Serendipitous discovery of novel bacterial methionine aminopeptidase inhibitors.

In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding.

NorA functions as a multidrug efflux protein in both cytoplasmic membrane vesicles and reconstituted proteoliposomes.

Overexpression of NorA, an endogenous efflux transporter of Staphylococcus aureus, confers resistance to certain fluoroquinolone antimicrobials and diverse other substrates. The norA gene was amplified by PCR and cloned in the expression vector pTrcHis2. Histidine-tagged NorA (NorA-His) was overexpressed in Escherichia coli cells to prepare two experimental systems, everted membrane vesicles enriched with NorA-His and proteoliposomes reconstituted with purified NorA-His. In membrane vesicles, NorA-His actively transported Hoechst 33342, a dye that is strongly fluorescent in the membrane but has low fluorescence in an aqueous environment. Transport was activated by the addition of ATP or lactate and reversed by the addition of nigericin, with the addition of K(+)-valinomycin having little effect. Transport of Hoechst 33342 was inhibited competitively by verapamil, a known inhibitor of NorA, and by other NorA substrates, including tetraphenyl phosphonium and the fluoroquinolones norfloxacin and ciprofloxacin. In contrast, sparfloxacin, a fluoroquinolone whose antimicrobial activity is not affected by NorA expression, exhibited noncompetitive inhibition. NorA induction and overexpression yielded 0.5 to 1 mg of a largely homogeneous 40- to 43-kDa protein per liter of culture. NorA-His incorporated into proteoliposomes retained the ability to transport Hoechst 33342 in response to an artificial proton gradient, and transport was blocked by nigericin and verapamil. These data provide the first experimental evidence of NorA functioning as a self-sufficient multidrug transporter.

Profiling of drugs for membrane activity using liposomes as an in vitro model system.

The increasing size of chemical libraries being analyzed by high-throughput screening results in a growing number of active compounds that need to be assessed before moving forward in the drug development process. As a consequence, more rapid and highly sensitive strategies are required to accelerate the process of drug discovery without increasing the cost. Due to the fact that significant numbers of compounds from combinatorial libraries are hydrophobic in nature, approaches are needed to evaluate the potentialfor these compounds to interfere with the functions of biological membranes. The liposome system was used to detect agents that act as follows: (i) ionophores able to induce specific ion permeability, e.g., valinomycin for K+ and protonophoric uncouplers for H+; (ii) ion antiporters which exchange H+ for other ions, e.g., nigericin; (iii) agents that form low specificity ion channels in the membrane, e.g., gramicidin; and (iv) detergents and other membrane-disrupting agents. We propose using this liposome assay during the drug development process to identify compounds that have membrane activity and, as a consequence, produce a biological effect by altering the physico-chemical properties of the cell membrane rather than interacting with a protein target. Screening of a representative set of biologically-active compounds (198) indicated that the majority of systemic antimicrobial drugs, but not topical drugs, lack membrane activity in this model system.

Vancomycin tolerance induced by erythromycin but not by loss of vncRS, vex3, or pep27 function in Streptococcus pneumoniae.

Vancomycin-tolerant Streptococcus pneumoniae is a growing problem among drug-resistant human pathogens. Some vancomycin-tolerant pneumococci have been reported to carry mutations in loci encoding a two-component regulatory system designated VncRS or in a proximal ABC transporter, Vex. A model was advanced proposing that the tolerance phenotype resulted from the inability of a vncS mutant to respond to the Vex-transported Pep27 "death peptide" signal and dephosphorylate VncR, thereby preventing relief of repression of autolytic and other cell death functions in response to antibiotics. To explore this hypothesis, we constructed mutations in vncS, vncR, vex3, and pep27 in S. pneumoniae strain R6 and two additional genetic backgrounds. The lytic responses of the isogenic DeltavncS, Deltavex3, DeltavncR, and Deltapep27 mutants, but not a DeltalytA strain, to vancomycin were indistinguishable from that of the parent strain. DeltavncS strains also failed to exhibit tolerance to vancomycin at various doses in multiple media and showed wild-type sensitivity to other classes of autolysis-inducing antibiotics. In contrast, addition of subinhibitory levels of the antibiotic erythromycin led to tolerance to vancomycin during late, but not early, exponential-phase growth in a DeltavncS strain, in the parent strain R6, and in two other strains bearing erythromycin resistance markers, namely, a DeltavncR strain and an unrelated DeltacomD strain that is defective in competence-quorum sensing. Thus, this tolerance effect resulted from changes in cell growth or other erythromycin-dependent phenomena and not inactivation of vncS per se. Consistent with these results, and in contrast to a previous report, we found that a synthetic form of Pep27 did not elicit lytic or nonlytic killing of pneumococci. Finally, microarray transcriptional analysis and beta-galactosidase reporter assays revealed VncS-dependent regulation of the vex123 gene cluster but did not support a role for VncRS in the regulation of autolytic or other putative cell death loci. Based on these findings, we propose that vancomycin tolerance in S. pneumoniae does not result from loss of vncS function alone.

An evaluation of detergents for NMR structural studies of membrane proteins.

Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment. In this study 25 membrane mimetics were screened using 2D (1)H-(15)N heteronuclear single quantum correlation NMR experiments to establish sample homogeneity and predict fitness for structure determination. A single detergent, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG), yielded high quality NMR spectra with sample lifetimes greater than one month for the five proteins tested - R. sphaeroides LH1 alpha and beta subunits, E. coli and B. pseudofirmus OF4 ATP synthase c subunits, and S. aureus small multidrug resistance transporter - with 1, 2, or 4 membrane spanning alpha-helices, respectively. Site-specific spin labeling established interhelical distances in the drug transporter and genetically fused dimers of c subunits in LPPG consistent with in vivo distances. Optical spectroscopy showed that LH1 beta subunits form native-like complexes with bacteriochlorophyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of (15)N transverse, longitudinal and (15)N[(1)H] nuclear Overhauser effect relaxation measurements yielded overall rotational correlation times of 8 to 12 nsec, similar to a 15-20 kDalton protein tumbling isotropically in solution, and consistent with the high quality NMR data observed.