Synthesis of narrow-spectrum anti-mycobacterial molecules without effect on the diversity of gut microbiota in mice based on the structure of rifampicin.

Rifampicin (RIF) is a broad-spectrum antimicrobial agent that is also a first-line drug for treating tuberculosis (TB). Based on the naphthyl ring structure of RIF this study synthesized 16 narrow-spectrum antimicrobial molecules that were specifically anti-Mycobacterium tuberculosis (Mtb). The most potent candidate was 2-((6-hydroxynaphthalen-2-yl) methylene) hydrazine-1-carbothioamide (compound 3c) with minimum inhibitory concentration (MIC) of 1 µg/mL against Mtb. Synergistic anti-Mtb test indicated that none of the combinations of 3c with the major anti-TB drugs are antagonistic. Consistent with RIF, compound 3c induced large amounts of reactive oxygen radicals (ROS) in the cells of Mtb. The killing kinetics of compound 3c and RIF are very similar. Furthermore, molecular docking showed that compound 3c was able to access the RIF binding pocket of the ß subunit of Mtb RNA polymerase (RNAP). Experiments in mice showed that compound 3c increased the variety of intestinal flora in mice, while RIF significantly decreased the diversity of intestinal flora in mice. In addition, compound 3c is non-toxic to animal cells with a selection index (SI) much more than 10. The evidence from this study suggests that the further development of 3c could contribute to the development of novel drug for TB treatment.

Sugar-Phosphate Toxicities Attenuate Salmonella Fitness in the Gut.

Pathogens are becoming resistant to antimicrobials at an increasing rate, and novel therapeutic strategies are needed. Using Salmonella as a model, we have investigated the induction of sugar-phosphate toxicity as a potential therapeutic modality. The approach entails providing a nutrient while blocking the catabolism of that nutrient, resulting in the accumulation of a toxic intermediate. We hypothesize that this build-up will decrease the fitness of the organism during infection given nutrient availability. We tested this hypothesis using mutants lacking one of seven genes whose mutation is expected to cause the accumulation of a toxic metabolic intermediate. The araD, galE, rhaD, glpD, mtlD, manA, and galT mutants were then provided the appropriate sugars, either in vitro or during gastrointestinal infection of mice. All but the glpD mutant had nutrient-dependent growth defects in vitro, suggestive of sugar-phosphate toxicity. During gastrointestinal infection of mice, five mutants had decreased fitness. Providing the appropriate nutrient in the animal's drinking water was required to cause fitness defects with the rhaD and manA mutants and to enhance the fitness defect of the araD mutant. The galE and mtlD mutants were severely attenuated regardless of the nutrient being provided in the drinking water. Homologs of galE are widespread among bacteria and in humans, rendering the specific targeting of bacterial pathogens difficult. However, the araD, mtlD, and rhaD genes are not present in humans, appear to be rare in most phyla of bacteria, and are common in several genera of Enterobacteriaceae, making the encoded enzymes potential narrow-spectrum therapeutic targets. IMPORTANCE Bacterial pathogens are becoming increasingly resistant to antibiotics. There is an urgent need to identify novel drug targets and therapeutic strategies. In this work we have assembled and characterized a collection of mutations in our model pathogen, Salmonella enterica, that block a variety of sugar utilization pathways in such a way as to cause the accumulation of a toxic sugar-phosphate. Mutations in three genes, rhaD, araD, and mtlD, dramatically decrease the fitness of Salmonella in a mouse model of gastroenteritis, suggesting that RhaD, AraD, and MtlD may be good narrow-spectrum drug targets. The induction of sugar-phosphate toxicities may be a therapeutic strategy that is broadly relevant to other bacterial and fungal pathogens.

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy, Synergy, Resistance and Mechanistic Studies.

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.

Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target.

Fusobacterium nucleatum, a pathobiont inhabiting the oral cavity, contributes to opportunistic diseases, such as periodontal diseases and gastrointestinal cancers, which involve microbiota imbalance. Broad-spectrum antimicrobial agents, while effective against F. nucleatum infections, can exacerbate dysbiosis. This necessitates the discovery of more targeted narrow-spectrum antimicrobial agents. We therefore investigated the potential for the fusobacterial enoyl-ACP reductase II (ENR II) isoenzyme FnFabK (C4N14_ 04250) as a narrow-spectrum drug target. ENRs catalyze the rate-limiting step in the bacterial fatty acid synthesis pathway. Bioinformatics revealed that of the four distinct bacterial ENR isoforms, F. nucleatum specifically encodes FnFabK. Genetic studies revealed that fabK was indispensable for F. nucleatum growth, as the gene could not be deleted, and silencing of its mRNA inhibited growth under the test conditions. Remarkably, exogenous fatty acids failed to rescue growth inhibition caused by the silencing of fabK. Screening of synthetic phenylimidazole analogues of a known FabK inhibitor identified an inhibitor (i.e., 681) of FnFabK enzymatic activity and F. nucleatum growth, with an IC50 of 2.1 µM (1.0 µg/mL) and a MIC of 0.4 µg/mL, respectively. Exogenous fatty acids did not attenuate the activity of 681 against F. nucleatum. Furthermore, FnFabK was confirmed as the intracellular target of 681 based on the overexpression of FnFabK shifting MICs and 681-resistant mutants having amino acid substitutions in FnFabK or mutations in other genetic loci affecting fatty acid biosynthesis. 681 had minimal activity against a range of commensal flora, and it was less active against streptococci in physiologic fatty acids. Taken together, FnFabK is an essential enzyme that is amenable to drug targeting for the discovery and development of narrow-spectrum antimicrobial agents.