@article {8462, title = {A curated list of targeted optimized promiscuous ketoreductases (TOP-K). [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Biochem J}, volume = {480}, year = {2023}, month = {2023 Jul 12}, pages = {975-997}, abstract = {

Enzymes are either specific or promiscuous catalysts in nature. The latter is portrayed by protein families like CYP450Es, Aldo-ketoreductases and short/medium-chain dehydrogenases which participate in detoxification or secondary metabolite production. However, enzymes are evolutionarily {\textquoteright}blind{\textquoteright} to an ever-increasing synthetic substrate library. Industries and laboratories have circumvented this by high-throughput screening or site-specific engineering to synthesize the product of interest. However, this paradigm entails cost and time-intensive one-enzyme, one-substrate catalysis model. One of the superfamilies regularly used for chiral alcohol synthesis are short-chain dehydrogenases/reductases (SDRs). Our objective is to determine a superset of promiscuous SDRs that can catalyze multiple ketones. They are typically classified into shorter {\textquoteright}Classical{\textquoteright} and longer {\textquoteright}Extended{\textquoteright} type ketoreductases. However, current analysis of modelled SDRs reveals a length-independent conserved N-terminus Rossmann-fold and a variable substrate-binding C-terminus substrate-binding region for both categories. The latter is recognized to influence the enzyme{\textquoteright}s flexibility and substrate promiscuity and we hypothesize these properties are directly linked with each other. We tested this by catalyzing ketone intermediates with the essential and specific enzyme: FabG_E, as well as non-essential SDRs such as UcpA and IdnO. The experimental results confirmed this biochemical-biophysical association, making it an interesting filter for ascertaining promiscuous enzymes. Hence, we created a dataset of physicochemical properties derived from the protein sequences and employed machine learning algorithms to examine potential candidates. This resulted in 24 targeted optimized ketoreductases (TOP-K) from 81 014 members. The experimental validation of select TOP-Ks demonstrated the correlation between the C-terminal lid-loop structure, enzyme flexibility and turnover rate on pro-pharmaceutical substrates.

}, keywords = {ketoreductases, machine learning, medium-chain dehydrogenases, short-chain dehydrogenases}, issn = {1470-8728}, doi = {10.1042/BCJ20230051}, url = {https://pubmed.ncbi.nlm.nih.gov/37335080/}, author = {Shanbhag, Anirudh P and Rajagopal, Sreenath and Ghatak, Arindam and Katagihallimath, Nainesh and Subramanian, Ramaswamy and Datta, Santanu} } @article {2598, title = {Validated In Silico Model for Biofilm Formation in Escherichia coli [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {ACS Synthetic Biology}, year = {2022}, month = {01/2022}, abstract = {

Using\ Escherichia coli\ as the representative biofilm former, we report here the development of an in silico model built by simulating events that transform a free-living bacterial entity into self-encased multicellular biofilms. Published literature on \~{}300 genes associated with pathways involved in biofilm formation was curated, static maps were created, and suitably interconnected with their respective metabolites using ordinary differential equations. Precise interplay of genetic networks that regulate the transitory switching of bacterial growth pattern in response to environmental changes and the resultant multicomponent synthesis of the extracellular matrix were appropriately represented. Subsequently, the in silico model was analyzed by simulating time-dependent changes in the concentration of components by using the R and python environment. The model was validated by simulating and verifying the impact of key gene knockouts (KOs) and systematic knockdowns on biofilm formation, thus ensuring the outcomes were comparable with the reported literature. Similarly, specific gene KOs in laboratory and pathogenic\ E. coli\ were constructed and assessed. MiaA, YdeO, and YgiV were found to be crucial in biofilm development. Furthermore, qRT-PCR confirmed the elevation of expression in biofilm-forming clinical isolates. Findings reported in this study offer opportunities for identifying biofilm inhibitors with applications in multiple industries. The application of this model can be extended to the health care sector specifically to develop novel adjunct therapies that prevent biofilms in medical implants and reduce emergence of biofilm-associated resistant polymicrobial-chronic infections. The in silico framework reported here is open source and accessible for further enhancements.

}, doi = {10.1021/acssynbio.1c00445}, url = {https://doi.org/10.1021/acssynbio.1c00445}, author = {Bhowmik, Purnendu and Rajagopal, Sreenath and Hmar, Rothangamawi Victoria and Singh, Purnima and Saxena, Pragya and Amar, Prakruthi and Thomas, Teby and Ravishankar, Rajani and Nagaraj, Savitha and Katagihallimath, Nainesh and Sarangapani, Ramanujan Kadambi and Ramachandran, Vasanthi and Datta, Santanu} } @article {1858, title = {Azaindole Based Potentiator of Antibiotics against Gram-Negative Bacteria [C-CAMP Startup Bugworks]}, journal = {ACS Infectious Diseases}, year = {2021}, month = {10/2021}, type = {Journal Article}, abstract = {
We discovered azaindole-based compounds with weak innate activity that exhibit substantial potentiation of antibacterial activities of different antibiotics, viz., rifampicin, erythromycin, solithromycin, and novobiocin in Gram-negative bacteria. In the presence of the azaindole derivatives, these antibiotics exhibited submicromolar minimum inhibitory concentrations (MICs) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The fold improvements in MIC of these antibiotics that were otherwise weak or inactive on their own against these bacteria were also observed against drug-resistant clinical isolates. Our studies indicate that this selective potentiation is probably through destabilization of the outer membrane{\textquoteright}s integrity, known to be regulated by the lipopolysaccharides (LPS). Thus, the azaindole based compounds described here open opportunities for those antibiotics that are otherwise ineffective due to LPS mediated entry barriers in Gram-negative bacteria.
}, keywords = {Azaindole, Bacterial Permeability, Lipopolysachhardies (LPS), Polymyxin, Synergy}, doi = {10.1021/acsinfecdis.1c00171}, url = {https://pubs.acs.org/doi/abs/10.1021/acsinfecdis.1c00171$\#$}, author = {Sharma, Sreevalli and Rao, Ranga and Reeve, Stephanie M and Phelps, Gregory A and Bharatham, Nagakumar and Katagihallimath, Nainesh and Ramachandran, Vasanthi and Raveendran, Savitha and Sarma, Maitrayee and Nath, Anubha} } @article {764, title = {Nitrothiophene carboxamides, a novel narrow spectrum antibacterial series: Mechanism of action and Efficacy [Bugworks Res. Pvt. Ltd., a C-CAMP Startup]}, journal = {Sci Rep}, volume = {8}, year = {2018}, month = {2018 May 08}, pages = {7263}, abstract = {

The mechanism of efflux is a tour-de-force in the bacterial armoury that has thwarted the development of novel antibiotics. We report the discovery of a novel chemical series with potent antibacterial properties that was engineered to overcome efflux liability. Compounds liable to efflux specifically via the Resistance Nodulation and cell Division (RND) pump, AcrAB-TolC were chosen for a hit to lead progression. Using structure-based design, the compounds were optimised to lose their binding to the efflux pump, thereby making them potent on wild-type bacteria. We discovered these compounds to be pro-drugs that require activation in E. coli by specific bacterial nitroreductases NfsA and NfsB. Hit to lead chemistry led to the generation of compounds that were potent on wild-type and multi-drug resistant clinical isolates of E. coli, Shigella spp., and Salmonella spp. These compounds are bactericidal and efficacious in a mouse thigh infection model.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-25407-7}, author = {Hameed P, Shahul and Bharatham, Nagakumar and Katagihallimath, Nainesh and Sharma, Sreevalli and Nandishaiah, Radha and Shanbhag, Anirudh P and Thomas, Teby and Narjari, Riya and Sarma, Maitrayee and Bhowmik, Purnendu and Amar, Prakruthi and Ravishankar, Rajani and Jayaraman, Ramesh and Muthan, Kubendran and Subbiah, Ramesh and Ramachandran, Vasanthi and Balasubramanian, V and Datta, Santanu} } @article {889, title = {A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation [Bugworks Res. Pvt. Ltd., a C-CAMP Startup]}, journal = {Microb Cell Fact}, volume = {17}, year = {2018}, month = {2018 Dec 03}, pages = {192}, abstract = {

INTRODUCTION: Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E.\ coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure\ to predict product chirality.

RESULTS: Six enzymes originating from Sulfolobus\ sulfotaricus, Zygosaccharomyces\ rouxii, Hansenula\ polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC\ 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole-cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E.\ coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z.\ rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565\ (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95\% (S)-specific alcohol with a chemical yield of 23-79\% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85-99\%.

CONCLUSIONS: Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces\ rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole-cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.

}, issn = {1475-2859}, doi = {10.1186/s12934-018-1036-2}, author = {Haq, Saiful F and Shanbhag, Anirudh P and Karthikeyan, Subbulakshmi and Hassan, Imran and Thanukrishnan, Kannan and Ashok, Abhishek and Sukumaran, Sunilkumar and Ramaswamy, S and Bharatham, Nagakumar and Datta, Santanu and Samant, Shalaka and Katagihallimath, Nainesh} }