Design, Synthesis, and Evaluation of Novel Δ2-Thiazolino 2-Pyridone Derivatives That Potentiate Isoniazid Activity in an Isoniazid-Resistant Mycobacterium tuberculosis Mutant.

Mycobacterium tuberculosis (Mtb) drug resistance poses an alarming threat to global tuberculosis control. We previously reported that C10, a ring-fused thiazolo-2-pyridone, inhibits Mtb respiration, blocks biofilm formation, and restores the activity of the antibiotic isoniazid (INH) in INH-resistant Mtb isolates. This discovery revealed a new strategy to address INH resistance. Expanding upon this strategy, we identified C10 analogues with improved potency and drug-like properties. By exploring three heterocycle spacers (oxadiazole, 1,2,3-triazole, and isoxazole) on the ring-fused thiazolo-2-pyridone scaffold, we identified two novel isoxazoles, 17h and 17j. 17h and 17j inhibited Mtb respiration and biofilm formation more potently with a broader therapeutic window, were better potentiators of INH-mediated inhibition of an INH-resistant Mtb mutant, and more effectively inhibited intracellular Mtb replication than C10. The (-)17j enantiomer showed further enhanced activity compared to its enantiomer and the 17j racemic mixture. Our potent second-generation C10 analogues offer promise for therapeutic development against drug-resistant Mtb.

Understanding the contribution of metabolism to Mycobacterium tuberculosis drug tolerance.

Treatment of Mycobacterium tuberculosis (Mtb) infections is particularly arduous. One challenge to effectively treating tuberculosis is that drug efficacy in vivo often fails to match drug efficacy in vitro. This is due to multiple reasons, including inadequate drug concentrations reaching Mtb at the site of infection and physiological changes of Mtb in response to host derived stresses that render the bacteria more tolerant to antibiotics. To more effectively and efficiently treat tuberculosis, it is necessary to better understand the physiologic state of Mtb that promotes drug tolerance in the host. Towards this end, multiple studies have converged on bacterial central carbon metabolism as a critical contributor to Mtb drug tolerance. In this review, we present the evidence that changes in central carbon metabolism can promote drug tolerance, depending on the environment surrounding Mtb. We posit that these metabolic pathways could be potential drug targets to stymie the development of drug tolerance and enhance the efficacy of current antimicrobial therapy.

Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae.

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant pathogen Pseudomonas syringae pathovar tomato strain DC3000 possess characteristic patterns, including (i) greater than 10% serine within the first 50 amino acids, (ii) an aliphatic residue or proline at position 3 or 4, and (iii) a lack of acidic amino acids within the first 12 residues. Here, the functional significance of the P. syringae T3SS substrate compositional patterns was tested. A mutant AvrPto effector protein lacking all three patterns was secreted into culture and translocated into plant cells, suggesting that the compositional characteristics are not absolutely required for T3SS targeting and that other recognition mechanisms exist. To further analyze the unique properties of T3SS targeting signals, we developed a computational algorithm called TEREE (Type III Effector Relative Entropy Evaluation) that distinguishes DC3000 T3SS substrates from other proteins with a high sensitivity and specificity. Although TEREE did not efficiently identify T3SS substrates in Salmonella enterica, it was effective in another P. syringae strain and Ralstonia solanacearum. Thus, the TEREE algorithm may be a useful tool for identifying new effector genes in plant pathogens. The nature of T3SS targeting signals was additionally investigated by analyzing the N-terminus of FtsX, a putative membrane protein that was classified as a T3SS substrate by TEREE. Although the first 50 amino acids of FtsX were unable to target a reporter protein to the T3SS, an AvrPto protein substituted with the first 12 amino acids of FtsX was translocated into plant cells. These results show that the T3SS targeting signals are highly mutable and that secretion may be directed by multiple features of substrates.