Evaluation of Inhibition of Bacterial Acetohydroxyacid Synthase as an Antimicrobial Target

Abstract

The branched chain amino acids (BCAAs) are synthesized by plants, algae, fungi, and bacteria but not by animals. Thus, the enzymes of the BCAA biosynthetic pathway are potential targets for the development of herbicides, fungicides, and antimicrobial compounds. Acetohydroxyacid synthase (AHAS) is a ThDP and FADdependent enzyme which catalyzes the first step in the BCAAs biosynthesis pathway. AHAS catalyzes the synthesis of acetolactate from two molecules of pyruvate or 2-aceto-2-hydroxybutyrate from pyruvate and 2-ketobutyrate. All AHASs are composed of catalytic subunits (CSUs), as well as separate, regulatory subunits (RSUs). Interest in targeting AHAS arose after the findings of several structurally unrelated classes of herbicides, including the sulfonylureas, imidazolinones, triazolopyrimidines, and pyrimidyl-oxybenzoates, specifically inhibit the enzyme. Moreover, previous studies of leucine and isoleucine-valineleucine auxotrophs of Mycobacterium tuberculosisH37Rv, leucine auxotrophs of M. bovis BCG, and the AHAS mutant of Burkholderia pseudomallei have also shown that the AHAS of pathogenic microorganisms could be a target for antimicrobial agents. However, the development strategy of AHAS inhibitors as antimicrobial agents was underwhelming given that most, if not all, of the organisms may obtain BCAAs from their environment and perhaps able to reverse the effect of these inhibitors. In fact, in certain intracellular and respiratory pathogens, the inhibition of AHAS is proved to be a potential strategy in developing antimicrobial agents. BCAA biosynthesis is required for survival and virulence of these pathogens in their infection sites in the body where the limited availability of BCAAs was measured. Here, we used the strategy of targeting AHAS as a new antimicrobial target and focused on the development of AHAS inhibitors. To date, we used the AHAS of pathogenic bacteria, Bacillus anthracis (Bantx) and Haemophilus influenzae (Hin), as candidates and/or sample organisms. In this study, genes of AHAS from both the Bantx and Hin were cloned, over expressed in Escherichia coli, and purified to homogeneity. To develop novel anti-anthracis and anti-influenzae drugs that inhibit AHAS, a chemical library was screened against Bantx and Hin-AHASs by using a high-throughput screening, and identified potent inhibitors, each representing a structural class with an IC50 in the low micro molar range (1.0 � 14.6 �M). The most potent hit chemicals identified against Bantx-AHAS were, AVS2087, AVS2093, AVS2387, and AVS2236. The basic scaffold of the AVS group consists of 1-pyrimidin-2-yl-1H-[1,2,4]triazole-3-sulfonamide. Subsequently, the potent hit chemicals identified against Hin-AHAS were, oxa-1-thia-4-aza3 cyclopenta[b]naphthalene (KHG25229), phenyl-2,3-dihydro-isothiazole (KHG25386), and phenyl-pyrrolidine-3-carboxylic acid phenylamide (KHG25056). The inhibition kinetics and molecular docking of the candidate hit chemicals revealed the inhibition mechanism and possible binding modes, respectively. As the need for novel antibiotic classes to combat bacterial drug resistance increases, the screening of new compounds that act against Bantx and Hin-AHASs shows the possibility of AHAS as a promising target for new antimicrobial drugs. Although the strategy of AHAS inhibition was used here for Bantx and Hin as sample organisms, this approach may also be useful for the development of other pathogenic bacterial AHAS.