Stephen K. Wrigley

Natural products to drugs: daptomycin and related lipopeptide antibiotics

Daptomycin (Cubicin) is a lipopeptide antibiotic approved in the USA in 2003 for the treatment of skin and skin structure infections caused by Gram-positive pathogens. It is a member of the 10-membered cyclic lipopeptide family of antibiotics that includes A54145, calcium-dependent antibiotic (CDA), amphomycin, friulimicin, laspartomycin, and others. This review highlights research on this class of antibiotics from 1953 to 2005, focusing on more recent studies with particular emphasis on the interplay between structural features and antibacterial activities; chemical modifications to improve activity; the genetic organization and biosynthesis of lipopeptides; and the genetic engineering of the daptomycin biosynthetic pathway to produce novel derivatives for further chemical modification to develop candidates for clinical evaluation.

Heterologous production of daptomycin in Streptomyces lividans

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128xa0kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the ϕC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55xa0mg/l.

A glutamic acid 3‐methyltransferase encoded by an accessory gene locus important for daptomycin biosynthesis in Streptomyces roseosporus

In many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3‐methyl‐glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium‐dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non‐ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3‐dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu12 instead of 3mGlu12. Complementation by only dptI, or its S.u2003coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu‐containing compounds in the mutant. Compared with A21978C, the Glu12‐containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.

Combinatorial biosynthesis of lipopeptide antibiotics in Streptomyces roseosporus

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. Cubicin® (daptomycin-for-injection) was approved in 2003 by the FDA to treat skin and skin structure infections caused by Gram-positive pathogens. Daptomycin is particularly significant in that it represents the first new natural product antibacterial structural class approved for clinical use in three decades. The daptomycin gene cluster contains three very large genes (dptA, dptBC, and dptD) that encode the nonribosomal peptide synthetase (NRPS). The related cyclic lipopeptide A54145 has four NRPS genes (lptA, lptB, lptC, and lptD), and calcium dependent antibiotic (CDA) has three (cdaPS1, cdaPS2, and cdaPS3). Mutants of S. roseosporus containing deletions of one or more of the NRPS genes have been trans-complemented with dptA, dptBC, and dptD by inserting these genes under the control of the ermEp* promoter into separate conjugal cloning vectors containing ϕC31 or IS117 attachment (attP int) sites; delivering the plasmids into S. roseosporus by conjugation from Escherichia coli; and inserting the plasmids site-specifically into the chromosome at the corresponding attB sites. This trans-complementation system was used to generate subunit exchanges with lptD and cdaPS3 and the recombinants produced novel hybrid molecules. Module exchanges at positions d-Ala8 and d-Ser11 in the peptide have produced additional novel derivatives of daptomycin. The approaches of subunit exchanges and module exchanges were combined with amino acid modifications of Glu at position 12 and natural variations in lipid side chain starter units to generate a combinatorial library of antibiotics related to daptomycin. Many of the engineered strains produced levels of novel molecules amenable to isolation and antimicrobial testing, and most of the compounds displayed antibacterial activities.

On the biosynthesis of an inhibitor of the p53/MDM2 interaction

Abstract The biosynthesis of a fungal secondary metabolite, chlorofusin, which disrupts the interaction between the proteins p53 and MDM2, has been investigated; the acetogenic origin of the chromophore backbone as well as of an aminodecanoic acid residue is demonstrated.

CD4-binding compounds: An assay to detect new classes of immunopharmacological agents

The interaction of antibodies with protein antigens is accepted as a paradigm of protein-protein interactions. In searching for a new generation of immunomodulatory compounds based on the interaction of the T-cell surface glycoprotein CD4 with MHC class II antigens, a model assay has been developed in which MHC molecules have been substituted by a monoclonal antibody (anti-Leu3a) to the CD4 amino-terminal domain-specific epitope, Leu3a. This assay can detect diverse classes of molecules including proteins such as HIV envelope glycoprotein gp120 and low molecular weight compounds such as aurin tricarboxylic acid, dextran sulphate and Evans blue. The interaction of these molecules with CD4 in the assay appears to be identical to their interaction with native CD4 on intact cells. Other protein-antibody pairs could be substituted for CD4-anti-Leu3a enabling this assay format to be used for the detection of proteins or small organic compounds which interfere with a wide range of therapeutically-relevant macromolecular interactions.