibitor with PI3Kα (docking score -6.932 and ΔG -56.297).

Screening of compound library identifies novel inhibitors against the MurA enzyme of Escherichia coli.

Bacterial cell has always been an attractive target for anti-infective drug discovery. MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) enzyme of Escherichia coli (E.coli) is crucial for peptidoglycan biosynthetic pathway, as it is involved in the early stages of bacterial cell wall biosynthesis. In the present study we aim to identify novel chemical structures targeting the MurA enzyme. For screening purpose, we used in silico approach (pharmacophore based strategy) for 52,026 library compounds (Chembridge, Chemdiv and in house synthetics) which resulted in identification of 50 compounds. These compounds were screened in vitro against MurA enzyme and release of inorganic phosphate (Pi) was estimated. Two compounds (IN00152 and IN00156) were found to inhibit MurA enzyme > 70% in primary screening and IC50 of 14.03 to 32.30 µM respectively. These two hits were further evaluated for their mode of inhibition studies and whole-cell activity where we observed 2-4 folds increase in activity in presence of Permeabilizer EDTA (Ethylenediaminetetraacetic acid). Combination studies were also performed with known antibiotics in presence of EDTA. Hits are reported for the first time against this target and our report also support the use of OM permeabilizer in combination with antibacterial compounds to address the permeability and efficacy issue. These lead hits can be further optimized for drug discovery. KEY POINTS: • Emerging Gram negative resistant strains is a matter of concern. • Need for new screening strategies to cope with drying up antibiotics pipeline. • Outer membrane permeabilizers could be useful to improve potency of molecules to reach its target.

Comprehensive genome-wide identification, characterization, and expression profiling of MATE gene family in Nicotiana tabacum.

The transporters belonging to the MATE family are involved in the transportation of diverse ligands, including metal ions and small organic molecules, and, therefore, play an important role in plant biology. Our genome-wide analysis led to the identification of 138 MATE genes in N. tabacum, which were grouped into four major phylogenetic clades. The expression of several NtMATE genes was reported to be differential in different tissues, namely young leaf, mature leaf, stem, root, and mature flower. The upstream regions of the NtMATE genes were predicted to contain several cis-acting elements associated with hormonal, developmental, and stress responses. Some of the genes were found to display induced expression following methyl jasmonate treatment. The co-expression analysis revealed 126 candidate transcription factor genes that might be involved in the transcriptional regulation of 21 NtMATE genes. Certain MATE genes (NtMATE81, NtMATE82, NtMATE88, and NtMATE89) were predicted to be targeted by micro RNAs (nta-miR167a, nta-miR167b, nta-miR167c, nta-miR167d and nta-miR167e). The computational analysis of MATE transporters provided insights into the key amino acid residues involved in the binding of the alkaloids. Further, the putative function of some of the NtMATE transporters was also revealed. The present study develops a solid foundation for the functional characterization of MATE transporter genes in N. tabacum.

Discovery of a New Donepezil-like Acetylcholinesterase Inhibitor for Targeting Alzheimer's Disease: Computational Studies with Biological Validation.

Alzheimer's disorder is one of the most common worldwide health problems, and its prevalence continues to increase, thereby straining the healthcare budgets of both developed and developing countries. So far, donepezil is the only Food and Drug Administration-approved dual-binding site inhibitor of acetylcholinesterase (AChE) that can amplify the cholinergic activity and also decrease Aß aggregation in Alzheimer patients. We report herein a new donepezil-like natural compound derivative (D1) as a convincing AChE inhibitor. The in silico studies suggests that D1 exhibits a dual-binding mode of action and interacts with both the catalytic anionic site and peripheral anionic site (PAS) of human AChE. The biological studies confirm the dual-binding site character of D1 and revealed that D1 not only enhances the acetylcholine levels but also reduces the accumulation of Aß plaques in Caenorhabditis elegans. In fact, 5 µM D1 was seen more potent in elevating the acetylcholine expression than 25 µM donepezil. While most of the non-cholinergic functions of donepezil, associated with the PAS of AChE, were gradually lost at higher concentrations, D1 was more functional at similar doses. Promisingly, D1 also exerted an agonistic effect on the α7 nicotinic acetylcholine receptor.

Rottlerin is a pan phosphodiesterase inhibitor and can induce neurodifferentiation in IMR-32 human neuroblastoma cells.

Phosphodiesterases are promising targets for pharmacological intervention against various diseases. There are already inhibitors of PDE3, PDE4 and PDE5 as approved drugs. However there is an unmet need to discover new chemical scaffolds as PDE inhibitors. The main drawback of most of PDE inhibitors is their non specificity; owing to their structural resemblance to cAMP or cGMP. Natural product compounds offer high structural diversity hence may provide new PDE inhibitors. We decided to screen our institutional natural product compound library of nearly 900 molecules for PDE5 inhibition and explore the selectivity against PDE1-11 and cytotoxicity of the hit molecule/s. Rottlerin was identified as a PDE5 inhibitor. It was found to inhibit other PDEs with varying specificities. Structure activity relationship data and molecular dynamics studies showed that Tyr612, Asp764, Gln817 and Phe820 in the binding pocket of PDE5 play an important role in the activity of rottlerin. As a pan PDE inhibitor, rottlerin was also found to activate the AMPK pathway and induce neurodifferentiation in IMR-32 cells, with the effect more efficient in samples co-treated with cAMP activator Forskolin. Rottlerin at higher concentrations was shown to induce autophagy, apoptosis and G2/S cell cycle arrest in IMR-32 cells.

Structural and functional characterization of the Vindoline biosynthesis pathway enzymes of Catharanthus roseus.

Vinblastine and its related compound vincristine are important mono terpenoid indole alkaloids accumulated in the leaves of Catharanthus roseus (Madagascar periwinkle). They serve as major anticancer drugs. Vinblastine is formed by the condensation of vindoline and catharanthine. The vindoline moiety is derived from tabersonine via vindoline biosynthesis pathway. The reaction sequence from tabersonine to vindoline is now well established and the enzymes involved in this pathway are identified. However, to date, the structures of the enzymes involved in the vindoline biosynthesis pathway are not known, leading to limited mechanistic understanding of the substrate binding and catalysis. The purpose of this work is to provide structural insight regarding all the steps of the vindoline pathway via rigorous homology modeling, molecular docking, and molecular dynamics analyses. Substrate and cofactors required for each step were docked onto the computationally built and validated three-dimensional (3D) model of the corresponding enzyme, and the catalytic reaction was analyzed from the structural point of view. Possible binding modes of the substrates and cofactors were generated and corresponding binding residues were identified. Enzyme-substrate models were verified based on structure evaluation methods and molecular dynamics based approaches. Findings of our analysis would be useful in rational designing of these important enzymes aimed toward bio-production of vindoline.