The number of patients treated for antibiotics-resistant infections has increased drastically in recent years. What started as a problem primarily associated with hospital-acquired Enterococcus infections, has not only moved into the general community, but also grown to include a number of widespread and serious pathogens. Drug-resistant Streptococci, Staphylococci and Pseudomonas strains are quite common. Currently as many as 70% of hospital-acquired infections in the US are resistant to at least one antibiotic, and about 40% of S. aureus infections are multidrug-resistant. Even powerful drugs like Vancomycin and Teicoplanin, which for years represented the "agents of last resort" for treatment of antibiotics-resistant infections, are no longer efficacious against certain strains of bacteria. It is important to realize, that the loss of efficacy of these compounds leaves very few treatment options for patients with multi-drug resistant infections. Clearly, there is an immediate unmet need for new antibiotics with novel modes of action. Moreover, the overall market for antibacterial drugs is large and growing - world-wide sales are projected to reach $30 billion in 2006. Several currently used antibacterial drugs belong to a group of natural products compounds known as cyclic peptides. Cyclic peptides are isolated from microorganisms such as bacteria or fungi. They are typically very specific, potent and efficacious drugs, but their use can be associated with significant side effects, and for some compounds the side effects are severe enough to impede clinical use. Moreover, although it may be known that the side effects can be reduced or even abolished by alterations to the structure of a cyclic peptide, this is rarely done. The reason for this is that the complexity and chemical properties of most cyclic peptides makes them unsuitable for cost-effective modifications, using currently available synthetic chemistry approaches. Compounds altered by this approach simply become too expensive. The overall goal of the project outlined in this proposal is to use a novel Chemo-Enzymatic Synthesis (CES) approach to identify the structural modifications required to eliminate, or at least significantly reduce, the nephrotoxicity associated with a cyclic peptide antibiotic that currently cannot be used systemically. The CES approach combines automated solid-phase peptide synthesis of linear precursor peptides, with enzymatic cyclization using a recombinant thioesterase (TE) domain derived from the relevant Non Ribosomal Peptide Synthetase (NRPS) complex. The approach effectively circumvents the most daunting problem associated with preparing cyclic peptides by synthetic chemistry: efficient, regiospecific cyclization of the linear precursor. Using CES, the assembly of modified cyclic peptides is simplified considerably and can be done at a cost compatible with commercial development. Successful achievement of the project goals will allow a considerably broader, systemic use of a very potent, broad-spectrum antibiotic and also generate a valuable addition to the current small inventory of drugs capable of treating multi drug-resistant infections. Due to a drastic increase in multi-drug resistant infections, in the last two decades, there is an immediate unmet need for new antibacterial drugs with novel modes of action. The proposed project will add a potent, efficacious, well-tolerated and economical antibacterial drug to a currently quite limited inventory of compounds with efficacy towards essentially all clinically relevant Gram-positive organisms, including the multi drug- resistant pathogens
