Synthetic group A streptogramin antibiotics that overcome Vat resistance.

Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Modular, Scalable Synthesis of Group A Streptogramin Antibiotics.

Streptogramin antibiotics are used clinically to treat multidrug-resistant bacterial infections, but their poor physicochemical properties and narrow spectra of activity have limited their utility. New methods to chemically modify streptogramins would enable structural optimization to overcome these limitations as well as to combat growing resistance to the class. Here we report a modular, scalable synthesis of group A streptogramin antibiotics that proceeds in 6-8 linear steps from simple chemical building blocks. We have applied our route to the synthesis of four natural products in this class including two that have never before been accessed by fully synthetic routes. We anticipate that this work will lead to the discovery of new streptogramin antibiotics that overcome previous limitations of the class.

Identification of novel inhibitors of the streptogramin group A acetyltransferase via virtual screening.

Virginiamycin acetyltransferase D (VatD) plays a vital rule in streptogramins resistance by chemically inactivating streptogramin A. Therefore, it is desirable to discover novel small molecular weight inhibitors of VatD via state-of-the-art virtual screening techniques. This "cocktail" strategy by combining VatD inhibitor with streptogramins may provide new therapeutic opportunity for resistant bacteria infections. Structure-based virtual screening method (molecular docking) was applied to rank and score a chemical database containing 300 000 commercially available compounds against the VatD substrate binding site. Twenty six out of the 200 top scored compounds from the docking calculation were selected and submitted to the VatD enzymatic inhibition assay. The plasmid pRSET B/vatD was constructed and transformed into E. coli (trxB) host cells for over-expression, and VatD enzyme was purified and validated by showing acetyltransferase activity to Virginiamycin M1. Three out of these 26 tested compounds showed enzymatic inhibition on VatD with IC50 168.6, 91.0 and 55.2 micromol x L(-1), separately. Other compounds could not be dissolved in the system and/or had little effect on the enzyme (IC50 > 200 micromol x L(-1)). To our knowledge, it is first time that small molecular weight organic compounds were identified as VatD inhibitors. It is expected that the VatD inhibitors identified at present study could serve as lead compounds for the further development of the novel therapeutic agents to overcome streptogramins resistance.

Streptogramin antibiotics: mode of action and resistance.

The streptogramin antibiotics were discovered over 40 years ago but are only now emerging as important therapeutic agents for the treatment of infection caused by a variety of bacteria. The streptogramins consist of mixtures of two structurally distinct compounds, type A and type B, which are separately bacteriostatic, but bactericidal in appropriate ratios. These antibiotics act at the level of inhibition of translation through binding to the bacterial ribosome. Resistance to streptogramins occurs through a number of mechanisms including target modification, efflux, and enzyme catalyzed antibiotic modification. This review describes the current understanding of streptogramin function and resistance with emphasis on molecular mechanism and epidemiology.

Crystal structure of Vat(D): an acetyltransferase that inactivates streptogramin group A antibiotics.

The streptogramin class of antibiotics act to inhibit bacterial protein synthesis, and their semisynthetic derivatives, such as dalfopristin-quinupristin (Synercid), are used to treat serious or life-threatening infections due to multiply antibiotic resistant bacteria. Acquired resistance of the nosocomial pathogen Enterococcus faecium to the group A component of natural and semisynthetic streptogramin mixtures is a prerequisite for the streptogramin resistance phenotype and is mediated by a streptogramin acetyltransferase. The crystal structure of Vat(D), a streptogramin acetyltransferase from a human urinary isolate of E. faecium, has been determined as an apoenzyme and in complex with either acetyl-CoA or virginiamycin M1 and CoA. These structures illustrate the location and arrangement of residues at the active site, and point to His 82 as a residue that may function as a general base. The structural similarity of Vat(D) to the xenobiotic acetyltransferase from Pseudomonas aeruginosa indicates similarities in the catalytic mechanism for these enzymes as well as several shared and distinctive antibiotic binding interactions between these enzymes and their respective substrates. These results reveal the molecular basis for a reaction by which Gram-positive cocci acquire resistance to a last resort antibiotic.