Abstract
Bacterial resistance towards aminoglycoside antibiotics mainly occurs because of aminoglycoside phosphotransferases (APHs). It is thus necessary to provide a rationale for focusing inhibitor development against APHs. The nucleotide triphosphate (NTP) binding site of eukaryotic protein kinases (ePKs) is structurally conserved with APHs. However, ePK inhibitors cannot be used against APHs due to cross reactivity. Thus, understanding bacterial resistance at the atomic level could be useful to design new inhibitors against such resistant pathogens. Hence, we carried out in vitro studies of APH from newly deposited multidrug-resistant organism Bacillus subtilis subsp. subtilis strain RK. Enzymatic modification studies of different aminoglycoside antibiotics along with purification and characterization revealed a novel class of APH, i.e., APH(5), with molecular weight 27 kDa approximately. Biochemical analysis of virtually screened inhibitor ZINC71575479 by coupled spectrophotometric assay showed complete enzymatic inhibition of purified APH(5). In silico toxicity study comparison of ZINC71575479 with known inhibitor of APH, i.e., tyrphostin AG1478, predicted its acceptable values for 96 h fathead minnow LC50, 48 h Tetrahymena pyriformis IGC50, oral rat LD50, and developmental toxicity using different QSAR methodologies. Thus, the present study gives novel insight into the aminoglycoside resistance and inhibition mechanism of APH(5) by applying experimental and computational techniques synergistically.
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Acknowledgments
KDS is gratefully acknowledged the University Grants Commission, New Delhi for financial support under UGC SAP Phase II programme [vide letter No. F. 4-8/2015/DRS-II (SAP-II)] sanctioned to Department of Biochemistry, Shivaji University, Kolhapur. RSP is thankful to UGC for providing BSR fellowship under UGC SAP DRS Phase I programme [vide letter No. F.7-207/2009 (BSR)]. SSB is grateful to DST PURSE-II scheme sanctioned to Shivaji University, Kolhapur, for providing fellowship. KDS is also thankful to DST SERB, New Delhi for providing infrastructural facility under project (Ref. No. EMR/2017/002688/BBM dated 25th October 2018). The authors are thankful to Department of Microbiology, Shivaji University, Kolhapur, for providing infrastructural facilities. Authors gratefully acknowledge to Department of Biotechnology, Shivaji University, Kolhapur for providing LCMS facility.
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Parulekar, R.S., Barale, S.S. & Sonawane, K.D. Antibiotic resistance and inhibition mechanism of novel aminoglycoside phosphotransferase APH(5) from B. subtilis subsp. subtilis strain RK. Braz J Microbiol 50, 887–898 (2019). https://doi.org/10.1007/s42770-019-00132-z
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DOI: https://doi.org/10.1007/s42770-019-00132-z