Keith R. Marotti

Discovery and Characterization of QPT-1, the Progenitor of a New Class of Bacterial Topoisomerase Inhibitors

ABSTRACT QPT-1 was discovered in a compound library by high-throughput screening and triage for substances with whole-cell antibacterial activity. This totally synthetic compound is an unusual barbituric acid derivative whose activity resides in the (−)-enantiomer. QPT-1 had activity against a broad spectrum of pathogenic, antibiotic-resistant bacteria, was nontoxic to eukaryotic cells, and showed oral efficacy in a murine infection model, all before any medicinal chemistry optimization. Biochemical and genetic characterization showed that the QPT-1 targets the β subunit of bacterial type II topoisomerases via a mechanism of inhibition distinct from the mechanisms of fluoroquinolones and novobiocin. Given these attributes, this compound represents a promising new class of antibacterial agents. The success of this reverse genomics effort demonstrates the utility of exploring strategies that are alternatives to target-based screens in antibacterial drug discovery.

Allosteric inhibition of Staphylococcus aureus D-alanine:D-alanine ligase revealed by crystallographic studies

d-alanine:d-alanine ligase (DDl) is an essential enzyme in bacterial cell wall biosynthesis and an important target for developing new antibiotics. It catalyzes the formation of d-alanine:d-alanine dipeptide, sequentially by using one d-alanine and one ATP as substrates for the first-half reaction, and a second d-alanine substrate to complete the reaction. Some gain of function DDl mutants can use an alternate second substrate, causing resistance to vancomycin, one of the last lines of defense against life-threatening Gram-positive infections. Here, we report the crystal structure of Staphylococcus aureus DDl (StaDDl) and its cocrystal structures with 3-chloro-2,2-dimethyl-N-[4(trifluoromethyl)phenyl]propanamide (inhibitor 1) (Ki = 4 μM against StaDDl) and with ADP, one of the reaction products, at resolutions of 2.0, 2.2, and 2.6 Å, respectively. The overall structure of StaDDl can be divided into three distinct domains. The inhibitor binds to a hydrophobic pocket at the interface of the first and the third domain. This inhibitor-binding pocket is adjacent to the first d-alanine substrate site but does not overlap with any substrate sites. An allosteric inhibition mechanism of StaDDl by this compound was proposed. The mechanism provides the basis for developing new antibiotics targeting d-alanine:d-alanine ligase. Because this compound only interacts with residues from the first d-alanine site, inhibitors with this binding mode potentially could overcome vancomycin resistance.

Staphylococcus aureus Cell Extract Transcription-Translation Assay: Firefly Luciferase Reporter System for Evaluating Protein Translation Inhibitors

ABSTRACT The promoter for the Staphylococcus aureus capsule polysaccharide synthesis gene (cap1A) was cloned in front of the firefly luciferase gene for use in a cell extractS. aureus transcription-translation system. The assay is rapid, reproducible, and sensitive and has a lower background level than the radiolabeled amino acid incorporation translation assays. We present data evaluating a transcription inhibitor and a number of protein translation inhibitors in this system.

3-Arylpiperidines as potentiators of existing antibacterial agents

Important resistance patterns in Gram-negative pathogens include active efflux of antibiotics out of the cell via a cellular pump and decreased membrane permeability. A 3-arylpiperidine derivative (1) has been identified by high-throughput assay as a potentiator with an IC(50) approximately 90 microM. This report details the evaluation of the tether length, aryl substitution and the importance of the fluorine on antibiotic accumulation. Evaluation of various tether lengths demonstrated that the two-carbon tethered analogues are optimal. Removal of the fluorine has a modest effect on antibiotic accumulation and the defluorinated analogue 17 is equally potent to the original lead 1.

