Dylan C. Alexander

Combinatorial biosynthesis of novel antibiotics related to daptomycin

Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus, is the active ingredient of Cubicin (daptomycin-for-injection), a first-in-class antibiotic approved for treatment of skin and skin-structure infections caused by Gram-positive pathogens and bacteremia and endocarditis caused by Staphylococcus aureus, including methicillin-resistant strains. Genetic engineering of the nonribosomal peptide synthetase (NRPS) in the daptomycin biosynthetic pathway was exploited for the biosynthesis of novel active antibiotics. λ-Red-mediated recombination was used to exchange single or multiple modules in the DptBC subunit of the NRPS to modify the daptomycin cyclic peptide core. We combined module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variations of the lipid tail to generate a library of novel lipopeptides, some of which were as active as daptomycin against Gram-positive bacteria. One compound was more potent against an Escherichia coli imp mutant that has increased outer membrane permeability. This study established a robust combinatorial biosynthesis platform to produce novel peptide antibiotics in sufficient quantities for antimicrobial screening and drug development.

Genetically Engineered Lipopeptide Antibiotics Related to A54145 and Daptomycin with Improved Properties

ABSTRACT Daptomycin is a cyclic lipopeptide antibiotic approved for the treatment of skin and skin structure infections caused by Gram-positive pathogens and for that of bacteremia and right-sided endocarditis caused by Staphylococcus aureus. Daptomycin failed to meet noninferiority criteria for the treatment of community-acquired pneumonia, likely due to sequestration in pulmonary surfactant. Many analogues of daptomycin have been generated by combinatorial biosynthesis, but only two displayed improved activity in the presence of bovine surfactant, and neither was as active as daptomycin in vitro. In the present study, we generated hybrid molecules of the structurally related lipopeptide A54145 in Streptomyces fradiae and tested them for antibacterial activity in the presence of bovine surfactant. Hybrid A54145 nonribosomal peptide synthetase (NRPS) biosynthetic genes were constructed by genetic engineering and were expressed in combination with a deletion of the lptI methyltransferase gene, which is involved in the formation of the 3-methyl-glutamic acid (3mGlu) residue at position 12. Some of the compounds were very active against S. aureus and other Gram-positive pathogens; one compound was also highly active in the presence of bovine surfactant, had low acute toxicity, and showed some efficacy against Streptococcus pneumoniae in a mouse model of pulmonary infection.

Development of a Genetic System for Combinatorial Biosynthesis of Lipopeptides in Streptomyces fradiae and Heterologous Expression of the A54145 Biosynthesis Gene Cluster

ABSTRACT A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu12), at the expense of factors containing 3-methyl-glutamic acid (3mGlu12). This provided a practical route to produce high levels of pure Glu12-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.

Production of novel lipopeptide antibiotics related to A54145 by Streptomyces fradiae mutants blocked in biosynthesis of modified amino acids and assignment of lptJ , lptK and lptL gene functions

A54145 is a complex of lipopeptide antibiotics produced by Streptomyces fradiae. A54145 factors are structurally related to daptomycin, with four modified amino acids, only one of which is present in daptomycin. We generated three mutants defective in lptJ, lptK or lptL, whose gene products are involved in the formation of hydroxy-Asn3 (hAsn3) and methoxy-Asp9 (moAsp9). Each of the mutants produced novel lipopeptides related to A54145 and the profiles allowed assignment of functions for those genes. We constructed strains carrying different combinations of these genes coupled with a mutation in the lptI gene involved in the biosynthesis of 3-methyl-Glu12 (3mGlu12), and all recombinants produced novel lipopeptides. One of the compounds displayed very good antibacterial activity in the presence of bovine surfactant, which interacts with daptomycin or A54145E to inhibit their antibacterial activities.

Structural characterization of a lipopeptide antibiotic A54145E(Asn3Asp9) produced by a genetically engineered strain of Streptomyces fradiae.

A potent new lipopeptide antibiotic, A54145E(Asn3Asp9), was isolated from the fermentation broth of Streptomyces fradiae DA1489 engineered to delete genes encoding enzymes involved in hydroxylation of Asn3 and methoxylation of Asp9. The chemical structure predicted from the genetic changes in the biosynthetic pathway was determined by analyses of chemical transformations, D, L-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS and 2D NMR techniques. These studies confirmed the primary amino acid sequence of A54145E(Asn3Asp9) predicted from the genetic engineering strategy, and also confirmed the structure and locations of three D-amino acids predicted from bioinformatic studies.

