Exploring the Antibacterial Potential of Semisynthetic Phytocannabinoid: Tetrahydrocannabidiol (THCBD) as a Potential Antibacterial Agent against Sensitive and Resistant Strains of Staphylococcus aureus.

Antimicrobial resistance (AMR) is one of the most challenging problems and is responsible for millions of deaths every year. We therefore urgently require new chemical entities with novel mechanisms of action. Phytocannabinoids have been adequately reported for the antimicrobial effect but not seriously pursued because of either stringent regulatory issues or poor drug-like properties. In this regard, the current work demonstrated the antibacterial potential of tetrahydrocannabidiol (THCBD, 4), a semisynthetic phytocannabinoid, against Staphylococcus aureus, the second-most widespread bug recognized by the WHO. THCBD (4) was generated from cannabidiol and subjected to extensive antibacterial screening. In in vitro studies, THCBD (4) demonstrated a potent MIC of 0.25 µg/mL against Gram-positive bacteria, S. aureus ATCC-29213. It is interesting to note that THCBD (4) has demonstrated strong effectiveness against efflux pump-overexpressing (SA-1199B, SA-K2191, SA-K2192, and Mupr-1) and multidrug-resistant (MRSA-15187) S. aureus strains. THCBD (4) has also shown a good effect in kill kinetic assays against ATCC-29213 and MRSA-15187. In the checkerboard assay, THCBD (4) has shown additive/indifference effects with several well-known clinically used antibiotics, tetracycline, mupirocin, penicillin G, and ciprofloxacin. THCBD (4) also exhibited good permeability in the artificial skin model. Most importantly, THCBD (4) has significantly reduced CFU in mice's in vivo skin infection models and also demonstrated decent plasma exposure with 16-17% oral bioavailability. Acute dermal toxicity of THCBD (4) suggests no marked treatment-related impact on gross pathophysiology. This attractive in vitro and in vivo profile of plant-based compounds opens a new direction for new-generation antibiotics and warrants further detailed investigation.

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.

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.

Potential Inhibitors Targeting Escherichia coli UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA): An Overview.

Antibiotic resistance is one of the biggest challenges that is escalating and affecting humanity across the globe. To overcome this increasing burden of resistance, discovering novel hits by targeting the enzymes involved in peptidoglycan (murein) biosynthesis has always been considered better in antimicrobial drug discovery. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme has been identified as essential for Escherichia coli survival and catalyzes the early-stage step in bacterial cell wall synthesis. The present article gives a brief overview of the role of enzymes in peptidoglycan synthesis and MurA enzyme (previously known as MurZ in E. coli), in particular, including its structural and active site features. This review also provides an insight into the current knowledge of the reported MurA inhibitors, their mechanism of action and drawbacks of these hits that hinder their clinical trials, which would be helpful for synthesis and discovering potent molecules. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00988-6.

A critical review on exploitation of agro-industrial biomass as substrates for the therapeutic microbial enzymes production and implemented protein purification techniques.

Annually, a huge amount of waste is generated by the industries that use agricultural biomass. Researchers have looked into employing this cheap and renewable agro-biomass as a substrate for enzyme production via fermentation processes to meet the ever-increasing worldwide need. Although there are a number of sources for enzyme extraction, microbial sources have dominated industrial sectors due to their easy availability and rapid growth. Microbial enzymes are currently used in a variety of industries, including pharmaceuticals, food, biofuels, textiles, paper, detergents, and so on, and using these nutritious feedstocks not only reduces production costs but also helps to reduce environmental concerns. The present review focuses on the therapeutic microbial enzymes produced using different agro-industrial biomass as raw materials, with down-streaming techniques for obtaining a final pure product. Additionally, the article also discussed biomass pretreatment processes, including physical, chemical and biological. The type of pretreatment method to be used is mostly governed by the intended use of the major molecular components of biomass (cellulose, hemicelluloses and lignin). Finally, purification challenges are included. All of this information will be useful in the industrial synthesis of high-purity targeted enzymes if the crucial aspects that have been discussed are taken into account.

Recent advances in biodiesel production: Challenges and solutions.

Mono alkyl fatty acid ester or methyl ethyl esters (biodiesel) are the promising alternative for fossil fuel or petroleum derived diesel with similar properties and could reduce the carbon foot print and the greenhouse gas emissions. Biodiesel can be produced from renewable and sustainable feedstocks like plant derived oils, and it is biodegradable and non-toxic to the ecosystem. The process for the biodiesel production is either through traditional chemical catalysts (Acid or Alkali Transesterification) or enzyme mediated transesterification, but as enzymes are natural catalysts with environmentally friendly working conditions, the process with enzymes are proposed to overcome the drawbacks of chemical synthesis. At present 95% of the biodiesel production is contributed by edible oils worldwide whereas recycled oils and animal fats contribute 10% and 6% respectively. Although every process has its own limitations, the enzyme efficiency, resistance to alcohols, and recovery rate are the crucial factors to be addressed. Without any benefit of doubt, production of biodiesel using renewable feedstocks and enzymes as the catalysts could be recommended for the commercial purpose, but further research on improving the efficiency could be an advantage.

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.

Screening of antitubercular compound library identifies novel ATP synthase inhibitors of Mycobacterium tuberculosis.

A limited number of anti-tuberculosis drug candidates with novel mode of action have entered clinical trials in recent years. ATP synthase is one such validated drug target which has yielded a drug recently. The aim of this study was to identify the novel chemical scaffolds targeting the Mycobacterium tuberculosis (M. tuberculosis) ATP synthase. In this study, inverted membrane vesicles of Mycobacterium smegmatis were prepared to establish luciferin based ATP estimation assay. This assay was used to screen 700 compounds which were earlier found to be active on the whole cell of M. tuberculosis. Antibacterial activity of hits against various susceptible and drug-resistant strains of M. tuberculosis was evaluated using the microplate alamar blue assay and their cytotoxicity was also determined to select the safe compounds for further study. Screening of 700 compounds resulted in the identification of two compounds (5228485 and 5220632) exhibiting an IC50 of 0.32 and 4.0 µg/ml respectively. Both compounds showed excellent anti-TB activity (MIC of 0.5-2.0 µg/ml against Mtb H37Rv) and low cytotoxicity in human cell line and sub-mitochondrial particles. The three-dimensional structure of M. tuberculosis ATPase was predicted using in-silico approach and docking studies were performed with the active compounds. The interaction between compounds and bacterial ATP synthase was confirmed by molecular docking analysis. In conclusion screening of compound library has resulted in the identification of two novel chemical scaffolds targeting mycobacterial ATP synthase.