The in silico investigation of the perplexity of synergistic duality: inter-molecular mechanisms of communication in Bcr–Abl.

Doctoral Degree. University of KwaZulu-Natal, Durban.Due to their important role in normal cellular physiology, protein kinase activity is tightly regulated and their aberrant activation can lead to cancer. Chronic myeloid leukaemia (CML) is a blood cancer described by unregulated growth of myeloid cells caused by a fusion protein, Bcr-Abl, a constitutively active form of the Abelson tyrosine kinase (Abl). Drug targeting of either the ATP binding pocket or allosteric pocket has led to durable therapeutic response, however the development of drug resistance still poses a major clinical challenge. Recent studies exploring synergistic inhibition as an effective approach, by dual targeting of Bcr-Abl using both catalytic and allosteric binding inhibitors. This thesis implements the use of advanced computational tools to unravel molecular insights to aid in the suppression of the emergence of resistance to Bcr-Abl when Nilotinib and ABL001 are co-administered to target both the catalytic and allosteric binding site of Bcr-Abl protein, respectively. Our studies revealed co-binding induced a stable Bcr-Abl protein structure, increased the degree of compactness of binding site residues around Nilotinib and subsequently improved the binding affinity of Nilotinib. Findings in this thesis further provide an atomistic perspective underlying the developed resistance of Nilotinib by point mutation at the catalytic active site only and both catalytic and activation loop sites. We also recognized and rationalized the structural interplay of this single and double mutation upon co-binding of Nilotinib with the novel inhibitor, ABL001. Our findings report the distortion of the overall conformational landscape of Bcr-Abl fusion oncoprotein caused by the mutation, resulting in a reduction of binding affinity of Nilotinib upon single binding. Interesting, co-administration with ABL001 impacted by the mutation results in a more compact and stable protein conformation. Findings reveal a structural mechanism by which the novel inhibitor ABL001 stabilizes Bcr-Abl fusion oncoprotein upon co-binding with Nilotinib, thus suppressing Nilotinib resistance. We also provide vital conformational dynamics and structural mechanisms of the mutant enzyme at the catalytic site-ligand interaction and mutant enzyme at both catalytic and activation loop ligand interactions which could potentially shift the current therapeutic protocol in chronic myeloid leukemia treatment, thus aiding in the design of novel inhibitors with improved therapeutic features against the mutant proteins

Structure-fonction des transporteurs transmembranaires de la famille MmpL3 de Mycobacterium tuberculosis