Ribosomes from an Oxazolidinone-Resistant Mutant Confer Resistance to Eperezolid in a Staphylococcus aureus Cell-Free Transcription-Translation Assay

ABSTRACT Oxazolidinone-resistant mutants of Staphylococcus aureus, isolated with a spiral plating technique, had a 16-fold higher MIC (2 versus 32 μg/ml) of eperezolid when compared to the parental sensitive strain. Eperezolid inhibited in vitro protein translation with 50% inhibitory concentrations of 30 μM for the oxazolidinone-sensitive S30 extract and 75 μM for the resistant extract. Experiments mixing various combinations of S100 and crude ribosome preparations from oxazolidinone-sensitive and -resistant S. aureus strains in a transcription-translation assay demonstrated that the resistant determinant resided within the ribosomal fraction. Ribosomes from the oxazolidinone-resistant strain bound less drug than ribosomes from the sensitive strain, indicating that the ribosome is the site of action for the oxazolidinones. These experiments demonstrate that an alteration of the ribosome is responsible for some or all of the oxazolidinone resistance observed in the S. aureusmutant.

Remodeling of the HDL in NIDDM: a fundamental role for cholesteryl ester transfer protein

When the Ay gene is expressed in KK mice, the yellow offspring (KKAy mice) become obese, insulin resistant, hyperglycemic, and severely hypertriglyceridemic, yet they maintain extraordinarily high plasma high-density lipoprotein (HDL) levels. Mice lack the ability to redistribute neutral lipids among circulating lipoproteins, a process catalyzed in humans by cholesteryl ester transfer protein (CETP). To test the hypothesis that it is the absence of CETP that allows these hypertriglyceridemic mice to maintain high plasma HDL levels, simian CETP was expressed in the KKAymouse. The KKAy-CETP mice retained the principal characteristics of KKAy mice except that their plasma HDL levels were reduced (from 159 ± 25 to 25 ± 6 mg/dl) and their free apolipoprotein A-I concentrations increased (from 7 ± 3 to 22 ± 6 mg/dl). These changes appeared to result from a CETP-induced enrichment of the HDL with triglyceride (from 6 ± 2 to 60 ± 18 mol of triglyceride/mol of HDL), an alteration that renders HDL susceptible to destruction by lipases. These data support the premise that CETP-mediated remodeling of the HDL is responsible for the low levels of that lipoprotein that accompany hypertriglyceridemic non-insulin-dependent diabetes mellitus.When the Ay gene is expressed in KK mice, the yellow offspring (KKAy mice) become obese, insulin resistant, hyperglycemic, and severely hypertriglyceridemic, yet they maintain extraordinarily high plasma high-density lipoprotein (HDL) levels. Mice lack the ability to redistribute neutral lipids among circulating lipoproteins, a process catalyzed in humans by cholesteryl ester transfer protein (CETP). To test the hypothesis that it is the absence of CETP that allows these hypertriglyceridemic mice to maintain high plasma HDL levels, simian CETP was expressed in the KKAy mouse. The KKAy-CETP mice retained the principal characteristics of KKAy mice except that their plasma HDL levels were reduced (from 159 +/- 25 to 25 +/- 6 mg/dl) and their free apolipoprotein A-I concentrations increased (from 7 +/- 3 to 22 +/- 6 mg/dl). These changes appeared to result from a CETP-induced enrichment of the HDL with triglyceride (from 6 +/- 2 to 60 +/- 18 mol of triglyceride/mol of HDL), an alteration that renders HDL susceptible to destruction by lipases. These data support the premise that CETP-mediated remodeling of the HDL is responsible for the low levels of that lipoprotein that accompany hypertriglyceridemic non-insulin-dependent diabetes mellitus.

Bacterial translation inhibitors, 1-acylindazol-3-ols as anthranilic acid mimics.

The discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents has been described in a recent series of papers. This paper describes the discovery of 1-acylindazol-3-ols as a novel bioisostere of an anthranilic acid. The synthesis and structure-activity relationships of the indazol bioisosteres are described herein.

Enols as potent antibacterial agents.

This paper describes the discovery of alpha-trifluoroketoacetamides as potent antibacterial agents against Gram-positive organisms. The initial SAR indicates that the aryl ethyl side chain is essential in maintaining antibacterial activity. The SAR observations have been utilized to design a bioisostere for the alpha-trifluoroketoacetamide with good activity against Gram-positive organisms.