STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF PROTEIN DRUG TARGETS FROM HUMAN PATHOGENS

Abstract

My Ph.D. research objectives mainly focus on the characterization of the structure-function relationships of few of the protein drug targets from human pathogens including multidrug-resistant gram-negative bacteria and protozoans that may direct us towards the development of novel, potential, broad-spectrum antimicrobials with new modes of actions or improved efficacy. Current human society is completely dominated by the MDR/XDR/PDR pathogenic bacteria and several other infectious agents for which there is dearth of treatment strategies (antibiotics/drugs/vaccines) and hence there is a demand for new drug molecules or vaccines with alternative modes of action or improved efficacy and characterization of new drug targets. As proteins are the driving horses of the cellular life of any living organism, it is a good idea to target the protein components (involving in the any of several essential metabolic/signaling/structural pathways) of the pathogen of any disease/infection. In this course of Ph.D. research, I have worked on three promising drug targets including two bacterial proteins enoyl-ACP Reductase (ENR) from Moraxella catarrhalis (McFabI) and Oxa-58 carbapenemase from Acinetobacter baumannii, and one protozoan protein deoxyhypusine hydroxylase (DOHH) from Leishmania donovani. M. catarrhalis and A. baumannii are known to cause severe nosocomial infections and several other pathological conditions. Several clinical strains of these two gram-negative pathogenic bacteria also exhibit resistance to many known classes of antibiotics and hence designated as multidrug-resistant bacteria. L. donovani causes visceral leishmaniasis (VL), the lethal form of leishmaniasis, in several economically poor countries and there are no parasite-specific drugs/vaccines. The proteins FabI and Oxa-58 are successfully characterized from the respective pathogens, using structural and biochemical techniques, and provided us with crucial structural information which may be useful in the development of novel structure based antimicrobial development. We are working on several methods to crystallize the LdDOHH protein and it is structurally modeled, followed by the search for new structural compounds which may exhibit anti-leishmanial activity. The bacterial type II fatty acid biosynthesis pathway, which is differing very much from the animal and human type I fatty acid biosynthesis pathway in its structural organization, has been validated to be a potential drug target pathway. All its catalytic steps are being catalyzed by individual proteins and many of these are proven as promising drug targets including the enoyl acyl carrier protein reductase (ENR). Moraxella catarrhalis is possessing the FabI (ENR I) isoform of the ENR and is a validated drug target against which many of the currently known inhibitors including triclosan, isoniazid and diazaborines are exhibiting antimicrobial activities. In my current work I have structurally and biochemically characterized the FabI protein from M. catarrhalis (McFabI) to elucidate its structural elements essential for the substrate binding and catalysis, which can be subsequently exploited for the structure-based drug designing. The structure of the McFabI is obtained in its apo form and NAD and triclosan bound ternary complex form also. Virtual screening of the libraries of small druggable molecules was carried out using these structural features and obtained few lead molecules. We also observed that 17-β estradiol (E2), a human major female sex hormone, is also binding with higher score and energy, and hence we have characterized its interactions with the McFabI enzyme. The biochemical analysis revealed that E2 is potentially binding to this enzyme with a kD value of ~0.5 μM and inhibiting the enzyme activity with a ki value of 38.1 μM. E2 was also observed to be exhibiting antimicrobial properties against M. catarrhalis. These results altogether are indicating that the direct antimicrobial activities of E2 might be contributing to the sex differences in resistance to infectious diseases in many organisms, including humans, and supporting the fact that females are more resistant than their male counterparts. The β-lactamase enzymes have been the major contributors of the antibiotic resistance in many of the multidrug resistance bacteria including Acinetobacter baumannii, one of the leading cause of nosocomial infections and deaths worldwide. Among these enzymes also, the carbapenem hydrolyzing class D β-lactamases (CHDLs) are the major concern in the context of constantly accelerating antimicrobial resistance, as they are able to hydrolyze the carbapenem antibiotics which have been the first line of defence against these MDR bacterial pathogens. A. baumannii is producing several groups of CHDLs including OXA-23, OXA-24, OXA-58 and OXA-48, and few others. In our current study, we are aiming at elucidating the active site elements that are crucial in facilitating the substrate recognition and subsequent catalysis. We determined the crystal structure of OXA-58 from A. baumannii (AbOXA-58) in complex with one of the carbapenem mimetic 6α-hydroxymethyl penicillin (6α-HMP) and obtained a stable acyl-enzyme complex intermediate. Analysis of these structures revealed that the active site of OXA-58 is exhibiting great amount of plasticity during its substrate recognition and a hydrophobic bridge formed over the active site cleft is very crucial in the carbapenem substrate recognition and its hydrolysis. The structural analysis of the point mutation variants of AbOXA-58 protein also revealed that the enzyme can use alternative active site residues to form the hydrophobic bridge in the absence of any of the earlier. These discoveries are very iii important in understanding the carbapenem hydrolysis and helpful in designing structure-based inhibitors against these β-lactamase enzymes. The hypusine biosynthesis pathway, participating in the posttranslational modification of a lysine residue on eukaryotic initiation factor 5A (eIF5A) leading to its maturation by having the hypusine residue, from many parasitic organisms was validated as a potential drug target. This pathway includes two enzymatic steps catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH), and both these enzymes are validated drug targets. Our study focuses on the characterization of the structural and biochemical elements of the DOHH enzyme from Leishmania donovani (LdDOHH), a protozoan parasite responsible for causing many forms of leishmaniasis, including its most lethal form ―visceral leishmaniasis (VL)‖, disease in humans and other animals. This enzyme was analyzed biochemically for its oligomeric forms in solution and secondary structural features. Crystallization trials were made rigorously to obtain its 3-dimensional structure and the protein was modelled using computational tools. The LdDOHH model structure was analysed and used for virtual screening of small molecule libraries in search of new anti-leishmanial drug molecules. We found few hit compounds, including the compounds 43, 712 and 1366, and analysed for their binding modes and strengths, their interaction patterns and conformational stability in bound state. These analyses showed that these compounds were binding tightly in the active site and may be promising molecules for testing their anti-leishmanial activities.