Academic literature on the topic 'Tuberculosis argininosuccinate lyase'

Pantothenate kinase (PanK) is a ubiquitous and essential enzyme that catalyses the first step in the universal Coenzyme A (CoA) biosynthetic pathway. In this step pantothenate is converted to phosphopantothentate, which subsequently forms CoA in four enzymatic steps. Three types of PanKs have been identified in bacteria, with variations in distribution, mechanism of regulation, cofactor requirement and affinity for substrates. As part of a major programme on mycobacterial proteins in our laboratory, studies on type I PanK from Mycobacterium tuberculosis (MtPanK) have been carried out previously. This investigation involved, apart from biochemical studies, structure determination of twenty-one independent crystals of binary and ternary complexes of MtPanK involving CoA, the ATP analogue AMPPCP, the GTP analogue GMPPCP, ADP, GDP, pantothenate, phosphopantothenate, citrate, pantothenol and nonyl pantothenamide. Analysis of these structures brought out the robustness of the mycobacterial PanK when compared to its E. coli homologue. It was observed that while the protein structure remained relatively rigid in all the MtPanK structures, the ligands exhibited substantial movement in the pre-formed pocket during the course of catalysis. This observation was unlike that seen in EcPanK, where the protein molecule underwent conformational changes during enzyme action. Also, the feedback inhibitor, CoA, showed a higher binding affinity to MtPanK compared to that to EcPanK. The differences exhibited by these homologous proteins despite sharing 52% sequence identity were surprising and merited further study. To this end, mutants of MtPanK were prepared, their structures solved, and solution studies related to binding and activity of these mutants were carried out. Apart from this, supplemental molecular dynamics (MD) studies were carried out on MtPanK and EcPanK and the mutants of MtPanK. Structural studies were carried out using conventional tools and techniques of macromolecular crystallography. The microbatch under-oil method was used for crystallisation in all cases. Data were collected at a home-source on a MAR 345 image plate mounted on a Bruker MICROSTAR ULTRA II Cu Kα rotating-anode X-ray generator or using a CCD detector (MARMosaic 225) on the synchrotron X-ray beamline BM14 at the European Synchrotron Radiation Facility, Grenoble, France. Data were processed using MOSFLM and SCALA and the structures were solved by the molecular replacement method using PHASER from the CCP4 suite. Refinement was carried out using REFMAC and manual model building was performed employing COOT. Structures were validated using PROCHECK. Thermal shift assay was used to study binding of CoA to the mutants. A radioactive assay and an enzyme coupled assay were employed to measure the activity of the mutants. To begin with, the high affinity of CoA to MtPanK was sought to be disturbed by disrupting the binding site using mutations. Therefore, two conserved phenylalanine residues of the hydrophobic binding site were targeted and two point mutants and a double mutant were constructed. Solution studies on the three mutants confirmed the reduction in CoA binding affinity and also that of activity to some extent. Structure solution of the mutants showed that apart from local rearrangements, the mutations led to partial or complete transition of the structure to that seen in EcPanK. Concerted movement was observed in the dimerisation region and the nucleotide binding region. To further understand this transformation and as a complementary effort to the studies on the CoA binding region mutants, mutations were made in the substrate binding regions of MtPanK such that non-conservatively substituted residues were replaced with those found in EcPanK. Solution studies on these mutants showed that CoA binding affinity was minimally affected by the mutations, while activity was reduced to some extent. In all, six structures were solved, half of which were CoA-free and showed partial or complete transformation to an Ec-like conformational state. Concerted movement was seen in another loop along with that seen in the dimerisation interface and the nucleotide binding region. The structures brought to light the changes in conformations of certain residues and their interactions that makes the Ec-like state feasible in MtPanK. Put together, these studies showed how small perturbations like those caused by point mutations could bring about global transformations in the structure of MtPanK. They also suggest that MtPanK may be able to utilise the nucleotide binding pocket as seen in EcPanK in the transformed structures. This is an aspect that may be important in relation to drug designing. The results obtained from the mutational studies were supplemented by MD simulations on MtPanK, EcPanK and mutants of MtPanK. These studies helped delineate an invariant core common to MtPanK, EcPanK and the MtPanK mutants. They also showed that wild-type EcPanK is indeed more flexible than wild-type MtPanK. Furthermore, MD simulations showed the impact of sequence on the structure of the Mt enzyme. Minor sequence changes appeared to influence different structural elements, including those far away from the sites of mutation. Thus, it would seem that an ensemble of structures is accessible to the PanK molecule and the selection of an appropriate conformation is based on the requirement brought about by mutations or ligand binding. Apart from the studies on MtPanK, structural studies on argininosuccinate lyase from Mycobacterium tuberculosis were also carried out. The native structure along with that bound to the substrate and products helped propose a catalytic mechanism based on previously available information and present studies. This investigation is presented in an appendix.

