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Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported that some dioxane-linked amide NBTIs induced double-stranded DNA breaks mediated by Staphylococcus aureus gyrase. To further explore the ability of this NBTI subclass to increase double-stranded DNA breaks, we examined the effects of OSUAB-185 on DNA cleavage mediated by Neisseria gonorrhoeae gyrase and topoisomerase IV. OSUAB-185 induced single-stranded and suppressed double-stranded DNA breaks mediated by N. gonorrhoeae gyrase. However, the compound stabilized both single- and double-stranded DNA breaks mediated by topoisomerase IV. The induction of double-stranded breaks does not appear to correlate with the binding of a second OSUAB-185 molecule and extends to fluoroquinolone-resistant N. gonorrhoeae topoisomerase IV, as well as type II enzymes from other bacteria and humans. The double-stranded DNA cleavage activity of OSUAB-185 and other dioxane-linked NBTIs represents a paradigm shift in a hallmark characteristic of NBTIs and suggests that some members of this subclass may have alternative binding motifs in the cleavage complex.

Background. The oxazolidinone PNU-100480 is superior to linezolid against experimental murine tuberculosis. Two metabolites contribute to but do not fully account for its superiority. This study examined the safety, tolerability, pharmacokinetics, and mycobactericidal activity of single ascending doses of PNU-100480. Methods. Nineteen healthy volunteers received 2 escalating single oral doses (35–1500 µg) of PNU-100480 or placebo. Eight subjects received 4 daily doses of 300 mg of linezolid. Drug concentrations and bactericidal activity against Mycobacterium tuberculosis in whole-blood bactericidal culture were measured. Results. All doses were safe and well tolerated. PNU-100480 doses to 1000 mg were well absorbed and showed approximately proportional increases in exposures of parent and metabolites. The geometric mean maximal concentrations of PNU-100480, PNU-101603, and PNU-101244 (sulfoxide and sulfone metabolites) at 1000 mg were 839, 3558, and 54 ng/mL, respectively. The maximal whole-blood bactericidal activity (−0.37 ± .06 log/day) occurred at combined PNU levels ⩾2 times the minimum inhibitory concentration. The observed geometric mean maximal concentration for linezolid was 6425 ng/mL. Its maximal whole-blood bactericidal activity also occurred at ⩾2 times the minimum inhibitory concentration, but it was only −0.16 ± .05 log/day (P<.001). Neither drug showed enhanced activity at higher concentrations. Conclusions. Single doses of PNU-100480 to 1000 mg were well tolerated and exhibited antimycobacterial activity superior to 300 mg of linezolid at steady state. Additional studies are warranted to define its role in drugresistant tuberculosis. Clinical trial registration ClinicalTrials.gov identifiers NCT00871949 and NCT00990990.

There presently is no rapid method to assess the bactericidal activity of new regimens for tuberculosis. This study examined PNU-100480, TMC207, PA-824, SQ109, and pyrazinamide, singly and in various combinations, against intracellular M. tuberculosis, using whole blood culture (WBA). The addition of 1,25-dihydroxy vitamin D facilitated detection of the activity of TMC207 in the 3-day cultures. Pyrazinamide failed to show significant activity against a PZA-resistant strain (M. bovis BCG), and was not further considered. Low, mid, and high therapeutic concentrations of each remaining drug were tested individually and in a paired checkerboard fashion. Observed bactericidal activity was compared to that predicted by the sum of the effects of individual drugs. Combinations of PNU-100480, TMC207, and SQ109 were fully additive, whereas those including PA-824 were less than additive or antagonistic. The cumulative activities of 2, 3, and 4 drug combinations were predicted based on the observed concentration-activity relationship, published pharmacokinetic data, and, for PNU-100480, published WBA data after oral dosing. The most active regimens, including PNU-100480, TMC207, and SQ109, were predicted to have cumulative activity comparable to standard TB therapy. Further testing of regimens including these compounds is warranted. Measurement of whole blood bactericidal activity can accelerate the development of novel TB regimens.

Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (KI ~ 3 μM). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery.

Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported that some dioxane-linked amide NBTIs induced double-stranded DNA breaks mediated by Staphylococcus aureus gyrase. To further explore the ability of this NBTI subclass to increase double-stranded DNA breaks, we examined the effects of OSUAB-185 on DNA cleavage mediated by Neisseria gonorrhoeae gyrase and topoisomerase IV. OSUAB-185 induced single-stranded and suppressed double-stranded DNA breaks mediated by N. gonorrhoeae gyrase. However, the compound stabilized both single- and double-stranded DNA breaks mediated by topoisomerase IV. The induction of double-stranded breaks does not appear to correlate with the binding of a second OSUAB-185 molecule and extends to fluoroquinolone-resistant N. gonorrhoeae topoisomerase IV, as well as type II enzymes from other bacteria and humans. The double-stranded DNA cleavage activity of OSUAB-185 and other dioxane-linked NBTIs represents a paradigm shift in a hallmark characteristic of NBTIs and suggests that some members of this subclass may have alternative binding motifs in the cleavage complex.

Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (K[sub.I] ~ 3 μM). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery.

There presently is no rapid method to assess the bactericidal activity of new regimens for tuberculosis. This study examined PNU-100480, TMC207, PA-824, SQ109, and pyrazinamide, singly and in various combinations, against intracellular M. tuberculosis, using whole blood culture (WBA). The addition of 1,25-dihydroxy vitamin D facilitated detection of the activity of TMC207 in the 3-day cultures. Pyrazinamide failed to show significant activity against a PZA-resistant strain (M. bovis BCG), and was not further considered. Low, mid, and high therapeutic concentrations of each remaining drug were tested individually and in a paired checkerboard fashion. Observed bactericidal activity was compared to that predicted by the sum of the effects of individual drugs. Combinations of PNU-100480, TMC207, and SQ109 were fully additive, whereas those including PA-824 were less than additive or antagonistic. The cumulative activities of 2, 3, and 4 drug combinations were predicted based on the observed concentration-activity relationship, published pharmacokinetic data, and, for PNU-100480, published WBA data after oral dosing. The most active regimens, including PNU-100480, TMC207, and SQ109, were predicted to have cumulative activity comparable to standard TB therapy. Further testing of regimens including these compounds is warranted. Measurement of whole blood bactericidal activity can accelerate the development of novel TB regimens.

This dissertation details synthetic efforts directed toward two oxo polyene macrolide antibiotics, dermostatin A and RK-397. The research was focused on the development of novel methods for the assembly of skipped polyol chains. This work culminated in the convergent synthesis of the full polyol domain of RK-397, including multigram quantities of each of the two key intermediates. The C22–C33 segment was synthesized in just ten steps, using a single catalytic enantioselective reaction followed by internal stereorelay to install the four tetrahedral stereocenters. A chemo- and stereoselective elimination was also developed for the installation of the C28–C29 olefin. The C10–C 21 fragment was synthesized in twelve steps using a two directional chain synthesis approach. The full polyol was assembled using an aldol reaction with remote asymmetric induction from the nucleophilic partner. A detailed investigation of the effect of additives on Keck's catalytic asymmetric allylation reaction is also presented.