RESUMEN

IMPORTANCE: Secreted virulence factors play a critical role in bacterial pathogenesis. Virulence effectors not only help bacteria to overcome the host immune system but also aid in establishing infection. Mtb, which causes tuberculosis in humans, encodes various virulence effectors. Triggers that modulate the secretion of virulence effectors in Mtb are yet to be fully understood. To gain mechanistic insight into the secretion of virulence effectors, we performed high-throughput proteomic studies. With the help of system-level protein-protein interaction network analysis and empirical validations, we unravelled a link between phosphorylation and secretion. Taking the example of the well-known virulence factor of CFP10, we show that the dynamics of CFP10 phosphorylation strongly influenced bacterial virulence and survival ex vivo and in vivo. This study presents the role of phosphorylation in modulating the secretion of virulence factors.

Asunto(s)

Mycobacterium tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Antígenos Bacterianos/metabolismo , Fosforilación , Virulencia , Proteómica , Factores de Virulencia

RESUMEN

Background: Multidrug-resistant tuberculosis (MDR-TB) is one of the world's most devastating contagious diseases and is caused by the MDR-Mycobacterium tuberculosis (MDR-Mtb) bacteria. It is therefore essential to identify novel anti-TB drug candidates and target proteins to treat MDR-TB. Here, in vitro and in silico studies were used to investigate the anti-TB potential of two newly sourced actinomycins, actinomycin-X2 (act-X2) and actinomycin-D (act-D), from the Streptomyces smyrnaeus strain UKAQ_23 (isolated from the Jubail industrial city of Saudi Arabia). Methods: The anti-TB activity of the isolated actinomycins was assessed in vitro using the Mtb H37Ra, Mycobacterium bovis (BCG), and Mtb H37Rv bacterial strains, using the Microplate Alamar Blue Assay (MABA) method. In silico molecular docking studies were conducted using sixteen anti-TB drug target proteins using the AutoDock Vina 1.1.2 tool. The molecular dynamics (MD) simulations for both actinomycins were then performed with the most suitable target proteins, using the GROningen MAchine For Chemical Simulations (GROMACS) simulation software (GROMACS 2020.4), with the Chemistry at HARvard Macromolecular Mechanics 36m (CHARMM36m) forcefield for proteins and the CHARMM General Force Field (CGenFF) for ligands. Results: In vitro results for the Mtb H37Ra, BCG, and Mtb H37Rv strains showed that act-X2 had minimum inhibitory concentration (MIC) values of 1.56 ± 0.0, 1.56 ± 0.0, and 2.64 ± 0.07 µg/mL and act-D had MIC values of 1.56 ± 0.0, 1.56 ± 0.0, and 1.80 ± 0.24 µg/mL respectively. The in silico molecular docking results showed that protein kinase PknB was the preferred target for both actinomycins, while KasA and pantothenate synthetase were the least preferred targets for act-X2and act-D respectively. The molecular dynamics (MD) results demonstrated that act-X2 and act-D remained stable inside the binding region of PknB throughout the simulation period. The MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) binding energy calculations showed that act-X2 was more potent than act-D. Conclusion: In conclusion, our results suggest that both actinomycins X2 and D are highly potent anti-TB drug candidates. We show that act-X2is better able to antagonistically interact with the protein kinase PknB target than act-D, and thus has more potential as a new anti-TB drug candidate.

Asunto(s)

Antituberculosos , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Antituberculosos/farmacología , Vacuna BCG/uso terapéutico , Dactinomicina/farmacología , Simulación del Acoplamiento Molecular , Proteínas Quinasas , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico

RESUMEN

Serine/threonine protein kinase B (PknB) is essential to Mycobacterium tuberculosis (M. tuberculosis) cell division and metabolism and a potential anti-tuberculosis drug target. Here we apply Hologram Quantitative Structure Activity Relationship (HQSAR) and three-dimensional QSAR (Comparative Molecular Similarity Indices Analysis (CoMSIA)) methods to investigate structural requirements for PknB inhibition by a series of previously described quinazoline derivatives. PknB binding of quinazolines was evaluated by molecular dynamics (MD) simulations of the catalytic domain and binding energies calculated by Molecular Mechanics/Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) methods. Evaluation of a training set against experimental data showed both HQSAR and CoMSIA models to reliably predict quinazoline binding to PknB, and identified the quinazoline core and overall hydrophobicity as the major contributors to affinity. Calculated binding energies also agreed with experiment, and MD simulations identified hydrogen bonds to Glu93 and Val95, and hydrophobic interactions with Gly18, Phe19, Gly20, Val25, Thr99 and Met155, as crucial to PknB binding. Based on these results, additional quinazolines were designed and evaluated in silico, with HQSAR and CoMSIA models identifying sixteen compounds, with predicted PknB binding superior to the template, whose activity spectra and physicochemical, pharmacokinetic, and anti-M. tuberculosis properties were assessed. Compound, D060, bearing additional ortho- and meta-methyl groups on its R2 substituent, was superior to template regarding PknB inhibition and % caseum fraction unbound, and equivalent in other aspects, although predictions identified hepatotoxicity as a likely issue with the quinazoline series. These data provide a structural basis for rational design of quinazoline derivatives with more potent PknB inhibitory activity as candidate anti-tuberculosis agents.

Asunto(s)

Mycobacterium tuberculosis , Relación Estructura-Actividad Cuantitativa , Antituberculosos/química , Antituberculosos/farmacología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/química , Quinazolinas/farmacología

RESUMEN

Serine/threonine kinase PknB and its corresponding phosphatase Stp are important regulators of many cell functions in the pathogen S. aureus. Genome-scale gene expression data of S. aureus strain NewHG (sigB+) elucidated their effect on physiological functions. Moreover, metabolic modelling from these data inferred metabolic adaptations. We compared wild-type to deletion strains lacking pknB, stp or both. Ser/Thr phosphorylation of target proteins by PknB switched amino acid catabolism off and gluconeogenesis on to provide the cell with sufficient components. We revealed a significant impact of PknB and Stp on peptidoglycan, nucleotide and aromatic amino acid synthesis, as well as catabolism involving aspartate transaminase. Moreover, pyrimidine synthesis was dramatically impaired by stp deletion but only slightly by functional loss of PknB. In double knockouts, higher activity concerned genes involved in peptidoglycan, purine and aromatic amino acid synthesis from glucose but lower activity of pyrimidine synthesis from glucose compared to the wild type. A second transcriptome dataset from S. aureus NCTC 8325 (sigB-) validated the predictions. For this metabolic adaptation, PknB was found to interact with CdaA and the yvcK/glmR regulon. The involved GlmR structure and the GlmS riboswitch were modelled. Furthermore, PknB phosphorylation lowered the expression of many virulence factors, and the study shed light on S. aureus infection processes.

RESUMEN

With tuberculosis still being one of leading causes of death in the world and the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), researchers have been seeking to find further therapeutic strategies or more specific molecular targets. PknB is one of the 11 Ser/Thr protein kinases of Mtb and is responsible for phosphorylation-mediated signaling, mainly involved in cell wall synthesis, cell division and metabolism. With the amount of structural information available and the great interest in protein kinases, PknB has become an attractive target for drug development. This work describes the optimization and application of an in silico computational protocol to find new PknB inhibitors. This multi-level computational approach combines protein-ligand docking, structure-based virtual screening, molecular dynamics simulations and free energy calculations. The optimized protocol was applied to screen a large dataset containing 129,650 molecules, obtained from the ZINC/FDA-Approved database, Mu.Ta.Lig Virtual Chemotheca and Chimiothèque Nationale. It was observed that the most promising compounds selected occupy the adenine-binding pocket in PknB, and the main interacting residues are Leu17, Val26, Tyr94 and Met155. Only one of the compounds was able to move the active site residues into an open conformation. It was also observed that the P-loop and magnesium position loops change according to the characteristics of the ligand. This protocol led to the identification of six compounds for further experimental testing while also providing additional structural information for the design of more specific and more effective derivatives.

