Hijacking intrinsic resistance in Mycobacterium abscessus with small molecule prodrugs
This proposal aims to develop narrow-spectrum prodrug-activated protein synthesis inhibitors to treat Mycobacterium abscessus (Mabs) infections. It builds on a recent discovery in our program, which elucidated a unique mechanism for prodrug activation of florfenicol amine (FFA) by hijacking the protective methods Mabs uses to block antibiotic action. Non-tuberculous mycobacteria (NTM) are emerging pathogens with high intrinsic drug resistance.  Mabs is the most pathogenic refractory NTM member,1-3 and infections with this pathogen are associated with especially poor clinical outcomes, similar to XDR-TB (extensively drug-resistant tuberculosis)1.  Florfenicol (FF) is a broad-spectrum analog of chloramphenicol (CAM), is orally bioavailable, safely used in agriculture as it lacks some of the pharmacological liabilities of CAM, and it mitigates plasmid-borne resistance by blocking the primary-hydroxy site of inactivation. FFA is the primary metabolite found in many host species after FF administration. Through routine MIC screening of synthetic intermediates, we discovered that FFA on its own had appreciable WhiB7 transcription factor-dependent MIC activity against Mabs.  Further studies demonstrated that in Mabs FFA acts as a prodrug that is acylated into the active form (FFac) within Mabs by Eis2, a Mabs specific acetyltransferase. eis2 is located in the whiB7 resistome, which results in a feed-forward mechanism of action between FFA, eis2, and whiB7, ultimately increasing the conversion of FFA to FFac, resulting in increased sensitivity. FFA was found to be highly synergistic with other protein synthesis inhibitors and resistance to FFA by inactivation of whiB7 or eis2 results in increased sensitivity to aminoglycosides and macrolides. Since eis2 is only present in Mabs and other closely related species, FFA has a narrow spectrum of activity and avoids the potential for mitochondrial cytotoxicity. Preliminary pharmacological studies demonstrate FFA is noncytotoxic and orally bioavailable. Overall design: mRNA profiles of Mycobacterium abscessus ATCC19977 strain after exposure to 1/2 minimal inhibitory concentration of chloramphenicol (256 mg/L) for 30 minutes, florfenicol and florfenicol amine at 25 µM for 30 minutes and 3 hours, and vehicle control at 30 minutes and 3 hours.