Despite nearly 85 years of successful antibacterial chemotherapy, bacterial infections still pose a serious threat to human health. Continually emerging drug-resistant bacteria make existing agents less effective, and a paucity of new agents and decreased development effort from the pharmaceutical industry provide little hope of replenishing the arsenal (1, 2). Complications and mortality due to bacterial infections continue to increase, already reaching epidemic proportions in some areas of the world. If humans are to regain the upper hand in fighting bacterial infections, then innovation, investment, and new antibacterial agents will be needed. Although some of the barriers to the discovery and approval of new compounds are economic or policy related, there is also a desperate need for new targets and new mechanisms of action. A renewed effort to expand our knowledge of bacterial physiology and to translate discoveries into the clinic will be needed to address these challenges and to reinvigorate antibiotic development pipelines.Recent advances in our understanding of how bacteria maintain physiological homeostasis revealed a promising class of potential antibiotic targets called riboswitches-noncoding mRNAs that form a structured receptor (or aptamer) which can directly bind to a specific small-molecule ligand or ion and thereby regulate gene expression (3-5). Ligand binding to a riboswitch aptamer stabilizes a conformationally distinct architecture in the mRNA that modulates the expression of the adjacent coding region(s) (4-8). To date, more than 35 riboswitch classes have been discovered and characterized (7). Three of these riboswitch classes have been revealed as important cellular targets of antibacterial small molecules whose mechanism of action had not been previously defined (9-13). More recently, several publications have demonstrated that novel small molecules that bind to selected riboswitch aptamers with affinities comparable to that of the cognate ligand can be rationally identified and optimized (14-21).In some cases, synthetic or natural riboswitch ligand analogs have demonstrated potent antibacterial activity (12-15, 20, 22). For example, the phosphorylated form of roseoflavin (RoF) (

The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMettRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.A novel class of antimicrobial agents, the oxazolidinones, target a wide spectrum of gram-positive and anaerobic bacteria (4, 6, 9, 28). These compounds act by inhibiting protein synthesis and have no effect on replication or transcription (8). Cell extracts exposed to oxazolidinones are impaired in protein synthesis when programmed by native mRNAs but do not appear to be inhibited when programmed by synthetic mRNAs that lack the signals required for initiation and termination of translation (7,8,26). This suggested that these compounds may target the initiation reaction. The oxazolidinone PNU-100766 (linezolid; Fig. 1) inhibits binding of the initiator fMettRNA to the 70S ribosomal particle programmed with a synthetic mRNA that harbors a Shine-Dalgarno sequence and a properly spaced initiation codon (29).Mutations to oxazolidinone resistance map to domain V of the 23S rRNA in Halobacterium halobium (18), Staphylococcus aureus (27), and the enterococci while mapping to domains IV and V in Escherichia coli (33). The position of these PNU-100766 resistance mutations suggested to us that the oxazolidinones may target peptidyl transferase indirectly by affecting the binding of the initiator tRNA. Since the P site accommodates the initiator tRNA and the nascent protein, these drugs could also affect the affinity of the peptidyl-tRNA for the ribosome.Recent studies have indicated that the oxazolidinones bind to 70S ribosomes, as well as to 50S subunits (19), but not to 30S subunits. In contrast, a report by Matassova et al. (20) demonstrated that oxazolidinone footprints map to the central domain of the 16S rRNA whereas the 23S rRNA ...

A series of pyrimidine thioethers was synthesized and evaluated for inhibitory properties against wild-type HIV-1 reverse transcriptase (RT) and an RT carrying the resistance-conferring mutation P236L. Modifications of both the pyrimidine and the functionality attached through the thioether yielded several analogues, which demonstrated activity against both enzyme types, with IC50 values as low as 190 nM against wild-type and 66 nM against P236L RT. Evaluation of a select number of pyrimidine thioethers in cell culture showed that these compounds have excellent activity against HIV-1IIIB-WT and retain good activity against a laboratory-derived HIV-1MF delavirdine-resistant variant.

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The (alkylamino)piperidine bis(heteroaryl)piperizines (AAP-BHAPs) are a new class of human immunodeficiency virus type 1 (HIV-1)-specific inhibitors which were identified by targeted screening of recombinant reverse transcriptase (RT) enzymes carrying key nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-conferring mutations and NNRTI-resistant variants of HIV-1. Phenotypic profiling of the two most potent AAP-BHAPs, U-95133 and U-104489, against in vitro-selected drug-resistant HIV-1 variants carrying the NNRTI resistance-conferring mutation (Tyr3Cys) at position 181 of the HIV-1 RT revealed submicromolar 90% inhibitory concentration estimates for these compounds. Moreover, U-104489 demonstrated potent activity against BHAP-resistant HIV-1 MF harboring the Pro-2363Leu RT substitution and significantly suppressed the replication of clinical isolates of HIV-1 resistant to both delavirdine (BHAP U-90152T) and zidovudine. Biochemical and phenotypic characterization of AAP-BHAP-resistant HIV-1 IIIB variants revealed that high-level resistance to the AAP-BHAPs was mediated by a Gly-1903Glu substitution in RT, which had a deleterious effect on the integrity and enzymatic activity of virion-associated RT heterodimers, as well as the replication capacity of these resistant viruses.

The major route of metabolism of the bis(heteroaryl)piperazine (BHAP) class of reverse transcriptase inhibitors (RTIs), atevirdine and delavirdine, is via oxidative N-dealkylation of the 3-ethyl- or 3-isopropylamino substituent on the pyridine ring. This metabolic pathway is also the predominant mode of metabolism of (alkylamino)piperidine BHAP analogs (AAP-BHAPs), compounds wherein a 4-(alkylamino)piperidine replaces the piperazine ring of the BHAPs. The novel AAP-BHAPs possess the ability to inhibit non-nucleoside reverse transcriptase inhibitor (NNRTI) resistant recombinant HIV-1 RT and NNRTI resistant variants of HIV-1. This report describes an approach to preventing this degradation which involves the replacement of the 3-ethyl- or 3-isopropylamino substituent with either a 3-tert-butylamino substituent or a 3-alkoxy substituent. The synthesis, bioactivity and metabolic stability of these analogs is described. The majority of analogs retain inhibitory activities in enzyme and cell culture assays. In general, a 3-ethoxy or 3-isopropoxy substituent on the pyridine ring, as in compounds 10, 20, or 21, resulted in enhanced stabilities. The 3-tert-butylamino substituent was somewhat beneficial in the AAP-BHAP series of analogs, but did not exert a significant effect in the BHAP series. Lastly, the nature of the indole substitution sometimes plays a significant role in metabolic stability, particularly in the BHAP series of analogs.

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