Asunto(s)

Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Bacterianas/química , Biología Computacional/métodos , Simulación por Computador , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Tuberculosis/tratamiento farmacológico

RESUMEN

With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) and extensive drug-resistant strains (XDR-TB), there is an urgent need to develop novel drugs for the treatment of tuberculosis. Here, we designed and synthesized a series of 5-methylpyrimidopyridone analogues as potential antitubercular agents. The most potent compound 6q exhibited a MIC value of 4 µM in vitro against Mycobacterium tuberculosis. The antitubercular activities of the synthesized compounds were impacted by the amantadine and 2-chlorophenyl groups, and were enhanced by the presence of 3-methyl(4-dimethylamino)piperidinylphenyl. Molecular modeling and binding studies suggest that PknB is the potential molecular target of 5-methylpyrimidopyridone compounds. This study provides insights for the future development of new antimycobacterial agents with novel mechanisms of action.

Asunto(s)

Antituberculosos/farmacología , Descubrimiento de Drogas , Mycobacterium tuberculosis/efectos de los fármacos , Piridonas/farmacología , Animales , Antituberculosos/síntesis química , Antituberculosos/química , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Piridonas/síntesis química , Piridonas/química , Relación Estructura-Actividad , Células Vero

RESUMEN

Ser/Thr phosphorylation by serine/threonine protein kinases (STPKs) plays significant roles in molecular regulation, which allows Mycobacteria to adapt their cell wall structure in response to the environment changes. Identifying direct targets of STPKs and determining their activities are therefore critical to revealing their function in Mycobacteria, for example, in cell wall formation and virulence. Herein, we reported that RmlA, a crucial L-rhamnose biosynthesis enzyme, is a substrate of STPK PknB in Mycobacterium tuberculosis (M. tuberculosis). Mass spectrometry analysis revealed that RmlA is phosphorylated at Thr-12, Thr-54, Thr-197, and Thr-12 is located close to the catalytic triad of RmlA. Biochemical and phenotypic analysis of two RmlA mutants, T12A/T12D, showed that their activities were reduced, and cell wall formation was negatively affected. Moreover, virulence of RmlA T12D mutant was attenuated in a macrophage model. Overall, these results provide the first evidence for the role of PknB-dependent RmlA phosphorylation in regulating cell wall formation in Mycobacteria, with significant implications for pathogenicity.

RESUMEN

Machine learning that generates biological hypotheses has transformative potential, but most learning algorithms are susceptible to pathological failure when exploring regimes beyond the training data distribution. A solution to address this issue is to quantify prediction uncertainty so that algorithms can gracefully handle novel phenomena that confound standard methods. Here, we demonstrate the broad utility of robust uncertainty prediction in biological discovery. By leveraging Gaussian process-based uncertainty prediction on modern pre-trained features, we train a model on just 72 compounds to make predictions over a 10,833-compound library, identifying and experimentally validating compounds with nanomolar affinity for diverse kinases and whole-cell growth inhibition of Mycobacterium tuberculosis. Uncertainty facilitates a tight iterative loop between computation and experimentation and generalizes across biological domains as diverse as protein engineering and single-cell transcriptomics. More broadly, our work demonstrates that uncertainty should play a key role in the increasing adoption of machine learning algorithms into the experimental lifecycle.

