Structure of ThiM from Vitamin B1 biosynthetic pathway of Staphylococcus aureus - Insights into a novel pro-drug approach addressing MRSA infections.

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.

MRSA infections: from classical treatment to suicide drugs.

Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today a major burden in nosocomial disease control. The global trend shows an alarming increase of MRSA infections as well as multi-drug resistance (MDR). The problem is exacerbated by the fact that infections with community-associated (CA) MRSA strains showing increased virulence and fitness add to infections with multi-drug resistant hospital-associated (HA) MRSA. The toxicity of pathogens and limited effectiveness of available treatment have led to high mortality rates and vast expenses caused by prolonged hospitalization and usage of additional antibiotics. Recently approved drugs still have classical targets and upcoming resistance can be expected. In a new approach by targeting co-factor syntheses of bacteria, the drug target and the affected pathways are uncoupled. This novel strategy is based on the thought of a classical pro-drug which has to be metabolized before becoming toxic for the bacterium as a dysfunctional co-factor, named suicide drug. Ideally these metabolizing pathways are solely present in the bacterium and absent in the human host, such as vitamin biosyntheses. This mini-review discusses current ways of MRSA infection treatment using new approaches including suicide drugs targeting co-factor biosyntheses.

Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution.

To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several "shift" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics® technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution.