Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13.

Widespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is ∼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection. PAPERCLIP.

Wide-ranging and unexpected consequences of altered Pol II catalytic activity in vivo.

Here we employ a set of RNA Polymerase II (Pol II) activity mutants to determine the consequences of increased or decreased Pol II catalysis on gene expression in Saccharomyces cerevisiae. We find that alteration of Pol II catalytic rate, either fast or slow, leads to decreased Pol II occupancy and apparent reduction in elongation rate in vivo. However, we also find that determination of elongation rate in vivo by chromatin immunoprecipitation can be confounded by the kinetics and conditions of transcriptional shutoff in the assay. We identify promoter and template-specific effects on severity of gene expression defects for both fast and slow Pol II mutants. We show that mRNA half-lives for a reporter gene are increased in both fast and slow Pol II mutant strains and the magnitude of half-life changes correlate both with mutants' growth and reporter expression defects. Finally, we tested a model that altered Pol II activity sensitizes cells to nucleotide depletion. In contrast to model predictions, mutated Pol II retains normal sensitivity to altered nucleotide levels. Our experiments establish a framework for understanding the diversity of transcription defects derived from altered Pol II activity mutants, essential for their use as probes of transcription mechanisms.

Target-based discovery of antagonists of the tick (Rhipicephalus microplus) kinin receptor identifies small molecules that inhibit midgut contractions.

BACKGROUND: A GPCR (G protein-coupled receptor) target-based approach was applied to identify antagonists of the arthropod-specific tick kinin receptor. These small molecules were expected to reproduce the detrimental phenotypic effects that had been observed in Rhipicephalus microplus females when the kinin receptor was silenced by RNA interference. Rhipicephalus microplus, the southern cattle tick, cattle fever tick, or Asian blue tick, is the vector of pathogenic microorganisms causing the deadly bovine babesiosis and anaplasmosis. The widespread resistance to acaricides in tick populations worldwide emphasizes that exploring novel targets for effective tick control is imperative. RESULTS: Fifty-three structural analogs of previously identified tick kinin antagonists were screened in a 'dual-addition' calcium fluorescence assay using a CHO-K1 cell line expressing the tick kinin receptor. Seven molecules were validated as non-cytotoxic antagonists, four of which were partial (SACC-0428764, SACC-0428780, SACC-0428800, and SACC-0428803), and three were full antagonists (SACC-0428799, SACC-0428801, and SACC-0428815). Four of these antagonists (SACC-0428764, SACC-0428780, SACC-0428799, and SACC-0428815) also inhibited the tick midgut contractions induced by the myotropic kinin agonist analog 1728, verifying their antagonistic bioactivity. The small molecules were tested on recombinant human neurokinin (NK) receptors, the one most similar to the invertebrate kinin receptors. Most molecules were inhibitors of the NK1 receptor, except SACC-0412066, a previously identified tick kinin receptor antagonist, which inhibited the NK1 receptor only at the highest concentration tested (25 µm). None of the molecules inhibited the NK3 human receptor. CONCLUSION: Molecules identified through this approach could be useful probes for studying the tick kinin signaling system and midgut physiology. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Elesclomol alleviates Menkes pathology and mortality by escorting Cu to cuproenzymes in mice.

Loss-of-function mutations in the copper (Cu) transporter ATP7A cause Menkes disease. Menkes is an infantile, fatal, hereditary copper-deficiency disorder that is characterized by progressive neurological injury culminating in death, typically by 3 years of age. Severe copper deficiency leads to multiple pathologies, including impaired energy generation caused by cytochrome c oxidase dysfunction in the mitochondria. Here we report that the small molecule elesclomol escorted copper to the mitochondria and increased cytochrome c oxidase levels in the brain. Through this mechanism, elesclomol prevented detrimental neurodegenerative changes and improved the survival of the mottled-brindled mouse-a murine model of severe Menkes disease. Thus, elesclomol holds promise for the treatment of Menkes and associated disorders of hereditary copper deficiency.

Structure-Guided Drug Design of 6-Substituted Adenosine Analogues as Potent Inhibitors of Mycobacterium tuberculosis Adenosine Kinase.

Mycobacterium tuberculosis adenosine kinase (MtbAdoK) is an essential enzyme of Mtb and forms part of the purine salvage pathway within mycobacteria. Evidence suggests that the purine salvage pathway might play a crucial role in Mtb survival and persistence during its latent phase of infection. In these studies, we adopted a structural approach to the discovery, structure-guided design, and synthesis of a series of adenosine analogues that displayed inhibition constants ranging from 5 to 120 nM against the enzyme. Two of these compounds exhibited low micromolar activity against Mtb with half maximal effective inhibitory concentrations of 1.7 and 4.0 µM. Our selectivity and preliminary pharmacokinetic studies showed that the compounds possess a higher degree of specificity against MtbAdoK when compared with the human counterpart and are well tolerated in rodents, respectively. Finally, crystallographic studies showed the molecular basis of inhibition, potency, and selectivity and revealed the presence of a potentially therapeutically relevant cavity unique to the MtbAdoK homodimer.

Photoinduced membrane damage of E. coli and S. aureus by the photosensitizer-antimicrobial peptide conjugate eosin-(KLAKLAK)2.

BACKGROUND/OBJECTIVES: Upon irradiation with visible light, the photosensitizer-peptide conjugate eosin-(KLAKLAK)2 kills a broad spectrum of bacteria without damaging human cells. Eosin-(KLAKLAK)2 therefore represents an interesting lead compound for the treatment of local infection by photodynamic bacterial inactivation. The mechanisms of cellular killing by eosin-(KLAKLAK)2, however, remain unclear and this lack of knowledge hampers the development of optimized therapeutic agents. Herein, we investigate the localization of eosin-(KLAKLAK)2 in bacteria prior to light treatment and examine the molecular basis for the photodynamic activity of this conjugate. METHODOLOGY/PRINCIPAL FINDINGS: By employing photooxidation of 3,3-diaminobenzidine (DAB), (scanning) transmission electron microscopy ((S)TEM), and energy dispersive X-ray spectroscopy (EDS) methodologies, eosin-(KLAKLAK)2 is visualized at the surface of E. coli and S. aureus prior to photodynamic irradiation. Subsequent irradiation leads to severe membrane damage. Consistent with these observations, eosin-(KLAKLAK)2 binds to liposomes of bacterial lipid composition and causes liposomal leakage upon irradiation. The eosin moiety of the conjugate mediates bacterial killing and lipid bilayer leakage by generating the reactive oxygen species singlet oxygen and superoxide. In contrast, the (KLAKLAK)2 moiety targets the photosensitizer to bacterial lipid bilayers. In addition, while (KLAKLAK)2 does not disrupt intact liposomes, the peptide accelerates the leakage of photo-oxidized liposomes. CONCLUSIONS/SIGNIFICANCE: Together, our results suggest that (KLAKLAK)2 promotes the binding of eosin Y to bacteria cell walls and lipid bilayers. Subsequent light irradiation results in membrane damage from the production of both Type I & II photodynamic products. Membrane damage by oxidation is then further aggravated by the (KLAKLAK)2 moiety and membrane lysis is accelerated by the peptide. These results therefore establish how photosensitizer and peptide act in synergy to achieve bacterial photo-inactivation. Learning how to exploit and optimize this synergy should lead to the development of future bacterial photoinactivation agents that are effective at low concentrations and at low light doses.