Asunto(s)

Biología Computacional/métodos , Predicción/métodos , Incertidumbre , Algoritmos , Aprendizaje Automático/tendencias , Distribución Normal

RESUMEN

Mycobacterium tuberculosis causes human tuberculosis, and a better understanding of its biology is required to identify vulnerabilities that might be exploited in developing new therapeutics. The iron-sulfur cluster of the essential M. tuberculosis central metabolic enzyme, aconitase (AcnA), disassembles when exposed to oxidative/nitrosative stress or iron chelators. The catalytically inactive apo-AcnA interacts with a sequence resembling an iron-responsive element (IRE) located within the transcript of another essential protein, CwlM, a regulator of peptidoglycan synthesis. A Mycobacterium smegmatis cwlM conditional mutant complemented with M. tuberculosis cwlM with a disrupted IRE is unable to recover from combinations of oxidative, nitrosative, and iron starvation stresses. An equivalent M. tuberculosis cwlM conditional mutant complemented with the cwlM gene lacking a functional IRE exhibits a growth defect in THP-1 macrophages. It appears that AcnA acts to couple peptidoglycan synthesis and central metabolism, and disruption of this coupling potentially leaves mycobacteria vulnerable to attack by macrophages.

Asunto(s)

Aconitato Hidratasa/metabolismo , Peptidoglicano/metabolismo , Humanos

RESUMEN

The emergence of increasingly drug-resistant Mycobacterium tuberculosis strains has become a crucial public health concern. In order to effectively treat tuberculosis, it is imperative to find newer drug targets, which are important for the in vivo bacterial survival and persistence. Phosphorylation based signaling cascades modulated by eukaryotic-like serine/threonine protein kinases and phosphatase in M. tuberculosis, transduce extracellular stimuli to a cellular response ensuing pathogen's growth, persistence and pathogenesis. Of the 11 STPKs that M. tuberculosis genome encodes, three kinases, namely PknA, PknB and PknG and the sole serine/threonine phosphatase PstP are crucial for the intracellular survival of the bacteria. PknA and PknB regulates cell growth, cell wall synthesis and morphological changes during bacterial cell division; while PknG modulates metabolic changes in response to stress and aids in bacterial survival during latency like conditions. PstP functions to dephosphorylate STPKs and their substrates and hence is important at nearly all stages of infection. Here, we review the current knowledge on PstP, PknA, PknB and PknG based on the genetic, biochemical, and functional studies in M. tuberculosis physiopathology. We further explore the potential of these molecules as targets for therapeutic intervention and discuss the advancement made in the development of inhibitors against these targets. © 2018 IUBMB Life, 70(9):889-904, 2018.

Asunto(s)

Antituberculosos/farmacología , Desarrollo de Medicamentos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Tuberculosis/tratamiento farmacológico , Animales , Humanos , Mycobacterium tuberculosis/enzimología , Fosforilación , Tuberculosis/enzimología , Tuberculosis/microbiología

RESUMEN

In recent years, several small natural cyclopeptides and cyclodepsipeptides were reported to have antimycobacterial activity. Following this lead, a synthetic pathway was developed for a small series of 12-membered ring compounds with one amide and two ester bonds (cyclotridepsipeptides). Within the series, the ring system proved to be necessary for growth inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis in the low micromolar range. Open-chain precursors and analogues were inactive. The compounds modulated autophosphorylation of the mycobacterial protein kinase B (PknB). PknB inhibitors were active at µM concentration against mycobacteria while inducers were inactive. PknB regulates the activity of the mycobacterial reductase InhA, the target of isoniazid. The activity of the series against Mycobacterium bovis BCG InhA overexpressing strains was indistinguishable from that of the parental strain suggesting that they do not inhibit InhA. All substances were not cytotoxic (HeLa > 5 µg/ml) and did not show any significant antiproliferative effect (HUVEC > 5 µg/ml; K-562 > 5 µg/ml). Within the scope of this study, the molecular target of this new type of small cyclodepsipeptide was not identified, but the data suggest interaction with PknB or other kinases may partly cause the activity.