Characterization of High-Level Daptomycin Resistance in Viridans Group Streptococci Developed upon In Vitro Exposure to Daptomycin

ABSTRACT Viridans group streptococci (VGS) are part of the normal flora that may cause bacteremia, often leading to endocarditis. We evaluated daptomycin against four clinical strains of VGS (MICs = 1 or 2 μg/ml) using an in vitro-simulated endocardial vegetation model, a simulated bacteremia model, and kill curves. Daptomycin exposure was simulated at 6 mg/kg of body weight and 8 mg/kg every 24 h for endocardial and bacteremia models. Total drug concentrations were used for analyses containing protein (albumin and pooled human serum), and free (unbound) drug concentrations (93% protein bound) were used for analyses not containing protein. Daptomycin MICs in the presence of protein were significantly higher than those in the absence of protein. Despite MICs below or at the susceptible breakpoint, all daptomycin regimens demonstrated limited kill in both pharmacodynamic models. A reduction of approximately 1 to 2 log10 CFU was seen for all isolates and dosages except daptomycin at 6 mg/kg, which achieved a reduction of 2.7 log10 CFU/g against one strain (Streptococcus gordonii 1649) in the endocardial model. Activity was similar in both pharmacodynamic models in the presence or absence of protein. Similar activity was noted in the kill curves over all multiples of the MIC. Regrowth by 24 h was seen even at 8× MIC. Postexposure daptomycin MICs for both pharmacodynamic models increased to >256 μg/ml for all isolates by 24 and 72 h. Despite susceptibility to daptomycin by standard MIC methods, these VGS developed high-level daptomycin resistance (HLDR) after a short duration following drug exposure not attributed to modification or inactivation of daptomycin. Further evaluation is warranted to determine the mechanism of resistance and clinical implications.

Pharmacokinetics-Pharmacodynamics of a Novel β-Lactamase Inhibitor, CB-618, in Combination with Meropenem in an In Vitro Infection Model

ABSTRACT The usefulness of β-lactam antimicrobial agents is threatened as never before by β-lactamase-producing bacteria. For this reason, there has been renewed interest in the development of broad-spectrum β-lactamase inhibitors. Herein we describe the results of dose fractionation and dose-ranging studies carried out using a one-compartment in vitro infection model to determine the exposure measure for CB-618, a novel β-lactamase inhibitor, most predictive of the efficacy when given in combination with meropenem. The challenge panel included Enterobacteriaceae clinical isolates, which collectively produced a wide range of β-lactamase enzymes (KPC-2, KPC-3, FOX-5, OXA-48, SHV-11, SHV-27, and TEM-1). Human concentration-time profiles were simulated for each drug, and samples were collected for drug concentration and bacterial density determinations. Using data from dose fractionation studies and a challenge Klebsiella pneumoniae isolate (CB-618-potentiated meropenem MIC = 1 mg/liter), relationships between change from baseline in log10 CFU/ml at 24 h and each of CB-618 area under the concentration-time curve over 24 h (AUC0–24), maximum concentration (Cmax), and percentage of the dosing interval that CB-618 concentrations remained above a given threshold were evaluated in combination with meropenem at 2 g every 8 h (q8h). The exposure measures most closely associated with CB-618 efficacy in combination with meropenem were the CB-618 AUC0–24 (r2 = 0.835) and Cmax (r2 = 0.826). Using the CB-618 AUC0–24 indexed to the CB-618-potentiated meropenem MIC value, the relationship between change from baseline in log10 CFU/ml at 24 h and CB-618 AUC0–24/MIC ratio in combination with meropenem was evaluated using the pooled data from five challenge isolates; the CB-618 AUC0–24/MIC ratio associated with net bacterial stasis and the 1- and 2-log10 CFU/ml reductions from baseline at 24 h were 27.3, 86.1, and 444.8, respectively. These data provide a pharmacokinetics-pharmacodynamics (PK-PD) basis for evaluating potential CB-618 dosing regimens in combination with meropenem in future studies.

Pharmacokinetics-Pharmacodynamics of CB-618 in Combination with Cefepime, Ceftazidime, Ceftolozane, or Meropenem: the Pharmacological Basis for a Stand-Alone β-Lactamase Inhibitor

ABSTRACT A major challenge in treating patients is the selection of the “right” antibiotic regimen. Given that the optimal β-lactam/β-lactamase inhibitor pair is dependent upon the spectrum of β-lactamase enzymes produced and the frequency of resistance to the β-lactamase inhibitor, it might be useful if a stand-alone were available for the clinician to pair with the “right” β-lactam rather than only in a fixed combination. We describe herein a one-compartment in vitro infection model studies conducted to identify the magnitudes of the pharmacokinetic-pharmacodynamic (PK-PD) index for a β-lactamase inhibitor, CB-618, that would restore the activity of four β-lactam partner agents (cefepime, ceftazidime, ceftolozane, and meropenem) with various doses (1 or 2 g) and dosing intervals (8 or 12 h). The challenge panel included Klebsiella pneumoniae (n = 5), Escherichia coli (n = 2), and Enterobacter cloacae (n = 1) strains, which produced a wide variety of β-lactamase enzymes (AmpC, CTXM-15, KPC-2, KPC-3, FOX-5, OXA-1/30, OXA-48, SHV-1, SHV-11, SHV-27, and TEM-1). Free-drug human concentration-time profiles were simulated for each agent, and specimens were collected for drug concentration and bacterial density determinations. CB-618 restored the activity of each β-lactam partner. The magnitudes of the CB-618 ratio of the area under the concentration-time curve from 0 to 24 h to the MIC (i.e., the AUC/MIC ratio) associated with net bacterial stasis and 1- and 2-log10 CFU/ml reductions from baseline at 24 h were 11.2, 32.9, and 136.3, respectively. These data may provide a PK-PD basis for the development of a stand-alone β-lactamase inhibitor.