Tuberculosis (TB) is a deadly disease responsible for the death of approximately 1.5 million people each year, with the highest being from developing nations. Tuberculosis affects mostly the lungs, and other parts of the body like nerves, bones and liver. Mycobacterium tuberculosis (Mtb) is the causative agent of TB in humans. The onset of infection is via the deposition of aerosol droplets containing the pathogen, M. tuberculosis, onto the lung alveolar surfaces. About one third of the world’s population asymptomatically harbors latent M. tuberculosis bacterium with a constant risk of disease activation. Due to the emergence of drug-resistant strains and the evolution through multi-drug resistance (MDR) to extensive drug resistance (XDR), the fight against TB has become extremely challenging. Standard treatment for TB comprises four first-line antimicrobials: isoniazid, rifampicin, pyrazinamide and ethambutol. However, resistance to all of these drugs has been observed in several MDR strains of Mtb. Despite the recent advances in target identification and drug discovery, there is a relentless need for novel inhibitors against vital pathways of Mtb. The novel drug-development regimens endorse strategies wherein the pre-approved drugs for other ailments could be re-purposed, thereby cutting down the cost and time associated with the process of drug discovery. Also, the target selection strategy requires to aim at the key enzymes from the essential biosynthetic pathways, keeping an eye on their underlying dissimilarities when compared to human host. The challenges in finding a suitable target for anti-Mtb drug discovery is it’s ever evolving stride and the conserved nature of the essential proteins. Many novel small molecule inhibitors of Mtb are undermined, during the course of studies, by cross reactivity with homologs proteins in the host. Traditionally, the replication machinery has been at the heart of drug discovery and the processes associated with logarithmic growth phase are vastly exploited for drug targeting. However, targeting these vital cellular components may result in some serious non-specific effects to the host. On the other hand, the intricate network of metabolic pathways provides novel avenues for specific targeting of pathogens, precisely for three main reasons: 1. There is an acute shortage of cellular nutrients due to the constant competition between the pathogen and the host, throughout the course of infection. 2. Infectious cycles often lead to the disruption of metabolic pathways, again leading to nutrient scarcity. 3. Survival of the pathogen within the hostile niche and under oxygen starvation conditions further potentiate the demands of crucial metabolites (amino acids, nitrogen bases, carbohydrates etc.) that are used as the building blocks for cellular machinery. 4. Metabolic pathways have evolved with time, to provide the much-required specificity for exclusive targeting of the pathogen, thereby limiting the cross-reactivity with the host pathways. In order to persist and efficiently replicate in host cells, intracellular pathogens must adapt their metabolism to the available nutrients and physical conditions (mainly pH, oxygen availability and osmotic pressure) in the host. Among the major metabolic, amino acid metabolism holds great importance; as they not only serve to meet the nutritional needs of the pathogen but also play a key role in the strategies employed during pathogenesis. Although the host and the pathogen compete for many metabolites, three amino acids, Arginine, Asparagine and Tryptophan seems to be a focus of this competition because the availability of these amino acids or their derivatives influence both pathogen behavior and the immune response. Arginine constitutes a major proportion of the total proteins in the cell and arginine and its precursor ornithine are used for the biosynthesis of the most common polyamines, putrescine and spermidine. These molecules are required for optimal growth of the organism and are involved in several physiological processes. Apart from being a critical amino acid for the synthesis of cellular proteins, arginine can also be used as a nitrogen source, under conditions of nitrogen starvation, hence crucial for pathogenesis. The glutamate and glutamine are the key metabolites in the central nitrogen metabolism; both serve as endogenous nitrogen acceptor as well as nitrogen donor. However, reports demonstrate that Mtb utilizes arginine and asparagine as the key sources of nitrogen during infection in mice models of tuberculosis. Therefore, our study focuses on the process of Arginine biosynthesis in M. tuberculosis, wherein it is essential for the survival and pathogenesis. Since the arginine metabolism is essential for both the host and the pathogen, and competition for arginine may shift the balance, and thus determine the outcome of the infection. The