Asunto(s)

Antituberculosos/síntesis química , Proteínas Bacterianas/antagonistas & inhibidores , Depsipéptidos/química , Oxidorreductasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Depsipéptidos/síntesis química , Depsipéptidos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Oxidorreductasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo

RESUMEN

Tuberculosis is the leading killer among infectious diseases worldwide. Increasing multidrug resistance has prompted new approaches for tuberculosis drug development, including targeted inhibition of virulence determinants and of signaling cascades that control many downstream pathways. We used a multisystem approach to determine the effects of a potent small-molecule inhibitor of the essential Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB. We observed differential levels of phosphorylation of many proteins and extensive changes in levels of gene expression, protein abundance, cell wall lipids, and intracellular metabolites. The patterns of these changes indicate regulation by PknA and PknB of several pathways required for cell growth, including ATP synthesis, DNA synthesis, and translation. These data also highlight effects on pathways for remodeling of the mycobacterial cell envelope via control of peptidoglycan turnover, lipid content, a SigE-mediated envelope stress response, transmembrane transport systems, and protein secretion systems. Integrated analysis of phosphoproteins, transcripts, proteins, and lipids identified an unexpected pathway whereby threonine phosphorylation of the essential response regulator MtrA decreases its DNA binding activity. Inhibition of this phosphorylation is linked to decreased expression of genes for peptidoglycan turnover, and of genes for mycolyl transferases, with concomitant changes in mycolates and glycolipids in the cell envelope. These findings reveal novel roles for PknA and PknB in regulating multiple essential cell functions and confirm that these kinases are potentially valuable targets for new antituberculosis drugs. In addition, the data from these linked multisystems provide a valuable resource for future targeted investigations into the pathways regulated by these kinases in the M. tuberculosis cell.IMPORTANCE Tuberculosis is the leading killer among infectious diseases worldwide. Increasing drug resistance threatens efforts to control this epidemic; thus, new antitubercular drugs are urgently needed. We performed an integrated, multisystem analysis of Mycobacterium tuberculosis responses to inhibition of its two essential serine/threonine protein kinases. These kinases allow the bacterium to adapt to its environment by phosphorylating cellular proteins in response to extracellular signals. We identified differentially phosphorylated proteins, downstream changes in levels of specific mRNA and protein abundance, and alterations in the metabolite and lipid content of the cell. These results include changes previously linked to growth arrest and also reveal new roles for these kinases in regulating essential processes, including growth, stress responses, transport of proteins and other molecules, and the structure of the mycobacterial cell envelope. Our multisystem data identify PknA and PknB as promising targets for drug development and provide a valuable resource for future investigation of their functions.

Asunto(s)

Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Mycobacterium tuberculosis/genética , Fosforilación/genética , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Transducción de Señal/fisiología

RESUMEN

BACKGROUND: The Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India. METHODS: A total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations. RESULTS: The sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding. CONCLUSIONS: Hence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein.

Asunto(s)

Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Tuberculosis/microbiología , Antituberculosos/farmacología , Secuencia de Bases , ADN Bacteriano/aislamiento & purificación , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Etambutol/farmacología , Variación Genética , Humanos , India , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Mitoxantrona , Simulación del Acoplamiento Molecular , Mutación , Ofloxacino/farmacología , Fenotipo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Estructura Terciaria de Proteína , Rifampin/farmacología , Análisis de Secuencia , Estreptomicina/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/genética

RESUMEN

New drugs are urgently needed to cure tuberculosis (TB) in a short period of time without causing any adverse effects since currently used drugs for the treatment of multi drug-resistant TB cause several adverse effects with poor success rate. Therefore, we aimed to prioritize known drugs towards repurposing for TB by employing bioinformatics approach in the present study. A total of 1554 FDA approved drugs were obtained from DrugBank. Serine/threonine-protein kinase, pknB (Rv0014c) of Mycobacterium tuberculosis (Mtb) was selected as the drug target since it involves in several vital functions of the Mtb. All of the 1554 drugs were subjected to molecular docking with pknB. Glide and AutoDock Vina were employed using rigid docking followed by induced fit docking protocol for prioritization of drugs. Out of 14 drugs prioritized, six are suggested as high-confident drugs towards repurposing for TB as they were consistently found within top 10 ranks of both methods, and strongly binding in the active site of the pknB. We also found atorvastatin as one of the high-confident drugs, which has already been demonstrated to be active against Mtb under in vitro conditions by other researchers. Therefore, we propose that the prioritized six high-confident drugs as potential candidates for repurposing for TB and suggest for further experimental studies. We also suggest that the bioinformatics procedure we have employed in this study could be effectively applied for prioritization of drugs for other diseases.

