Synthetic Simplification of Carolacton Enables Chemical Genetic Studies in Streptococcus mutans

Abstract

Understanding the broader biological impact of carolacton, a macrolactone natural product, has been ongoing for the past decade. Multiple studies have shown connections to regulatory systems, acid tolerance mechanisms, biofilm formation, and recently folate dehydrogenase (FolD). Progress elucidating the cause of biofilm-specific activity in Streptococcus mutans has been limited due to low-throughput analyses of carolacton-treated cells. We disclose the discovery of a simplified carolacton-inspired analog that demonstrates inhibitory activity against S. mutans biofilm cells. This discovery permitted a proof of concept chemical genetic screen of S. mutans mutants identifying the carbon catabolite protein A signaling pathway as a putative target.

A look around the West Indies: The spices of life are secondary metabolites

“… 16 This finding also showed the importance of chain length and, in a follow up paper in 2019, we were able to simplify the carolacton side chain while oversaturating the carolacton macrocycle to produce a drastically simplified analog, (+)-2 ( 3 ).This analog not only maintained activity with an IC 50 of 44 μM but also allowed for the first observance of a minimum inhibitory concentration (MIC) value for any compound structurally related to carolacton at 250 μM. 17 This analog effectuated the preliminary screening of S. mutans mutants, aiding in the identification of a carbon catabolite control protein (Ccpa) as the putative target of 3 . Hence, this analog provided an additional tool for which further biological data could be garnered in future assays.…”

An Update on Pharmacological Relevance and Chemical Synthesis of Natural Products and Derivatives with Anti SARS‐CoV‐2 Activity

“…A natural product Carolacton ( 14 ) (Figure 4), is a macrolide keto‐carboxylic acid produced by the myxobacterium Sorangium cellulosum with a wide biological profile. The comprehensive evaluations have shown an association of 14 with regulatory systems, acid tolerance mechanism, biofilm formation, and folate dehydrogenase [85–86] . It acts as a promising inhibitor of biofilm formation in the human pathogen Streptococcus mutant , a growth inhibitor of S. pneumonia , and an inhibitor of efflux pump mutant Escherichia coli ΔtolC [87–89] …”

Collaboration in Natural Product Total Synthesis: Carolacton – A Decade of Discovery

“…For our second generation, we aimed to make eight analogs that were designed to have a flexible sidechain by removing the aryl bioisostere (27) (Scheme 4). 22 Once again, the diverted synthesis strategy was utilized to access these analogs, and a simplified structure, coined 'analog 2', was identified. Structurally, carolacton and analog 2 shared key features such as the terminal carboxylic acid and length of the sidechain.…”

“…Through the investigation, we discovered that ΔccpA was not susceptible to analog 2. 22 This gene codes for carbon catabolite protein A, CcpA, and is responsible for the regulation of EPS formation, cell attachment, acetate metabolism branch-chain amino acid synthesis, oxidative stress tolerance, and other mechanisms tied to the acid tolerance response of S. mutans. In 2014, a transcriptomic screen completed by Sudhakar et al, showed that CcpA was one of seven transcriptional regulators to be highly coordinated to carolacton's biological activity.…”

“…11 These findings were emphasized further when Kirschning and co-workers published compounds 18-24. 12,19,20 By masking the carboxylic acid with a methyl ester (22) and by tethering the sidechain to form a secondary macrocyclic structure (24), they dis-covered that the ester would hydrolyze in cell culture conditions, revealing the carboxylic acid, which is paramount for activity. As a result, these analogs demonstrated biofilm-specific activity but only because the resulting active metabolite was the natural product carolacton.…”