Drug Discovery and Structural Studies on Mycobacterium tuberculosis Proteins Related to Drug Resistance and Persistence

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which is one of the leading infectious diseases worldwide. The current therapy for drug-sensitive TB is complex and lasts for at least six months. Improper use of antibiotics during this regimen has led to the emergence of drug resistance, which represents a grave threat to human health. This problem is further exacerbated by the ability of the bacterium to persist in the host in a non-replicating state despite the use of antibiotics. The majority of antibiotics currently used to treat tuberculosis only affect replicating bacteria. Therefore, it is critical to develop new antitubercular drugs that can shorten the current therapy while maintaining activity against persistent bacteria as well as the drug-resistant strains. In this dissertation, structural and drug discovery studies on Mtb proteins related to drug resistance and persistence are presented. InhA, the enoyl-ACP-reductase enzyme of the mycolic acid biosynthesis pathway, is the molecular target of the antitubercular prodrugs isoniazid and ethionamide, and it is one of the best validated targets for Mtb drug discovery. A target-based high throughput screening and a structure-based drug design were performed to identify potent activation-free InhA inhibitors that were effective against drug-resistant Mtb strains. The molecular basis of InhA inhibition by these inhibitors was revealed by X-ray crystallography. In addition, the mode of action for ethionamide was revealed by using a cell-based activation system and X-ray crystallography. Furthermore, it was demonstrated that InhA is the clinically relevant primary target of isoniazid. The regulation of InhA function was also studied, which revealed that phosphorylation of InhA occurs at its C-terminal. Phosphomimetic mutants showed that phosphorylation decreases InhA activity by decreasing the affinity toward cofactor NADH. The results of these studies are presented in Chapters II, III, IV, and V. It is essential to understand the physiology of the bacterium to target the persistent state of Mtb infection. In Chapter VI, I report our studies on CarD, an essential Mtb transcription regulator that is required for persistent infection. The structure of the CarD/RNAP complex was determined by X-ray crystallography and the CarD-DNA interactions were investigated

Mechanism of thioamide drug action against tuberculosis and leprosy

Dover developed molecular constructs and the initial in viro data that initiated a structural biology route to the determination of the mode of action of the important anti-TB agent ethionamide used to treat multi-drug resistant infections and its analogue prothionamide used to treat leprosy

Mycobacterium tuberculosis Dihydrofolate Reductase Is Not a Target Relevant to the Antitubercular Activity of Isoniazid

Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro, and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia coli cell-based system was designed that allowed coexpression of both M. tuberculosis katG and dfrA genes in the presence of INH. The DHFR protein isolated from the experimental sample was not found bound with any INH-NADP adduct by enzyme inhibition assay and mass spectroscopic analysis. We also used whole-genome sequencing to determine whether polymorphisms in dfrA could be detected in six INH-resistant clinical isolates known to lack mutations in inhA and katG, but no such mutations were found. The dfrA overexpression experiments, together with the biochemical and sequencing studies, conclusively demonstrate that DHFR is not a target relevant to the antitubercular activity of INH

Machine learning–driven multiscale modeling reveals lipid-dependent dynamics of RAS signaling proteins

RAS is a signaling protein associated with the cell membrane that is mutated in up to 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomistic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influence effector binding, and therefore may be a mechanism for regulating cell signaling cascades

Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor

AbstractDespite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment

Drug Discovery and Structural Studies on Mycobacterium tuberculosis Proteins Related to Drug Resistance and Persistence

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which is one of the leading infectious diseases worldwide. The current therapy for drug-sensitive TB is complex and lasts for at least six months. Improper use of antibiotics during this regimen has led to the emergence of drug resistance, which represents a grave threat to human health. This problem is further exacerbated by the ability of the bacterium to persist in the host in a non-replicating state despite the use of antibiotics. The majority of antibiotics currently used to treat tuberculosis only affect replicating bacteria. Therefore, it is critical to develop new antitubercular drugs that can shorten the current therapy while maintaining activity against persistent bacteria as well as the drug-resistant strains. In this dissertation, structural and drug discovery studies on Mtb proteins related to drug resistance and persistence are presented. InhA, the enoyl-ACP-reductase enzyme of the mycolic acid biosynthesis pathway, is the molecular target of the antitubercular prodrugs isoniazid and ethionamide, and it is one of the best validated targets for Mtb drug discovery. A target-based high throughput screening and a structure-based drug design were performed to identify potent activation-free InhA inhibitors that were effective against drug-resistant Mtb strains. The molecular basis of InhA inhibition by these inhibitors was revealed by X-ray crystallography. In addition, the mode of action for ethionamide was revealed by using a cell-based activation system and X-ray crystallography. Furthermore, it was demonstrated that InhA is the clinically relevant primary target of isoniazid. The regulation of InhA function was also studied, which revealed that phosphorylation of InhA occurs at its C-terminal. Phosphomimetic mutants showed that phosphorylation decreases InhA activity by decreasing the affinity toward cofactor NADH. The results of these studies are presented in Chapters II, III, IV, and V. It is essential to understand the physiology of the bacterium to target the persistent state of Mtb infection. In Chapter VI, I report our studies on CarD, an essential Mtb transcription regulator that is required for persistent infection. The structure of the CarD/RNAP complex was determined by X-ray crystallography and the CarD-DNA interactions were investigated

Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

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Structural dynamics of RAF1-HSP90-CDC37 and HSP90 complexes reveal asymmetric client interactions and key structural elements.

RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 μs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1s unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding

Structural dynamics of RAF1-HSP90-CDC37 and HSP90 complexes reveal asymmetric client interactions and key structural elements

Abstract RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 μs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1’s unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding

Standardization of ELISA protocols for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling

Understanding the infection parameters and host responses against SARS-CoV-2 require data from large cohorts using standardized methods. Here, the authors optimize a serum ELISA protocol that has minimal cross-reactivity and flexible sample collection workflow in an attempt to standardize data generation and help inform on COVID-19 pandemic and immunity