enzymes involved in this pathway will be a promising target for anti-TB drug development. Despite the acknowledged significance of Arginine biosynthesis in the pathogens like M. tuberculosis, inhibitors to target this pathway remain to be discovered. Moreover, inhibitors of this pathway may provide novel insights to the significance of arginine biosynthesis in Mtb associated stress responses and persistence. Ornithine acetyltransferase (MtArgJ), one of the crucial enzymes during the biosynthesis of arginine in Mtb, is essential for its survival and pathogenesis. MtArgJ lacks a homolog in human genome, thereby being a good target against Mtb. We hypothesize that a targeted inhibitor against this key player of mycobacterial metabolism has the potential to combat the Mtb survival and pathogenesis. In the present thesis, we have characterized the potential of MtArgJ from M. tuberculosis as a valuable target for drug design against tuberculosis. Most importantly, the approach is to specifically target a novel allosteric site identified in this study, on the MtArgJ surface. Since we are not using the age-old approach of substrate analog as an inhibitor, we hereby further minimize or even eliminate the chances of cross-reactivity with the host cellular proteins. In the later parts, we have identified an allosteric inhibitor of MtArgJ, that significantly reduces the survival of pathogenic Mtb through the pre-clinical models of tuberculosis. Chapter 1 of this thesis gives a detailed account of the history of Tuberculosis, and its pathogenesis. The chapter further elaborates on the metabolic pathways of Mycobacterium tuberculosis, with special emphasis on the arginine biosynthesis pathway. The drug discovery regime and therapeutic challenges associated with the disease are discussed in the later parts of the chapter. All the information discussed in this chapter serves a preface for the work done throughout the thesis, and outlinesthe objectives for rest of the chapters. Chapter 2 describes the characterization and kinetic analysis of MtArgH, the last enzyme from the arginine biosynthetic pathway in M. tuberculosis. This chapter demonstrates the importance of a c-terminal cysteine residue (Cys441) in the catalysis and thermal stability of the enzyme. We further propose the existence of a product mediated feed-back inhibition of MtArgH, wherein fumarate, one of the product of MtArgH, gradually modifies the Cys441 through succination. Chapter 3 to 5 discuss about the work carried out on the enzyme Ornithine acetyltransferase (MtArgJ), a crucial enzyme for arginine biosynthesis in M. tuberculosis. We have identified a selective allosteric inhibitor against this key player of mycobacterial metabolism, employing the below-mentioned strategy. First step was to characterize the target, followed by a structure based in-silico screen. The best hits were subjected to in-vitro validation, leading to the in-vivo testing of the potential molecule, in the pre-clinical model of tuberculosis. Target characterization In-silico screen In-vitro validation Pre-clinical testing Chapter 3 starts with the characterization of the MtArgJ, wherein we identified a novel hydrophobic pocket present on the enzyme surface. We further characterized the potential of this pocket in allosterically modulating the active site. This was then followed by a structure based in-silico screen with a library of FDA approved drugs, specifically targeting this novel allosteric pocket on MtArgJ. Chapter 4 deals with the in vitro validation of the identified compounds from in-silico screen. We here identified two lead molecules, Pranlukast (PRK) and Sorafenib (SRB), to have significant affinity for the allosteric site on MtArgJ, leading to the inhibition of its enzymatic activity. We further propose the key residues involved in this interaction, thereby suggesting a potential molecular mechanism of inhibition. Chapter 5 leads us to the in-vitro and in-vivo characterization of these compounds as a potent anti-tubercular agent. We first demonstrate its efficacy in deducing the survival of the pathogenic strains of Mtb in-vitro and in the macrophage models of infection. We also tested the efficacy of these compounds in combination with the standard of care TB therapy drugs, and found PRK to work efficiently in such combinations. Finally, we evidence the potency of PRK in compromising the survival and pathogenesis of Mtb in mice models of tuberculosis infection. PRK is presently being used as a drug against chronic asthma, therefore its human safety is already assured. This will facilitate its induction into the direct clinical trials against tuberculosis. Taken together, the work done in this thesis demonstrates a novel metabolic inhibitor of Mtb pathogenesis, through the pre-clinical models of infection with the potential for development of advanced combinatorial therapy against tuberculosis.