Protein reactivity of natural product-derived γ-butyrolactones.

The discovery of novel and unique target-drug pairs for the treatment of human diseases such as cancer and bacterial infections is an urgent goal of chemical and pharmaceutical sciences. Natural products represent an inspiring source of compounds for designing chemical biology methods with applications in target identification and characterization. Inspired by the huge structural diversity of γ-butyrolactones, which constitute up to 10% of all known compounds of natural origin, we extended the "activity-based protein profiling" (ABPP) target identification technology to this promising and so far unexplored natural compound class. We designed and synthesized a comprehensive set of natural product-derived γ-lactones and thiolactones that varied in protein reactivity. Several important bacterial enzymes that are involved in diverse cellular functions such as metabolism (dihydrolipoyl dehydrogenase and 6-phosphofructokinase), cell wall biosynthesis (MurA1 and MurA2), and protein folding (trigger factors) were obtained. Especially protein folding in bacteria could represent a novel strategy for antibiotic intervention and requires chemical tools for characterization and inhibition. Future studies that extend structural modifications to protein reactive α-methylene-γ-butyrolactone as well as to reversible binding γ-lactones and thiolactones will reveal if this premise holds true.

Beta-lactam probes as selective chemical-proteomic tools for the identification and functional characterization of resistance associated enzymes in MRSA.

With the development of antibiotic resistant bacterial strains, infectious diseases have become again a life threatening problem. One of the reasons for this dilemma is the limited number and breadth of current therapeutic targets for which several resistance strategies have evolved over time. To identify resistance associated targets and to understand their function, activity, and regulation, we utilized a novel strategy based on small synthetic beta-lactam molecules that were applied in activity based protein profiling experiments (ABPP) to comparatively profile in situ enzyme activities in antibiotic sensitive and resistant S. aureus strains (MRSA). Several enzyme activities which are unique to the MRSA strain including known resistant associated targets, involved in cell wall biosynthesis and antibiotic sensing, could be identified. In addition, we also identified uncharacterized enzymes which turned out to be capable of hydrolyzing beta-lactam antibiotics. This technology could therefore represent a valuable tool to monitor the activity and function of other yet unexplored resistance associated enzymes in pathogenic bacteria and help to discover new drug targets for customized therapeutic interventions.

Beta-lactams as selective chemical probes for the in vivo labeling of bacterial enzymes involved in cell wall biosynthesis, antibiotic resistance, and virulence.

With the development of antibiotic-resistant bacterial strains, infectious diseases have become again a life-threatening problem. One of the reasons for this dilemma is the limited number and breadth of current therapeutic targets for which several resistance strategies have evolved over time. To expand the number of addressable enzyme targets and to understand their function, activity, and regulation, we utilized a chemical proteomic strategy, called activity-based protein profiling (ABPP) pioneered by Cravatt, for the identification of beta-lactam-binding enzymes under in vivo conditions. In this two-tiered strategy, we first prepared a selection of conventional antibiotics for labeling diverse penicillin binding proteins (PBPs) and second introduced a new synthetic generation of beta-lactam probes, which labeled and inhibited a selection of additional PBP unrelated bacterial targets. Among these, the virulence-associated enzyme ClpP and a resistance-associated beta-lactamase were labeled and inhibited by selected probes, indicating that the specificity of beta-lactams can be adjusted to versatile enzyme families with important cellular functions.