Asunto(s)

Biología Computacional/métodos , Reposicionamiento de Medicamentos , Tuberculosis/tratamiento farmacológico , Atorvastatina/farmacología , Aprobación de Drogas , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Conformación Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estados Unidos , United States Food and Drug Administration

RESUMEN

Tuberculosis (TB), caused by Mycobacterium tuberculosis is one of the most lethal communicable disease globally. As per the WHO Global TB Report (2015), 9.6 million cases were reported in year 2014 alone. The receptor-like protein kinase, PknB is crucial for sustained mycobacterial growth. Therefore, PknB can be a potential target to develop anti-tuberculosis drugs. In present study, we performed a comparative study to investigate binding efficacies of three phytomolecules namely, Demethylcalabaxanthone, Cryptolepine hydrochloride and Ermanin. 3D structures of PknB and phytomolecules were retrieved from Protein Data Bank (PDB ID: 2FUM) and PubChem Chemical Compound Database, respectively. PknB was set to be rigid and phytochemicals were kept free to rotate. All computational simulations were carried out using Autodock 4.0 on Windows platform. In-silico study demonstrated a strong complex formation (large binding constants and low ΔG) between phytomolecules and target protein PknB of Mycobacterium tuberculosis. However, Demethylcalabaxanthone was able to bind PknB more strongly (Kb=6.8×105M-1, ΔG=-8.06kcal/mol) than Cryptolepine hydrochloride (Kb=3.06×105M-1, ΔG=-7.58kcal/mol) and Ermanin (Kb=9.8×104M-1, ΔG=-6.9kcal/mol). These in silico analysis indicate that phytomolecules are capable to target PknB protein efficiently which is vital for mycobacterial survival and therefore can be excellent alternatives to conventional anti-tuberculosis drugs.

Asunto(s)

Antituberculosos/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Flavonoides/química , Alcaloides Indólicos/química , Ligandos , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/enzimología , Relación Estructura-Actividad Cuantitativa , Quinolinas/química , Xantonas/química

RESUMEN

The Mycobacterium tuberculosis protein kinase B (PknB) is critical for growth and survival of M. tuberculosis within the host. The series of aminopyrimidine derivatives show impressive activity against PknB (IC50 < .5 µM). However, most of them show weak or no cellular activity against M. tuberculosis (MIC > 63 µM). Consequently, the key structural features related to activity against of both PknB and M. tuberculosis need to be investigated. Here, two- and three-dimensional quantitative structure-activity relationship (2D and 3D QSAR) analyses combined with molecular dynamics (MD) simulations were employed with the aim to evaluate these key structural features of aminopyrimidine derivatives. Hologram quantitative structure-activity relationship (HQSAR) and CoMSIA models constructed from IC50 and MIC values of aminopyrimidine compounds could establish the structural requirements for better activity against of both PknB and M. tuberculosis. The NH linker and the R1 substituent of the template compound are not only crucial for the biological activity against PknB but also for the biological activity against M. tuberculosis. Moreover, the results obtained from MD simulations show that these moieties are the key fragments for binding of aminopyrimidine compounds in PknB. The combination of QSAR analysis and MD simulations helps us to provide a structural concept that could guide future design of PknB inhibitors with improved potency against both the purified enzyme and whole M. tuberculosis cells.

