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Lipoic acid (LA) is an essential cofactor in prokaryotic and eukaryotic organisms, required for the function of several multienzyme complexes such as oxoacid dehydrogenases. Prokaryotes either synthesize LA or salvage it from the environment. The salvage pathway in Staphylococcus aureus includes two lipoate-protein ligases, LplA1 and LplA2, as well as the amidotransferase LipL. In this study, we intended to hijack the salvage pathway by LA analogues that are transferred via LplA2 and LipL to the E2 subunits of various dehydrogenases, thereby resulting in nonfunctional enzymes that eventually impair viability of the bacterium. Initially, a virtual screening campaign was carried out to identify potential LA analogues that bind to LplA2. Three selected compounds affected S. aureus USA300 growth in minimal medium at concentrations ranging from 2.5 to 10 μg/mL. Further analysis of the most potent compound ( Lpl-004 ) revealed its transfer to E2 subunits of dehydrogenase complexes and a negative impact on its functionality. Growth impairment caused by Lpl-004 treatment was restored by adding products of the lipoate-dependent enzyme complexes. In addition, Caenorhabditis elegans infected with LpL-004 -treated USA300 demonstrated a significantly expanded lifespan compared to worms infected with untreated bacteria. Our results provide evidence that LA analogues exploiting the LA salvage pathway represent an innovative strategy for the development of novel antimicrobial substances.

The serine protease Caseinolytic protease subunit P (ClpP) plays an important role for protein homeostasis in bacteria and contributes to various developmental processes, as well as virulence. Therefore, ClpP is considered as a potential drug target in Gram-positive and Gram-negative bacteria. In this study, we utilized a biochemical assay to screen several small molecule libraries of approved and investigational drugs for Escherichia coli ClpP inhibitors. The approved drugs bortezomib, cefmetazole, cisplatin, as well as the investigational drug cDPCP, and the protease inhibitor 3,4-dichloroisocoumarin (3,4-DIC) emerged as ClpP inhibitors with IC 50 values ranging between 0.04 and 31 µM. Compound profiling of the inhibitors revealed cefmetazole and cisplatin not to inhibit the serine protease bovine α-chymotrypsin, and for cefmetazole no cytotoxicity against three human cell lines was detected. Surface plasmon resonance studies demonstrated all novel ClpP inhibitors to bind covalently to ClpP. Investigation of the potential binding mode for cefmetazole using molecular docking suggested a dual covalent binding to Ser97 and Thr168. While only the antibiotic cefmetazole demonstrated an intrinsic antibacterial effect, cDPCP clearly delayed the bacterial growth recovery time upon chemically induced nitric oxide stress in a ClpP-dependent manner.

Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin). Thiamin in its activated form, thiamin pyrophosphate, is an essential co-factor for all organisms. Therefore, thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional co-factors by the bacterium can function as pro-drugs which thus block various co-factor dependent pathways. We characterized one of the key enzymes within the S. aureus vitamin B1 biosynthetic pathway, 5-(hydroxyethyl)-4-methylthiazole kinase (SaThiM; EC 2.7.1.50), a potential target for pro-drug compounds and analyzed the native structure of SaThiM and complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues.