The Synthesis and Evaluation of Heterocycles as Anti-Tuberculosis Agents

<p>Tuberculosis (TB) is the leading cause of death from a single infectious agent, Mycobacterium tuberculosis (Mtb), worldwide. Currently, the efficacy of TB treatment regimens has declined due to the rise in antibacterial resistance and the shortage of new TB drugs. Thus, much effort has been spent in anti-tuberculosis drug development and in identifying new therapeutic targets against Mtb. One such target is NADH dehydrogenase-II (NDH-II), an essential enzyme in the mycobacterial electron transport chain that is not present in mammalian cells. In this thesis, four classes of heterocyclic compounds that have the potential to target NDH-II and their evaluation as anti-tubercular agents, are described. An overview of TB drug development and NDH-II as a promising target for TB drugs are described in Chapter 1. In Chapters 2 and 3, the potential of anti-tubercular drugs based on the quinolinequinone (QQ) scaffold is described. QQs have previously shown promise as TB drugs by activating NDH-II to overproduce harmful reactive oxygen species leading to bacterial cell death. Chapter 2 describes the total synthesis of the QQ natural products ascidiathiazone A and ascidiathiazone B, and derivatives thereof, using a synthetic route that allows for high divergency and the efficient synthesis of the natural products and their intermediates. To this end, the first total synthesis of ascidiathiazone B is reported, as is the identification of ascidiathiazone A as a promising anti-tuberculosis drug with an MIC of 1.6 μM against Mtb. Insight into the ability of a representative quinone, 7-chloro-6-chloroethylamino-2-methyl-QQ, to increase NDH-II activity is also described. In Chapter 3, the syntheses of thirty-two simplified QQs with different functional groups at the 6- and 7-positions of the QQ scaffold are described. These compounds were screened against Mtb, with the lead compound from this library, 7-chloro-6-propargylamino-QQ, exhibiting an MIC of 8 μM against Mtb. Structure-activity data revealed diminished biological activity for QQs bearing tertiary amines, as compared to those with secondary amines, suggesting that the presence of a hydrogen bond donor at the 6- and 7-positions of QQs may play a critical role in antimycobacterial activity. In Chapter 4, the synthesis and anti-mycobacterial activity of chromonyl-pyrimidines is presented. Chromonyl-pyrimidines have a structural resemblance to quinolinyl pyrimidines, a class of known NDH-II inhibitors and anti-TB agents. Chromones have shown promise as TB drugs, though they have not yet been reported to bind NDH-II. Despite this, chromonyl-pyrimidines contain a ketone functionality that may be able to bind the quinone binding site. For the first eleven-member library of chromonyl pyrimidines synthesised, all but two of the compounds exhibited inhibitory activity against Mtb, however, the growth inhibition was modest (MIC = 36-684 M). Accordingly, a second generation of chromonyl pyrimidines was synthesised, which included six compounds with improved potency against Mtb – all with an MIC value of 12.5 μM. The activity of these chromonyl pyrimidines was attributed to the presence of aromatic rings both on the pyrimidine and the chromone scaffolds, though changes to the electronic properties of the aryl groups, i.e. the incorporations of electron-withdrawing and electron-donating groups, did not affect inhibitory activity. Finally, in Chapter 5, a library of phthalazinones and pyrimidinyl-phthalazinones with anti-tubercular activity is described. While phthalazinones have not yet been extensively explored as anti-mycobacterial agents, the phthalazinone scaffold has the potential to act as an uncoupler. Uncouplers are typically weak acids or bases that act on the electron transport chain by dissipating the proton motive force and ultimately preventing the generation of ATP. In Mtb, this uncoupling process is detrimental and leads to cell death. Phthalazinones are weakly basic and, due to their bicyclic ketone-bearing motif, has the potential to bind NDH-II at the proposed Q-site. Accordingly, a series of phthalazinones was synthesised to investigate their anti-tubercular activity and uncoupling activity. From the library of phthalazinones, N-tert-butyl- and nitro-substituted phthalazinones elicited high inhibitory activity, both with an MIC value of 3 μM. Of particular note among the pyrimidinyl-phthalazinones was the 4-fluorophenyl-pyrimidinyl-N-heptyl phthalazinone, which showed high potency against Mtb with an MIC of 1.6 μM. Further biological studies showed that some phthalazinones increased the rate of NADH oxidation in mycobacteria, which could be a result of uncoupling activity, while a number of pyrimidinyl-phthalazinones decreased NADH oxidation rates. These mechanistic results indicated that the two classes of compounds may have different modes of inhibition.</p>

The Synthesis and Evaluation of Heterocycles as Anti-Tuberculosis Agents

<p>Tuberculosis (TB) is the leading cause of death from a single infectious agent, Mycobacterium tuberculosis (Mtb), worldwide. Currently, the efficacy of TB treatment regimens has declined due to the rise in antibacterial resistance and the shortage of new TB drugs. Thus, much effort has been spent in anti-tuberculosis drug development and in identifying new therapeutic targets against Mtb. One such target is NADH dehydrogenase-II (NDH-II), an essential enzyme in the mycobacterial electron transport chain that is not present in mammalian cells. In this thesis, four classes of heterocyclic compounds that have the potential to target NDH-II and their evaluation as anti-tubercular agents, are described. An overview of TB drug development and NDH-II as a promising target for TB drugs are described in Chapter 1. In Chapters 2 and 3, the potential of anti-tubercular drugs based on the quinolinequinone (QQ) scaffold is described. QQs have previously shown promise as TB drugs by activating NDH-II to overproduce harmful reactive oxygen species leading to bacterial cell death. Chapter 2 describes the total synthesis of the QQ natural products ascidiathiazone A and ascidiathiazone B, and derivatives thereof, using a synthetic route that allows for high divergency and the efficient synthesis of the natural products and their intermediates. To this end, the first total synthesis of ascidiathiazone B is reported, as is the identification of ascidiathiazone A as a promising anti-tuberculosis drug with an MIC of 1.6 μM against Mtb. Insight into the ability of a representative quinone, 7-chloro-6-chloroethylamino-2-methyl-QQ, to increase NDH-II activity is also described. In Chapter 3, the syntheses of thirty-two simplified QQs with different functional groups at the 6- and 7-positions of the QQ scaffold are described. These compounds were screened against Mtb, with the lead compound from this library, 7-chloro-6-propargylamino-QQ, exhibiting an MIC of 8 μM against Mtb. Structure-activity data revealed diminished biological activity for QQs bearing tertiary amines, as compared to those with secondary amines, suggesting that the presence of a hydrogen bond donor at the 6- and 7-positions of QQs may play a critical role in antimycobacterial activity. In Chapter 4, the synthesis and anti-mycobacterial activity of chromonyl-pyrimidines is presented. Chromonyl-pyrimidines have a structural resemblance to quinolinyl pyrimidines, a class of known NDH-II inhibitors and anti-TB agents. Chromones have shown promise as TB drugs, though they have not yet been reported to bind NDH-II. Despite this, chromonyl-pyrimidines contain a ketone functionality that may be able to bind the quinone binding site. For the first eleven-member library of chromonyl pyrimidines synthesised, all but two of the compounds exhibited inhibitory activity against Mtb, however, the growth inhibition was modest (MIC = 36-684 M). Accordingly, a second generation of chromonyl pyrimidines was synthesised, which included six compounds with improved potency against Mtb – all with an MIC value of 12.5 μM. The activity of these chromonyl pyrimidines was attributed to the presence of aromatic rings both on the pyrimidine and the chromone scaffolds, though changes to the electronic properties of the aryl groups, i.e. the incorporations of electron-withdrawing and electron-donating groups, did not affect inhibitory activity. Finally, in Chapter 5, a library of phthalazinones and pyrimidinyl-phthalazinones with anti-tubercular activity is described. While phthalazinones have not yet been extensively explored as anti-mycobacterial agents, the phthalazinone scaffold has the potential to act as an uncoupler. Uncouplers are typically weak acids or bases that act on the electron transport chain by dissipating the proton motive force and ultimately preventing the generation of ATP. In Mtb, this uncoupling process is detrimental and leads to cell death. Phthalazinones are weakly basic and, due to their bicyclic ketone-bearing motif, has the potential to bind NDH-II at the proposed Q-site. Accordingly, a series of phthalazinones was synthesised to investigate their anti-tubercular activity and uncoupling activity. From the library of phthalazinones, N-tert-butyl- and nitro-substituted phthalazinones elicited high inhibitory activity, both with an MIC value of 3 μM. Of particular note among the pyrimidinyl-phthalazinones was the 4-fluorophenyl-pyrimidinyl-N-heptyl phthalazinone, which showed high potency against Mtb with an MIC of 1.6 μM. Further biological studies showed that some phthalazinones increased the rate of NADH oxidation in mycobacteria, which could be a result of uncoupling activity, while a number of pyrimidinyl-phthalazinones decreased NADH oxidation rates. These mechanistic results indicated that the two classes of compounds may have different modes of inhibition.</p>

CAR T-Cells Depend on the Coupling of NADH Oxidation with ATP Production

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD+ by the enzyme Lactobacillus brevis NADH Oxidase (LbNOX), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if LbNOX promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing LbNOX have enhanced oxygen as well as lactate consumption and increased pyruvate production. LbNOX renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell’s expressing LbNOX showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by LbNOX is insufficient to promote tumor clearance.

