Lynn L. Silver

Platensimycin is a selective FabF inhibitor with potent antibiotic properties

Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of β-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

Outbreak of Klebsiella pneumoniae Producing a New Carbapenem-Hydrolyzing Class A β-Lactamase, KPC-3, in a New York Medical Center

ABSTRACT From April 2000 to April 2001, 24 patients in intensive care units at Tisch Hospital, New York, N.Y., were infected or colonized by carbapenem-resistant Klebsiella pneumoniae. Pulsed-field gel electrophoresis identified a predominant outbreak strain, but other resistant strains were also recovered. Three representatives of the outbreak strain from separate patients were studied in detail. All were resistant or had reduced susceptibility to imipenem, meropenem, ceftazidime, piperacillin-tazobactam, and gentamicin but remained fully susceptible to tetracycline. PCR amplified a blaKPC allele encoding a novel variant, KPC-3, with a His(272)→Tyr substitution not found in KPC-2; other carbapenemase genes were absent. In the outbreak strain, KPC-3 was encoded by a 75-kb plasmid, which was transferred in vitro by electroporation and conjugation. The isolates lacked the OmpK35 porin but expressed OmpK36, implying reduced permeability as a cofactor in resistance. This is the third KPC carbapenem-hydrolyzing β-lactamase variant to have been reported in members of the Enterobacteriaceae, with others reported from the East Coast of the United States. Although producers of these enzymes remain rare, the progress of this enzyme group merits monitoring.

Antibacterial Agents That Inhibit Lipid A Biosynthesis

Lipid A constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and is essential for bacterial growth. Synthetic antibacterials were identified that inhibit the second enzyme (a unique deacetylase) of lipid A biosynthesis. The inhibitors are chiral hydroxamic acids bearing certain hydrophobic aromatic moieties. They may bind to a metal in the active site of the deacetylase. The most potent analog (with an inhibition constant of about 50 nM) displayed a minimal inhibitory concentration of about 1 microgram per milliliter against Escherichia coli, caused three logs of bacterial killing in 4 hours, and cured mice infected with a lethal intraperitoneal dose of E. coli.

Discovery of FabH/FabF Inhibitors from Natural Products

ABSTRACT Condensing enzymes are essential in type II fatty acid synthesis and are promising targets for antibacterial drug discovery. Recently, a new approach using a xylose-inducible plasmid to express antisense RNA in Staphylococcus aureus has been described; however, the actual mechanism was not delineated. In this paper, the mechanism of decreased target protein production by expression of antisense RNA was investigated using Northern blotting. This revealed that the antisense RNA acts posttranscriptionally by targeting mRNA, leading to 5′ mRNA degradation. Using this technology, a two-plate assay was developed in order to identify FabF/FabH target-specific cell-permeable inhibitors by screening of natural product extracts. Over 250,000 natural product fermentation broths were screened and then confirmed in biochemical assays, yielding a hit rate of 0.1%. All known natural product FabH and FabF inhibitors, including cerulenin, thiolactomycin, thiotetromycin, and Tü3010, were discovered using this whole-cell mechanism-based screening approach. Phomallenic acids, which are new inhibitors of FabF, were also discovered. These new inhibitors exhibited target selectivity in the gel elongation assay and in the whole-cell-based two-plate assay. Phomallenic acid C showed good antibacterial activity, about 20-fold better than that of thiolactomycin and cerulenin, against S. aureus. It exhibited a spectrum of antibacterial activity against clinically important pathogens including methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Haemophilus influenzae.

Determination of Selectivity and Efficacy of Fatty Acid Synthesis Inhibitors

Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics. In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development. To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. We demonstrated that the inverse t½ value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity. The Km values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 ± 0.3 and 10 ± 2.7 μm in Staphylococcus aureus and Bacillus subtilis, respectively. The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn2+ > Ca2+ > Mn2+ > Mg2+; no inhibition was found with β-mercaptoethanol or dithiothreitol. We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure. BABX showed IC50 values of 11.4 and 35.3 μg/ml in the S. aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S. aureus and permeable E. coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 μg/ml. Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.

Direct interaction of a vancomycin derivative with bacterial enzymes involved in cell wall biosynthesis

BACKGROUND The glycopeptide antibiotic vancomycin complexes DAla-DAla termini of bacterial cell walls and peptidoglycan precursors and interferes with enzymes involved in murein biosynthesis. Semisynthetic vancomycins incorporating hydrophobic sugar substituents exhibit efficacy against DAla-DLac-containing vancomycin-resistant enterococci, albeit by an undetermined mechanism. Contrasting models that invoke either cooperative dimerization and membrane anchoring or direct inhibition of bacterial transglycosylases have been proposed to explain the bioactivity of these glycopeptides. RESULTS Affinity chromatography has revealed direct interactions between a semisynthetic hydrophobic vancomycin (DCB-PV), and select Escherichia coli membrane proteins, including at least six enzymes involved in peptidoglycan assembly. The N(4)-vancosamine substituent is critical for protein binding. DCB-PV inhibits transglycosylation in permeabilized E. coli, consistent with the observed binding of the PBP-1B transglycosylase-transpeptidase. CONCLUSIONS Hydrophobic vancomycins interact directly with a select subset of bacterial membrane proteins, suggesting the existence of discrete protein targets. Transglycosylase inhibition may play a role in the enhanced bioactivity of semisynthetic glycopeptides.