L’émergence de la résistance à une multitude d’agents antimicrobiens chez des bactéries pathogènes est considérée comme une menace majeure pour la santé publique (2). Ces souches sont reconnues comme des organismes multirésistants aux médicaments ou MDR (multidrug-resistant) (4). Les recherches progressent chez les bactéries, à Gram positif, à Gram négatif et acido-alcoolo-résistantes au vu de l’ampleur de la menace pour la santé publique, ces bactéries multirésistantes sont devenues les cibles potentielles à cette fin de recherche. De ce fait, les objectifs de la présente étude ont consisté en la caractérisation structurale et fonctionnelle de différents transporteurs transmembranaires de la famille des RND (Resistance-Nodulation-Division) encore énigmatiques, à savoir: le MmpL3 chez Mycobacterium tuberculosis (Mtb) via l’étude de son orthologue CmpL1 chez Corynebacterium glutamicum (Cgl) et le TriAxBC chez Pseudomonas aeruginosa (P. aeruginosa). Ainsi, comme première démarche présentée dans le chapitre 2, la structure du transporteur MmpL3 Mtb (un transporteur d'acides mycoliques – sous forme de tréhalose de monomycolates (ou TMM) - essentiel pour la viabilité de Mtb) (5) et celle de son orthologue CmpL1 Cgl ont été prédites via le serveur I-TASSER (6-8). Ces structures ont été validées par la suite en comparant à la carte électronique générée pour CmpL1 (18 Å) par des analyses de microscopie électronique en transmission à coloration négative (TEM). La caractérisation du transporteur CmpL1 purifié par chromatographie à exclusion stérique a confirmé le complexe trimérique de taille avoisinant les 315 KDa (incluant la couronne du détergent) en accord avec des analyses par gel SDS-PAGE. Des études génétiques et biochimiques en collaboration ont d’autre part identifié des résidus engagés dans le transport du TMM chez MmpL3 ainsi que d’autres impliqués dans la résistance à des inhibiteurs ciblant ce transporteur. L’ensemble de ces données a mis en évidence la localisation des résidus essentiels au transport et à la résistance au niveau du canal central du modèle trimérique de MmpL3. La région de MmpL3 activant le transport par force protomotrice a été localisée au niveau d’une cavité centrale qui est une caractéristique intrinsèque de la famille des RND. Les cartes électroniques de faible résolution déjà obtenues pour la protéine CmpL1 font de ce projet une des directions futures du laboratoire. Dans le chapitre 3, nous illustrons le deuxième aspect du présent projet qui repose sur l’extension de l’étude du potentiel thérapeutique du ciblage du transporteur transmembranaire MmpL3 chez les différentes souches de Mycobacterium. Nos collaborateurs ont effectué une analyse biochimique de l’effet thérapeutique des inhibiteurs les plus prometteurs du transporteur MmpL3 Mtb sur certaines souches mycobactériennes non-tuberculeuses (NTB) multi-résistantes. Basés sur nos modélisations structurales comparatives obtenues par I-TASSER (6-8), nous avons pu complémenter les informations biochimiques en soulignant les similitudes et les différences de structure entre les souches TB et NTB ainsi que leurs impacts fonctionnels. Ce chapitre met en évidence l’intérêt du ciblage thérapeutique de MmpL3 chez les espèces NTB. En effet, l’efficacité de certains inhibiteurs de MmpL3 Mtb sélectionnés sur le traitement des infections pulmonaires NTB promet de pouvoir généraliser cette nouvelle voie de traitement pour d’autres souches multi-résistantes NTB voire à contribuer à remédier à la problématique de la résistance aux antibiotiques et décomplexifier le traitement actuel. D’autres études en collaboration entreprenant les mêmes approches d’études structurales ont été réalisées pour les transporteurs tripartites TriAxBC (P. aeruginosa), des pompes à efflux appartenant à la famille des RND. Le but du chapitre 4 était de générer une structure du complexe et de déchiffrer son mode d’assemblage et d’expulsion des antibiotiques vers le milieu externe. Un modèle à structure quaternaire de TriAxBC a été prédit par I-TASSER (6-8) et validé contre sa carte électronique à 4.3 Å générée en Cryo-EM. Le complexe TriAxBC a été également caractérisé par filtration sur gel confirmant une taille approximative de 620 KDa et sa composition en trimère par visualisation sur gel SDS-PAGE. En conclusion, nous avons pu à travers cette étude combiner différentes approches biochimiques, génétiques et structurales soutenant la nécessité d’une approche multidisciplinaire pour l’approfondissement de la compréhension de la structure et du mode de fonctionnement des transporteurs RND. Ces derniers demeurent toujours énigmatiques; toutefois, nos avancées et d’autres à venir permettront la génération de nouveaux médicaments spécifiques traitant les bactéries multirésistantes.The emergence of resistance to a multitude of antimicrobial agents in pathogenic bacteria is considered a major threat to public health (2). These strains are recognized as multidrug resistant organisms (MDR) (4). Research is progressing in Gram positive, Gram positive high GC and Gram negative bacteria, and given the scale of the public health threat, these MDR have become potential targets for this research. The objectives of the present study consist of the structural and functional characterization of various transmembrane transporters of the still enigmatic RND (Resistance-Nodulation-Division) family, namely: MmpL3 in Mycobacterium tuberculosis (Mtb) via the study of its ortholog CmpL1 in Corynebacterium glutamicum (Cgl) and TriAxBC in Pseudomonas aeruginosa (P. aeruginosa). The first component of this project, presented in Chapter 2, studies the structure of the transporter MmpL3 Mtb (a TMM mycolic acid transporter essential for the viability of Mtb (5) and that of its CmpL1 Cgl orthologue, which have been predicted via the I- Tasser Pack (6-8). These structures were subsequently validated by comparing to the electronic map generated for CmpL1 (18 Å) by negative staining transmission electron microscopy (TEM). Characterization of the purified CmpL1 transporter by size exclusion chromatography confirmed the trimeric complex size around 315 KDa (including the detergent crown) corroborated by SDS-PAGE gel analyses. Collaborative genetic and biochemical studies have also identified residues involved in the transport of TMM in MmpL3 as well as those residues conferring antibiotic resistance. This data highlighted the location of the essential residues of transport and resistance in the central channel of the trimeric Mmpl3 model. The MmpL3 region activating proto-motor transport has been located at a central cavity, which is an intrinsic feature of the RND family. The low-resolution electronic maps obtained for the protein CmpL1 may serve as the foundation of future studies. In Chapter 3 we explore the therapeutic potential of the targeting of the transmembrane transporter MmpL3 in different Mycobacterium strains. Our collaborators studied the therapeutic effect of the most promising inhibitors of the MmpL3 Mtb transporter on certain multi-resistant mycobacterial non-tuberculous (NTB) strains. Based on our comparative structural modeling obtained by I-TASSER (6-8), we supplemented the biochemical data by highlighting the structural similarities and differences between the TB and NTB strains as well as their functional impacts. This chapter highlights the interest of direct or indirect targeting of MmpL3 in NTB species. Indeed, the efficacy of certain selected MmpL3 Mtb inhibitors on the treatment of NTB pulmonary infection have potential as generalizable treatment options for other NTB multi-resistant strains, or even to help address the problem of resistance to antibiotics and simplify current combination approaches. Other collaborative studies undertaking the same structural approaches were carried out for TriAxBC tripartite carriers (P. aeruginosa), efflux pumps belonging to the RND family. The purpose of Chapter 4 was to generate a structure of the complex and decipher its mode of assembly and expulsion of antibiotics from the intracellular environment. A quaternary structure model of TriAxBC was predicted by I-TASSER (6-8) and validated against its 4.3 Å electronic map generated by Cryo-EM. The TriAxBC complex was also characterized by gel filtration confirming an approximate size of 620 KDa and its trimer composition by SDS-PAGE. In conclusion, this study is combining different biochemical, genetic and structural approaches to highlight the need for a multidisciplinary approach to characterizing the structure function of RND transporters. The latter remain enigmatic; however, our contribution and the progress of others will allow the generation of new specific drugs targeting multiresistant strains

Characterization of differentially culturable bacteria in exenic culture and from tuberculosis patients

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree of Doctor of Philosophy Johannesburg, March 2018.During tuberculosis (TB) disease, host-derived stresses and chemotherapy are thought to drive tubercle bacilli into differential growth states. This is evidenced by the presence of differentially culturable tubercle bacilli (DCTB) in the sputum of treatment naïve TB patients. These bacteria do not form colonies on solid media but can be cultured following supplementation of liquid media with culture filtrate as a source of growth stimulatory molecules. As DCTB are non-replicating and phenotypically drug tolerant, these organisms are thought to underpin the lengthy culture diagnosis and protracted treatment period required for TB disease. The purpose of this study was to investigate the use of culture filtrate in unmasking DCTB populations to: (1) quantify these populations in treatment naïve individuals, (2) assess the response of DCTB versus conventionally culturable bacteria to first-line treatment, (3) determine the relationship between DCTB cultured in the most probable number (MPN) assay with other TB culture methods and (4) to enhance currently employed culture diagnostic methods. The results from this study confirmed that treatment naïve individuals coinfected with HIV had significantly lower quanta of DCTB in their sputum compared to their HIV-negative counterparts. These findings implicate the host immune response in influencing the prevalence of DCTB in sputum. During treatment, four patterns of decline in DCTB were described. One quarter of the patient population accumulated DCTB during the first seven days of treatment, whilst approximately the same number of individuals displayed a rapid decline in DCTB during this period. The remaining individuals either displayed static or atypical patterns of DCTB over the first 14 days of treatment. Following treatment completion, residual DCTB was cultured in approximately two thirds of the patients analysed, suggesting that bacteriological sterilization of lungs was not achieved. These observations were confirmed using a novel fluorogenic probe specific for the detection of live Mycobacterium tuberculosis. DCTB cultured in the MPN assay was shown to directly correlate with current TB culture methods. These findings demonstrate a potential utility for the MPN assay in early bactericidal activity studies to assess the sterilising effect of new TB drugs on DCTB populations. Furthermore, the addition of culture filtrate to the BACTEC MGIT 960 assay reduced the rates of TB detection in smear-negative, HIV-positive individuals. Collectively, these observations demonstrate that the detection of DCTB in sputum can serve as a possible biomarker for treatment response. Further long term studies are required to determine if DCTB can use used to assess the risk of relapse disease and to test the efficacy of new drugs on persistent bacterial populations.LG201