Scholarly Work - Chemistry & Biochemistry
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/8714
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Item Carbon metabolism modulates the efficacy of drugs targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis(2019-06) Kalia, Nitin P.; Shi Lee, Bei; Ab Rahman, Nurlilah B.; Moraski, Garrett C.; Miller, Marvin J.; Pethe, KevinThe influence of carbon metabolism on oxidative phosphorylation is poorly understood in mycobacteria. M. tuberculosis expresses two respiratory terminal oxidases, the cytochrome bc1:aa3 and the cytochrome bd oxidase, which are jointly required for oxidative phosphorylation and mycobacterial viability. The essentiality of the cytochrome bc1:aa3 for optimum growth is illustrated by its vulnerability to chemical inhibition by the clinical drug candidate Q203 and several other chemical series. The cytochrome bd oxidase is not strictly essential for growth but is required to maintain bioenergetics when the function of the cytochrome bc1:aa3 is compromised. In this study, we observed that the potency of drugs targeting the cytochrome bc1:aa3 is influenced by carbon metabolism. The efficacy of Q203 and related derivatives was alleviated by glycerol supplementation. The negative effect of glycerol supplementation on Q203 potency correlated with an upregulation of the cytochrome bd oxidase-encoding cydABDC operon. Upon deletion of cydAB, the detrimental effect of glycerol on the potency of Q203 was abrogated. The same phenomenon was also observed in recent clinical isolates, but to a lesser extent compared to the laboratory-adapted strain H37Rv. This study reinforces the importance of optimizing in vitro culture conditions for drug evaluation in mycobacteria, a factor which appeared to be particularly essential for drugs targeting the cytochrome bc1:aa3 terminal oxidase.Item Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b(2018-03) Streit, Bennett R.; Celis Luna, Arianna I.; Moraski, Garrett C.; Shisler, Krista A.; Shepard, Eric M.; Rodgers, Kenton R.; Lukat-Rodgers, Gudrun S.; DuBois, Jennifer L.The H2O2-dependent oxidative decarboxylation of coproheme III is the final step in the biosynthesis of heme b in many microbes. However, the coproheme decarboxylase reaction mechanism is unclear. The structure of the decarboxylase in complex with coproheme III suggested that the substrate iron, reactive propionates, and an active-site tyrosine convey a net 2e-/2H+ from each propionate to an activated form of H2O2 Time-resolved EPR spectroscopy revealed that Tyr-145 forms a radical species within 30 sec of the reaction of the enzyme-coproheme complex with H2O2 This radical disappeared over the next 270 sec, consistent with a catalytic intermediate. Use of the harderoheme III intermediate as substrate or substitutions of redox-active side chains (W198F, W157F, or Y113S) did not strongly affect the appearance or intensity of the radical spectrum measured 30 sec after initiating the reaction with H2O2, nor did it change the ~270 sec required for the radical signal to recede to ≤ 10% of its initial intensity. These results suggested Tyr-145 as the site of a catalytic radical involved in decarboxylating both propionates. Tyr-145 was accompanied by partial loss of the initially present Fe(III) EPR signal intensity, consistent with the possible formation of Fe(IV)=O. Site-specifically deuterated coproheme gave rise to a kinetic isotope effect of ~2 on the decarboxylation rate constant, indicating that cleavage of the propionate Cbeta-H bond was partly rate limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H2O2 intermediate and then from the propionate Cbeta-H to Tyr-145.Item Preparation and Evaluation of Potent PentafluorosulfanylSubstituted Anti-Tuberculosis Compounds(2017-07) Moraski, Garrett C.; Bristol, Ryan; Seeger, Natalie; Boshoff, Helena I.; Tsang, Patricia Siu-Yee; Miller, Marvin J.The global fight to stop tuberculosis (TB) remains a great challenge, particularly with the increase in drug-resistant strains and a lack of funding to support the development of new treatments. To bolster a precarious drug pipeline, we prepared a focused panel of eight pentafluorosulfanyl (SF5 ) compounds which were screened for their activity against Mycobacterium tuberculosis (Mtb) H37Rv in three different assay conditions and media. All eight compounds had sub-micromolar potency, and four displayed MICs <100 nm. Seven compounds were evaluated against non-replicating and mono-drug-resistant Mtb, and for their ability to inhibit Mtb within the macrophage. The greatest potency was observed against intracellular Mtb (MIC <10 nm for three compounds), which is often the most challenging to target. In general, the SF5 -bearing compounds were very similar to their CF3 counterparts, with the major differences observed being their in vitro ADME properties. Two SF5 -bearing compounds were found to have greater protein binding than their corresponding CF3 counterparts, but were also less metabolized in human microsomes, resulting in longer half-lives.Item Imidazo[1,2-a]Pyridine-3-Carboxamides Are Active Antimicrobial Agents against Mycobacterium avium Infection In Vivo(2016-08) Moraski, Garrett C.; Cheng, Yong; Cho, Sanghyun; Cramer, Jeffrey W; Godfrey, Alexander; Masquelin, Thierry; Franzblau, Scott G.; Miller, Marvin J.; Schorey, JefferyA panel of six imidazo[1,2-a]pyridine-3-carboxamides (IAPs) were shown to have low-micromolar activity against Mycobacterium avium strains. Compound ND-10885 (compound 2) showed significant activity in the lung, spleen, and liver in a mouse M. avium infection model. A combined regimen consisting of ND-10885 (compound 2) and rifampin was additive in its anti-M. avium activity in the lung. Our data indicate that IAPs represent a new class of antibiotics that are active against M. avium and could potentially serve as an effective addition to a combined treatment regimen.Item Putting Tuberculosis (TB) To Rest: Transformation of the Sleep Aid, Ambien, and “Anagrams” Generated Potent Antituberculosis Agents(2014-12) Moraski, Garrett C.; Miller, Patricia; Bailey, Mai Ann; Ollinger, Juliane; Parish, Tanya; Boshoff, Helena I.; Cho, Sanghyun; Anderson, Jeffery; Mulugeta, Surafel; Franzblau, Scott G.; Miller, Marvin J.Zolpidem (Ambien, 1) is an imidazo[1,2-a]pyridine-3-acetamide and an approved drug for the treatment of insomnia. As medicinal chemists enamored by how structure imparts biological function, we found it to have strikingly similar structure to the antitubercular imidazo[1,2-a]pyridine-3-carboxyamides. Zolpidem was found to have antituberculosis activity (MIC of 10–50 μM) when screened against replicating Mycobacterium tuberculosis (Mtb) H37Rv. Manipulation of the Zolpidem structure, notably, to structural isomers (“anagrams”), attains remarkably improved potency (5, MIC of 0.004 μM) and impressive potency against clinically relevant drug-sensitive, multi- and extensively drug-resistant Mtb strains (MIC < 0.03 μM). Zolpidem anagrams and analogues were synthesized and evaluated for their antitubercular potency, toxicity, and spectrum of activity against nontubercular mycobacteria and Gram-positive and Gram-negative bacteria. These efforts toward the rational design of isomeric anagrams of a well-known sleep aid underscore the possibility that further optimization of the imidazo[1,2-a]pyridine core may well “put TB to rest”.