Novel illudins from Coprinopsis episcopalis (syn. Coprinus episcopalis), and the distribution of illudin-like compounds among filamentous fungi

The illudins are a family of fungal sesquiterpenes that have been studied as anti-tumor agents, and they also have antibacterial activity. Over a four-year period, 25 304 fungal isolates (approximately 97% ascomycetes and 3% basidiomycetes), were screened for antibacterial activity against methicillin-resistant Staphylococcus aureus. Illudin-like compounds with antibacterial and cytotoxic activity against tumor cell lines were observed in 10 basidiomycete strains. The isolates were recovered from different types of substrata using indirect methods and only formed sterile mycelia in pure culture. The isolates were genetically related but not identical, based on PCR-based fingerprinting techniques. DNA sequencing of the ITS1-5.8 S-ITS2 region of the strains revealed that nine had identical sequences, indicating that they were conspecific. The sequence of the remaining isolate was 96.34% similar, suggesting that it was a closely related species. The D1-D2 region of the 25 S rRNA gene of the two strain types was also sequenced. Both sequences were 99.39% similar, and Coprinopsis gonophylla (syn. Coprinus gonophyllus) was the closest match for both. Strains were grown in pure culture on a rice-based medium that allowed the development of basidiomata from one culture of the main strain type, which was identified as C. episcopalis, a close relative of C. gonophyllus. Both species (or strain types) produced different types of illudin-like compounds. Three novel illudins (I, I2 and J2) were found to be produced by the cultures identified as C. episcopalis, while only illudinic acid was produced by the other Coprinopsis sp. The taxonomical relationships of the Coprinops is species identified in this study with other illudin producers previously reported in the literature are discussed.

What is an "Ideal" Antibiotic? Discovery Challenges and Path forward.

An ideal antibiotic is an antibacterial agent that kills or inhibits the growth of all harmful bacteria in a host, regardless of site of infection without affecting beneficial gut microbes (gut flora) or causing undue toxicity to the host. Sadly, no such antibiotics exist. What exist are many effective Gram-positive antibacterial agents as well as broad-spectrum agents that provide treatment of certain Gram-negative bacteria but not holistic treatment of all bacteria. However effectiveness of all antibacterial agents is being rapidly eroded due to resistance. This viewpoint provides an overview of todays antibiotics, challenges and potential path forward of discovery and development of new (ideal) antibiotics.

Design and synthesis of novel antibacterial agents with inhibitory activity against DNA polymerase III.

4-Substituted 2-amino-6-(anilino)pyrimidines have been found to be selective inhibitors of DNA polymerase III, a replicative enzyme known to be essential in the DNA synthesis of Gram-positive bacteria. Among the analogues, 18 displayed an IC(50) of 10 microM against DNA polymerase III from Staphylococcus aureus.

Characterization of a Carbapenem-Hydrolyzing Enzyme, PoxB, in Pseudomonas aeruginosa PAO1

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe and life-threatening infections that are highly impervious to treatment. This microbe readily exhibits intrinsic and acquired resistance to varied antimicrobial drugs. Resistance to penicillin-like compounds is commonplace and provided by the chromosomal AmpC β-lactamase. A second, chromosomally encoded β-lactamase, PoxB, has previously been reported in P. aeruginosa. In the present work, the contribution of this class D enzyme was investigated using a series of clean in-frame ampC, poxB, and oprD deletions, as well as complementation by expression under the control of an inducible promoter. While poxB deletions failed to alter β-lactam sensitivities, expression of poxB in ampC-deficient backgrounds decreased susceptibility to both meropenem and doripenem but had no effect on imipenem, penicillin, and cephalosporin MICs. However, when expressed in an ampCpoxB-deficient background, that additionally lacked the outer membrane porin-encoding gene oprD, PoxB significantly increased the imipenem as well as the meropenem and doripenem MICs. Like other class D carbapenem-hydrolyzing β-lactamases, PoxB was only poorly inhibited by class A enzyme inhibitors, but a novel non-β-lactam compound, avibactam, was a slightly better inhibitor of PoxB activity. In vitro susceptibility testing with a clinical concentration of avibactam, however, failed to reduce PoxB activity against the carbapenems. In addition, poxB was found to be cotranscribed with an upstream open reading frame, poxA, which itself was shown to encode a 32-kDa protein of yet unknown function.