A sodium-translocating module linking succinate production to formation of a membrane potential in Prevotella bryantii

Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff which is accomplished by the microbial community in the rumen. Roughly 40% of the rumen microbiota belong to the family of Prevotellaceae which ferment sugars to organic acids such as acetate, propionate as well as succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na + -translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This S odium-translocating N ADH: F umarate oxido R eductase (SNFR) supercomplex was enriched by BN-PAGE and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min -1 mg -1 ), quinone reduction (490 nmol min -1 mg -1 ) and fumarate reduction (1200 nmol min -1 mg -1 ) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii . Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the FAD cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD + and succinate. We propose that the regeneration of NAD + in P. bryantii is intimately linked to the build-up of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. Importance Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella sp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium-gradient formation by a newly described supercomplex consisting of Na + -translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the S odium-translocating N ADH: F umarate oxido R eductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii . Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.

MDH2 is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle

The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD+ and ATP. In contrast to ADP, NADH metabolically produced in the cytosol is not transported across the inner mitochondrial membrane and must be indirectly transferred inside mitochondria through the malate-aspartate shuttle (MAS) to fuel RC with electrons. MAS is the major pathway maintaining cytosolic NADH/NAD+ redox balance in mammalian tissues such as liver and heart and its activity is crucial for cell metabolism, division and survival. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD+ redox stress have not been elucidated. The recent discovery that complex I (NADH dehydrogenase), and not complex II (Succinate dehydrogenase), can assemble with other RC complexes to form functional entities called respirasomes, led to the assumption that this supramolecular organisation would favour NADH oxidation. Surprisingly, our bioenergetic characterization of liver and heart mitochondria demonstrates that the RC systematically favours electrons provided by complex II. However, mitochondrial malate dehydrogenase (MDH2) mediated metabolic regulation can rewire respiratory chain electrons flow from succinate toward NADH oxidation in response to increase MAS activity. Interestingly, this new regulatory mechanism synergistically increases the NADH oxidative capacity of the RC and rewires MDH2 driven anaplerosis of the TCA, preventing malate production from succinate to favor oxidation of cytosolic malate. This discovery demonstrates that MAS does not only passively balance cytosolic and mitochondrial NADH but instead, in response to cytosolic redox stress, MAS actively rewires fuelling of the RC, inhibiting complex II to prioritize cytosolic NADH oxidation and increase complex I oxidative capacity.

Relevance of NADH Dehydrogenase and Alternative Two-Enzyme Systems for Growth of Corynebacterium glutamicum With Glucose, Lactate, and Acetate

The oxidation of NADH with the concomitant reduction of a quinone is a crucial step in the metabolism of respiring cells. In this study, we analyzed the relevance of three different NADH oxidation systems in the actinobacterial model organism Corynebacterium glutamicum by characterizing defined mutants lacking the non-proton-pumping NADH dehydrogenase Ndh (Δndh) and/or one of the alternative NADH-oxidizing enzymes, L-lactate dehydrogenase LdhA (ΔldhA) and malate dehydrogenase Mdh (Δmdh). Together with the menaquinone-dependent L-lactate dehydrogenase LldD and malate:quinone oxidoreductase Mqo, the LdhA-LldD and Mdh-Mqo couples can functionally replace Ndh activity. In glucose minimal medium the Δndh mutant, but not the ΔldhA and Δmdh strains, showed reduced growth and a lowered NAD+/NADH ratio, in line with Ndh being the major enzyme for NADH oxidation. Growth of the double mutants ΔndhΔmdh and ΔndhΔldhA, but not of strain ΔmdhΔldhA, in glucose medium was stronger impaired than that of the Δndh mutant, supporting an active role of the alternative Mdh-Mqo and LdhA-LldD systems in NADH oxidation and menaquinone reduction. In L-lactate minimal medium the Δndh mutant grew better than the wild type, probably due to a higher activity of the menaquinone-dependent L-lactate dehydrogenase LldD. The ΔndhΔmdh mutant failed to grow in L-lactate medium and acetate medium. Growth with L-lactate could be restored by additional deletion of sugR, suggesting that ldhA repression by the transcriptional regulator SugR prevented growth on L-lactate medium. Attempts to construct a ΔndhΔmdhΔldhA triple mutant were not successful, suggesting that Ndh, Mdh and LdhA cannot be replaced by other NADH-oxidizing enzymes in C. glutamicum.