CHEMICAL GENETIC APPROACHES TO COMBAT MULTIPLE DRUG RESISTANCE IN GRAM-NEGATIVE BACTERIA

Abstract

Decline in the antibacterial drug discovery along with the emergence of multiple drug resistance in Gram negative bacterial pathogens poses a serious threat to the human health. Over a period of last 50 years, only a few antibacterial drugs have been introduced to the market and most of the clinically useful old antibiotics have become ineffective against many bacterial pathogens. Many of the infectious diseases, in particularly nosocomial (hospital acquired) infections caused by the Gram negative bacteria are almost incurable. Acinetobacter baumannii,the leading cause of hospital acquired infection worldwide is an example of such a grave situation. Various mutants of the organism are resistant to almost all the antibiotics available in the market. Discovery of novel antibacterial molecules with new therapeutic targets and reviving the efficacy of old antibiotics are the only solution of the problem. This study is an endeavor to that direction by employing a small molecule library. Phenotype based whole cell screening was adopted for screening a small molecule library of 10956 molecules against Gram negative bacteria. Screening led to the identification of IITR06144, a small molecule which is effective against a broad range of bacterial pathogens. The mode of action of the molecule was investigated by using molecular genetic techniques. The broad spectrum molecule was found to interfere with bacterial cell division. It restricts two daughter cells to separate from each other during cell division by inhibiting FtsZ, a major protein involved in septum formation during cytokinesis. In another approach, 8000 molecules were screened to discover a novel efflux pump inhibitor (EPI) of AbeM, a proton driven multidrug transporter in A. baumannii. Fluoroquinolones are the major substrates of AbeM and expression of this efflux pump confer fluoroquinolone resistance in A.baumannii. The objective of the study was to identify a molecule which can potentiate the efficiency of fluoroquinolones against the drug resistant A.baumannii. Elevated resistance of E.coli KAM32 (devoid of major efflux pumps) harboring multiple copies of abeM in a multicopy plasmid has been exploited for the screening. IITR08027, a small molecule (not inhibitory by its own) has been identified which causes a significant fold (16-32 fold) of reduction in Minimum Inhibitory Concentrations (MICs) of both ciprofloxacin and norfloxacin against clinical isolate of A.baumannii express AbeM or its homologues. The small molecule has been characterized and established as an efflux pump II inhibitor. The advantage of using the molecule in combination with ciprofloxacin against fluoroquinolone resistant A.baumannii was evaluated by chemical genetic studies.