IPA-3: An Inhibitor of Diadenylate Cyclase of Streptococcus suis with Potent Antimicrobial Activity

Abstract

Antimicrobial resistance (AMR) poses a huge threat to public health. The development of novel antibiotics is an effective strategy to tackle AMR. Cyclic diadenylate monophosphate (c-di-AMP) has recently been identified as an essential signal molecule for some important bacterial pathogens involved in various bacterial physiological processes, leading to its synthase diadenylate cyclase becoming an attractive antimicrobial drug target. In this study, based on the enzymatic activity of diadenylate cyclase of Streptococcus suis (ssDacA), we established a high-throughput method of screening for ssDacA inhibitors. Primary screening with a compound library containing 1133 compounds identified IPA-3 (2,2′-dihydroxy-1,1′-dinapthyldisulfide) as an ssDacA inhibitor. High-performance liquid chromatography (HPLC) analysis further indicated that IPA-3 could inhibit the production of c-di-AMP by ssDacA in vitro in a dose-dependent manner. Notably, it was demonstrated that IPA-3 could significantly inhibit the growth of several Gram-positive bacteria which harbor an essential diadenylate cyclase but not E. coli, which is devoid of the enzyme, or Streptococcus mutans, in which the diadenylate cyclase is not essential. Additionally, the binding site in ssDacA for IPA-3 was predicted by molecular docking, and contains residues that are relatively conserved in diadenylate cyclase of Gram-positive bacteria. Collectively, our results illustrate the feasibility of ssDacA as an antimicrobial target and consider IPA-3 as a promising starting point for the development of a novel antibacterial.

Computer-aided design of a cyclic di-AMP synthesizing enzyme CdaA inhibitor

“…Despite the undeniable structural similarity between DAC domains of DisA and CdaA, the majority of DisA inhibitors do not show inhibitory effects on CdaA, except of bromophenol (Chen et al 2018 ), which was shown to inhibit both DAC domain containing enzymes. The second known CdaA inhibitor, IPA-3 (2,2′-dihydroxy-1,1′-dinapthyldisulfide) (Li et al 2022 ), containing naphthyl groups, has revealed potent antimicrobial activity. Unfortunately, no structural data has been obtained for any of those inhibitors, hence structure-based optimization of those compounds is not yet feasible.…”

Cyclic-di-AMP signalling in lactic acid bacteria

“…A DAC inhibitor compound called ST056083, which was identified from a 1000 compound library (Zheng et al 2014 ), was shown to inhibit c-di-AMP levels, EPS production, and biofilm formation in E. faecalis (Chen et al 2018 ). In other work, an inhibitor called IPA-3 was identified from a screen of 1133 compounds using the Streptococcus suis CdaA enzyme (Li et al 2022 ). This compound was able to inhibit the growth of a number of different c-di-AMP producing Gram-positive bacteria, but not E. coli , which does not make c-di-AMP (Li et al 2022 ).…”

“…In other work, an inhibitor called IPA-3 was identified from a screen of 1133 compounds using the Streptococcus suis CdaA enzyme (Li et al 2022 ). This compound was able to inhibit the growth of a number of different c-di-AMP producing Gram-positive bacteria, but not E. coli , which does not make c-di-AMP (Li et al 2022 ).…”

Structure-based Search for novel c-di-AMP Synthase Inhibiting Fragments

“…Moreover, the usage of kinase inhibitors as starting points for the development of new antibiotics will be discussed with a special focus on the inhibitory effect of Ruxolitinib on CdaA. Furthermore, inhibition by Ruxolitinib will be compared to known DAC inhibitors IPA-3 and Br-TH (Chen et al, 2018;Li et al, 2022). Finally, the success of a fragment screen campaign based on CdaA from Bacillus subtilis is discussed, with an outlook as to which methods could be applied to develop the identified compounds into a possible lead compound.…”

“…To put this into context, it would be insightful to carry out similar experiments with the published CdaA inhibitors Bromophenol-Thiohydantoin (Br-TH) and IPA-3 (Chen et al, 2018;Li et al, 2022). While IPA-3 was identified using a kinase inhibitor library, bromophenol derivatives are also known for their inhibitory effects on kinases (Wang et al, 2015).…”