Richard H. Baltz

Renaissance in antibacterial discovery from actinomycetes.

The soil actinomycetes have been important sources of antibiotics, but were nearly abandoned in recent years in favor of high-throughput target-based screening of chemical libraries. The latter approach has not been productive, so it is time to reinvigorate the discovery of new antibiotics from a proven source. Recent progress has been made on antibiotic discovery from actinomycetes by using high-throughput fermentation, isolation of marine actinomycetes, mining genomes for cryptic pathways, and combinatorial biosynthesis to generate new secondary metabolites related to existing pharmacophores.

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.

Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration?

There are few new antibiotics in the pipeline today. The reasons may include starvation at the front of the pipeline due to inadequate sources of suitable compounds to screen coupled with poorly validated discovery methodologies. A successful antibiotic discovery approach in the past, based upon whole cell antibiotic screening of natural products from actinomycetes and fungi, eventually suffered from constipation in the middle of the pipeline due to rediscovery of known compounds, even though low throughput methodology was employed at the front end. The current lack of productivity may be attributed to the poor choice of strategies to address the discovery of new antibiotics. Recent applications of high throughput in vitro screening of individual antibacterial targets to identify lead compounds from combinatorial chemical libraries, traditional chemical libraries, and partially purified natural product extracts has not produced any significant clinical candidates. The solution to the current dilemma may be to return to natural product whole cell screening. For this approach to work in the current millennium, the process needs to be miniaturized to increase the throughput by orders of magnitude over traditional screening, and the rediscovery of known antibiotics needs to be minimized by methods that can be readily monitored and improved over time.

Daptomycin: mechanisms of action and resistance, and biosynthetic engineering.

Daptomycin is a lipopeptide antibiotic used clinically to treat infections caused by Gram-positive bacteria. Laboratory studies have shown that Staphylococcus aureus resistance to daptomycin occurs stepwise and slowly. Mutations associated with decreased susceptibility were mapped in mprF, yycG, rpoB, and rpoC, each giving about twofold increases in the minimal inhibitory concentration (MIC) and combinations giving higher MICs. The mprF gene encodes a dual functional enzyme that couples lysine to phosphatidylglycerol (PG) and transfers the lysyl-PG (LPG) to the outer leaflet of the membrane. LPG is less acidic than PG, and thus reduces the binding of Ca(++)-bound daptomycin to bacterial membranes. The mprF mutants have higher LPG/PG ratios in the membrane outer leaflet and bind less daptomycin than the wild-type strain. YycG is a sensor histidine kinase of a two component signal transduction system required for viability in many low G+C Gram-positive bacteria. The observation of DapR mutations in yycG suggests that YycG may be a target for daptomycin antibacterial activity. Daptomycin inserts into PG rich membrane at the cell division septum, but also inserts into lung surfactant, explaining why it failed to meet non-inferiority criteria in clinical trials for community acquired pneumonia (CAP). Recent advances in biosynthetic engineering have provided new tools to generate novel lipopeptides with modifications in the core peptide: several were very potent antibiotics against Gram-positive pathogens, and some were active in the presence of surfactant.

Molecular engineering approaches to peptide, polyketide and other antibiotics

Molecular engineering approaches to producing new antibiotics have been in development for about 25 years. Advances in cloning and analysis of antibiotic gene clusters, engineering biosynthetic pathways in Escherichia coli, transfer of engineered pathways from E. coli into Streptomyces expression hosts, and stable maintenance and expression of cloned genes have streamlined the process in recent years. Advances in understanding mechanisms and substrate specificities during assembly by polyketide synthases, nonribosomal peptide synthetases, glycosyltransferases and other enzymes have made molecular engineering design and outcomes more predictable. Complex molecular scaffolds not amenable to synthesis by medicinal chemistry (for example, vancomycin (Vancocin), daptomycin (Cubicin) and erythromycin) are now tractable by molecular engineering. Medicinal chemistry can further embellish the properties of engineered antibiotics, making the two disciplines complementary.

Daptomycin: from the mountain to the clinic, with essential help from Francis Tally, MD.

Daptomycin has been approved and successfully launched for the treatment of complicated skin and skin-structure infections caused by gram-positive pathogens [1] and bacteremia and right-sided endocarditis due to Staphylococcus aureus, including strains that are resistant to methicillin or other antibiotics [2]. The development of the drug, however, was not straightforward; it involved a cast of characters, including scientists at Eli Lilly and at Cubist Pharmaceuticals. Of most importance, the development of daptomycin involved the tenacious leadership of Dr. Francis Tally. As a tribute to Dr. Tally, we attempt to reconstruct the path of daptomycin from the mountain to the clinic.

Non-ribosomal peptide synthetase module fusions to produce derivatives of daptomycin in Streptomyces roseosporus

Genetic engineering has been applied to reprogramme non-ribosomal peptide synthetases (NRPSs) to produce novel antibiotics, but little is known about what determines the efficiency of production. We explored module exchanges at nucleotide sequences encoding interpeptide linkers in dptD, a gene encoding a di-modular NRPS subunit that incorporates 3-methylglutamic acid (3mGlu(12)) and kynurenine (Kyn(13)) into daptomycin. Mutations causing amino acid substitutions, deletions or insertions in the inter-module linker had no negative effects on lipopeptide yields. Hybrid DptD subunits were generated by fusing the 3mGlu(12) module to terminal modules from calcium-dependent antibiotic (CDA) or A54145 NRPSs, and recombinants produced daptomycin analogues with Trp(13) or Ile(13) at high efficiencies. A recombinant expressing DptD with a hybrid Kyn(13) module containing a di-domain from a d-Asn module caused the production of a new daptomycin analogue containing Asn(13).

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. coelicolor 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.

Chapter 20. Biosynthesis and genetic engineering of lipopeptides in Streptomyces roseosporus.

Daptomycin is an acidic cyclic lipopeptide antibiotic approved for treatment of infections caused by Gram-positive pathogens, including Staphylococcus aureus strains resistant to other antibiotics. Daptomycin biosynthesis is carried out by a giant multisubunit, multienzyme nonribosomal peptide synthetase (NRPS). The daptomycin (dpt) biosynthetic genes have been cloned in a bacterial artificial chromosome (BAC) vector, sequenced, and expressed in Streptomyces lividans. Several of the dpt genes, including the three NRPS genes, are transcribed as a lengthy polycistronic message. The daptomycin-producing strain, Streptomyces roseosporus, can be genetically manipulated, and a number of deletion mutants encompassing one or more of the dpt genes have been constructed. Several of the dpt genes have been expressed from ectopic chromosomal loci (varphiC31 or IS117 attB sites) under the transcriptional control of the strong constitutive ermEp* promoter, and recombinant strains produced high levels of lipopeptides, thus establishing a trans-complementation system for combinatorial biosynthesis. A number of hybrid NRPS subunits have been generated by lambda-Red-mediated recombination, and combinatorial libraries of lipopeptides have been generated by NRPS subunit exchanges, module exchanges, multidomain exchanges, deletion mutagenesis, and multiple natural lipidations, using the ectopic trans-complementation system in S. roseosporus.