SummaryTo address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.

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 methicillinresistant Staphylococcus aureus, Bacillus subtilis, and Haemophilus influenzae.Hundreds of essential proteins have been identified in bacteria as potential drug targets (1,16,18,23). Of these, only a few are targets of therapeutically useful drugs. These include penicillin binding proteins, D-Ala-D-Ala ligase, MurA, undecaprenyl pyrophosphate, and alanine racemase for cell wall; 30S and 50S ribosomal subunits, elongation factor G, and IletRNA synthetase for protein synthesis; RNA polymerase for RNA synthesis; InhA (FabI) for fatty acid synthesis; dihydrofolate reductase (FolA) and p-aminobenzoic acid synthase (FolP) for metabolism; and DNA gyrase and topoisomerase IV for DNA synthesis. In past decades, extensive chemical modification of existing antibiotics has afforded improved activity against their targets. This strategy served well to develop new and effective antibiotics; however, such modification is becoming increasingly difficult and identification of new classes of compounds with different modes of action is critical to combat emerging resistance and meet clinical needs.

TR-701 is the orally active prodrug of TR-700, a novel oxazolidinone that demonstrates four-to eightfoldgreater activity than linezolid (LZD) against Staphylococcus and Enterococcus spp. In this study evaluating the in vitro sensitivity of LZD-resistant isolates, TR-700 demonstrated 8-to 16-fold-greater potency than LZD against all strains tested, including methicillin-resistant Staphylococcus aureus (MRSA), strains of MRSA carrying the mobile cfr methyltransferase gene, and vancomycin-resistant enterococci. The MIC 90 for TR-700 against LZD-resistant S. aureus was 2 g/ml, demonstrating the utility of TR-700 against LZD-resistant strains. A model of TR-700 binding to 23S rRNA suggests that the increased potency of TR-700 is due to additional target site interactions and that TR-700 binding is less reliant on target residues associated with resistance to LZD.Oxazolidinone antibiotics are one of the newest classes of antibiotics developed within the past 30 years, with linezolid (LZD) representing the only marketed member of this class. In 2000, LZD (Zyvox) was granted approval for the treatment of infections associated with vancomycin-resistant Enterococcus faecium, nosocomial pneumonia, community-acquired pneumonia due to Streptococcus pneumoniae and methicillin-sensitive Staphylococcus aureus (MSSA), and complicated skin and skin structure infections, including cases due to methicillinresistant Staphylococcus aureus (MRSA) (1). Later approvals included pediatric use, pneumonia due to multidrugresistant S. pneumoniae, and treatment of diabetic foot infections, without osteomyelitis, caused by gram-positive bacteria. These approvals represent important milestones for the novel oxazolidinone class in the treatment of serious infections.Oxazolidinones have been shown to bind to the 50S ribosomal subunit and inhibit protein translation (31). A model of the binding of LZD to the 23S rRNA peptidyl transferase region has been previously published, based upon in vivo crosslinking experiments (18). This model predicts that LZD would specifically interfere with the binding of the amino acid portion of the aminoacyl tRNA to the ribosomal A site. The recent crystal structure of LZD bound to the 50S ribosomal subunit confirms these findings and suggests that the mechanism of inhibition involves competition with the incoming A site substrates (13). Mutations in the 23S rRNA central loop of domain V, the peptidyl transferase center (PTC), are associated with the development of LZD resistance.

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.

We describe our initial application of a biochemical strategy, comprising combinatorial screening and rational optimization, which directly identifies oligonucleotides with maximum affinity (per unit length), specificity, and rates of hybridization to structurally preferred sites on folded RNA, to the problem of design of antisense oligonucleotides active against the hepatitis C virus (HCV). A fully randomized sequence DNA oligonucleotide (10-mer) library was equilibrated with each of two folded RNA fragments (200 and 370 nucleotides (nt)), together spanning the 5 440 nt of an HCV transcript (by overlapping 130 nt), which were varied over a range of concentrations. The equilibrations were performed in solution under conditions determined to preserve RNA structure and to limit all RNA-DNA library oligonucleotide interactions to 1:1 stoichiometry. Subsequent Escherichia coli RNase H (endoribonuclease H: EC 3.1.26.4) cleavage analysis identified two preferred sites of highest affinity heteroduplex hybridization. The lengths and sequences of different substitute chemistry oligonucleotides complementary to these sites were rationally optimized using an iterative and quantitative analysis of binding affinity and specificity. Thus, DNA oligonucleotides that hybridized with the same affinity to the preferred sites in the folded RNA fragments found by screening as to short ( 25 nt) RNA complements were identified but were found to vary in length (10 -18 nt) from site to site. Phosphorothioate (P‫؍‬S) and 2-fluoro (2-F) uniformly substituted oligonucleotides also were found, which hybridized optimally to these sites, supporting the design of short (10 -15-nt) and maximally specific oligonucleotides that are more nuclease-resistant (via P‫؍‬S) and have higher affinity (via 2-F) than DNA. Finally, the affinities of DNA and uniform 2-F-, P‫؍‬S-substituted 10 -20-mer oligonucleotide complements for the best hybridization site, from HCV nt 355 to nt 364 -374, closely corresponded to antisense mechanism inhibition activities in an in vitro translation assay and in a human cell-based HCV core protein expression assay, respectively. These results validate our strategy for the selection of hybridization-optimized and biologically active antisense oligonucleotides targeting HCV RNA and support the potential for utility in further applications.In order to ensure sequence-unique recognition of antisense and ribozyme oligonucleotide drug candidates with a chosen site on any given transcript or transcript precursor RNA, a necessary and sufficient requirement for a minimum of 15-17 complementary base pairs has been proposed (1, 2), and typically 20 are sought (3, 4). However, both the kinetics and the thermodynamics of antisense oligonucleotide hybridization may be profoundly attenuated when an energetic cost is incurred to disrupt secondary and tertiary structures of folded RNA that block the hybridization (5-8). Further, the complexity of folded RNA suggests that there should be great variability of RNA structure-dependent hybridizati...