Selection and characterization of alanine racemase inhibitors against Aeromonas hydrophila

Abstract

BackgroundCombining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors.ResultsWe identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 μM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 μg/ml. These compounds have no activity on horseradish peroxidase and d-amino acid oxidase at a concentration of 50 μM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 μM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 μM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase.ConclusionsOur findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-1010-x) contains supplementary material, which is available to authorized users.

Cloning, Biochemical Characterization and Inhibition of Alanine racemase fromStreptococcus iniae

“…Many studies have focused on alanine racemase to develop antibacterial drugs for multiple bacterial species (Scaletti et al, 2012;Shrestha et al, 2017). Unlike D-cycloserine which is a cyclic analog of alanine and exerted its inhibitory effect through interaction with the enzymebound PLP cofactor (Batson et al, 2017), both homogentisic acid and hydroquinone are not structural analogs of Alr as such they are not interfering with other PLP dependent enzymes (Wang et al, 2017). According to the results of antimicrobial activity assay, the two inhibitors are capable of inhibiting both gram-positive and gram-negative bacteria with varying efficacies except Pseudomonas aeruginosa.…”

“…Many inhibitors of alanine racemase have been discovered (Wang et al, 2017). The enzyme almost completely lost its activity after treatment with 1 and 10 mM hydroxylamine.…”

“…In our previous study, we found that homogentisic acid and hydroquinone are two alanine racemase inhibitors with minimal cytotoxicity against mammalian cells and can be utilized as potential agents of antibiotics (Wang et al, 2017). In this study we investigated the inhibitory effects of homogentisic acid and hydroquinone on SiAlr, DMSO was used as the blank control and DCS, a known alanine racemase inhibitor, as the positive control.…”

“…Alanine racemase racemization assay was conducted in two coupled enzyme reactions, using standard racemization mixture (Wang et al, 2017).…”

“…The bacterial cell wall is indispensable for the survival and viability of bacteria and has always been an interesting target for many antibiotics and antimicrobial agents (Anthony et al, 2011). Alanine racemase is ubiquitous among bacteria and rare in eukaryotes but absent in humans (Kawakami et al, 2018), hence it emerges as an attractive and potential therapeutic target for the antimicrobial drug (Wang et al, 2017). Numerous inhibitors were identified as able to affect the activity of alanine racemase (Kim et al, 2003a;Kim et al, 2003b).…”