Asunto(s)

Antituberculosos/química , Antituberculosos/farmacología , Computadores Moleculares , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Pirimidinas/química , Pirimidinas/farmacología , Relación Estructura-Actividad Cuantitativa , Sustitución de Aminoácidos , Descubrimiento de Drogas , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica

RESUMEN

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to ß-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.

Asunto(s)

Mycobacterium tuberculosis/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/fisiología , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , beta-Lactamas/farmacología

RESUMEN

Isocitrate dehydrogenase (IDH) gene from Staphylococcus aureus ATCC12600 was cloned, sequenced and characterized (HM067707). PknB site was observed in the active site of IDH; thus, it was predicted as IDH may be regulated by phosphorylation. Therefore, in this study, PknB, alkaline phosphatase III (SAOV 2675) and IDH genes (JN695616, JN645811 and HM067707) of S. aureus ATCC12600 were over expressed from clones PV 1, UVPALP-3 and UVIDH 1. On passing the cytosloic fractions through nickel metal chelate column, pure enzymes were obtained. Phosphorylation of pure IDH by PknB resulted in the complete loss of activity and was restored upon dephosphorylation with SAOV 2675 which indicated that phosphorylation and dephosphorylation regulate IDH activity in S. aureus. Further, when S. aureus ATCC12600 was grown in BHI broth, decreased IDH activity and increased biofilm units were observed; therefore, this regulation of IDH alters redox status in this pathogen favouring biofilm formation.

Asunto(s)

Biopelículas , Isocitrato Deshidrogenasa/metabolismo , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/genética , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de Aminoácido

RESUMEN

Staphylococcus aureus, a natural inhabitant of nasopharyngeal tract, survives mainly as biofilms. Previously we have observed that S. aureus ATCC 12600 grown under anaerobic conditions exhibited high rate of biofilm formation and L-lactate dehydrogenase activity. Thus, the concentration of pyruvate plays a critical role in S. aureus, which is primarily catalyzed by pyruvate kinase (PK). Analyses of the PK gene sequence (JN645815) revealed presence of PknB site in PK gene indicating that phosphorylation may be influencing the functioning of PK. To establish this hypothesis the pure enzymes of S. aureus ATCC 12600 were obtained by expressing these genes in PK 1 and PV 1 (JN695616) clones and passing the cytosolic fractions through nickel metal chelate column. The molecular weights of pure recombinant PK and PknB are 63 and 73 kDa, respectively. The enzyme kinetics of pure PK showed K M of 0.69 ± 0.02 µM, while the K M of PknB for stpks (stpks = NLCNIPCSALLSSDITASVNCAK) substrate was 0.720 ± 0.08 mM and 0.380 ± 0.07 mM for autophosphorylation. The phosphorylated PK exhibited 40 % reduced activity (PK = 0.2 ± 0.015 µM NADH/min/ml to P-PK = 0.12 ± 0.01 µM NADH/min/ml). Elevated synthesis of pyruvate kinase was observed in S. aureus ATCC 12600 grown in anaerobic conditions suggesting that the formed pyruvate is more utilized in the synthesis phase, supporting increased rate of biofilm formation.

RESUMEN

The Mycobacterium tuberculosis protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. The present study describes the comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Although PknB-mediated kinase activity is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants not expressing PknB. By creating chimeric kinases, we find that the intracellular kinase domain has unique functions in the virulent strain, which cannot be substituted by other kinases. Interestingly, we find that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The differential behavior of PknB vis-à-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. Mouse infection studies performed to determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB is not only critical for growth of the pathogen in vitro but is also essential for the survival of the pathogen in the host.

Asunto(s)

Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/enzimología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Membrana Celular/metabolismo , Proliferación Celular , Espacio Extracelular/metabolismo , Regulación Bacteriana de la Expresión Génica , Espacio Intracelular/metabolismo , Ratones , Viabilidad Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiología , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Transporte de Proteínas