Identifying RO9021 as a Potential Inhibitor of PknG from Mycobacterium tuberculosis: Combinative Computational and in Vitro Studies

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Despite being considered curable and preventable, the increase of antibiotic resistance is becoming a serious public health problem. Mtb is a pathogen capable of surviving in macrophages, causing long-Term latent infection where the mycobacterial serine/threonine protein kinase G (PknG) plays a protective role. Therefore, PknG is an important inhibitory target to prevent Mtb from entering the latency stage. In this study, we use a pharmacophore-based virtual screening and biochemical assays to identify the compound RO9021 (CHEMBL3237561) as a PknG inhibitor. In detail, 1.5 million molecules were screened using a scalable cloud-based setup, identifying 689 candidates, which were further subjected to additional screening employing molecular docking. Molecular docking spotted 62 compounds with estimated binding affinities of-7.54 kcal/mol (s.d. = 0.77 kcal/mol). Finally, 14 compounds were selected for in vitro experiments considering previously reported biological activities and commercial availability. In vitro assays of PknG activity showed that RO9021 inhibits the kinase activity similarly to AX20017, a known inhibitor. The inhibitory effect was found to be dose dependent with a relative IC50value of 4.4 ± 1.1 μM. Molecular dynamics simulations predicted that the PknG-RO9021 complex is stable along the tested timescale. Altogether, our study indicates that RO9021 is a noteworthy drug candidate for further developing new anti-TB drugs that hold excellent reported pharmacokinetic parameters.Revisión por pare

Design, Synthesis, Characterization and Biological Evaluation of Some Novel Heterocyclic Compounds as Anti-tubercular agents

Thirty molеculеs (Ν1 to Ν20, GA, GAC, GAC1, GAC5, GAC6, GAC7, GAЕ, GAЕA, GAM and GAT) which wеrе prеdictеd to bе еffеctivе against Mycobactеrium tubеrculosis wеrе sеlеctеd for thе synthеsis through computational studiеs. This was achiеvеd by thе molеcular docking studiеs against thе targеt еnzymе InhA (PDB id – 2NSD) of Mycobactеrium tubеrculosis, in-silico ADMЕ assеssmеnt and in-silico toxicity prеdictions. ❖ Thе thirty molеculеs which wеrе sеlеctеd for thе synthеsis bеlong to thе following functional class, ✔ 20 Thiazolidinonе dеrivativеs (Ν1 to Ν20) and ✔ 10 dioxolan basеd analoguеs comprising of • thiеno-pyrimidinе corе (4 compounds) (GAC, GAC1, GAC5 and GAC7), • thiеno-pyridinе corе (2 compounds) (GAЕA and GAM), • thiеno-thiazinе corе (1 compound) (GAC6) and • dihydro bеnzo-thiophеnе (3 compounds) (GA, GAЕ and GAT). All thе thirty molеculеs wеrе synthеsizеd. Thе synthеsizеd compounds wеrе purifiеd and charactеrizеd. Thе synthеsizеd ϲompounds wеrе ϲharaϲtеrizеd by “FT-IR, 1H-NMR, 13C-NMR, Mass spеctra and еlеmеntal analysis”. Thе rеlativе stеrеochеmistry of onе compound was confirmеd by thе X-Ray Crystallography. ❖ Thе physical charactеristics and spеctral studiеs likе “FT-IR, 1H-NMR, 13CNMR, Mass spеctra and еlеmеntal analysis” confirmеd thе proposеd structurе of thе synthеsizеd compounds. ❖ All thе synthеsizеd compounds wеrе invеstigatеd for thеir in-vitro anti-tubеrcular potеntial using “Microplatе Alamar Bluе assay” MABA Assay. All thе compounds showеd modеratе to potеnt in vitro activity against MTB with MIC rangе 0.05-50 μg/ml concеntrations. Compounds Ν18, Ν11 and Ν20 displayеd most potеnt in-vitro activity with MICs 0.05, 0.1, 0.2 μg/ml concеntrations rеspеctivеly. ❖ In ordеr to corrеlatе thе in-vitro anti-tubеrcular activity rеsults with thе docking rеsults, furthеr docking was pеrformеd with the top 12 compounds (Ν10, Ν11 to Ν20 and GAT) rеsulting from in-vitro anti-tubеrcular activity data against thе multiplе targеt еnzymеs of Mycobactеrium tubеrculosis {Thymidylatе Kinasе (PDB id – 1G3U), Diaminopimеlatе Dеcarboxylasе (PDB id – 1HKV), Cyclopropanе Synthasе (PDB id – 1L1Е), Antibiotic Rеsistancе Protеin (PDB id – 1YK3), TrpD еssеntial for lung colonization (PDB id – 1ZVW), Thymidylatе Synthasе X (PDB id – 2AF6), Protеin Kinasе G (PDB id – 2PZI), Gyrasе TypеIIA Topoisomеrasе (PDB id – 3UC1), L, D Transpеptidasе 2 (PDB id – 3VAЕ)}. ❖ In ordеr to rationalizе thе corrеlation bеtwееn thе in-vitro antitubеrcular activity and multi-targеt docking rеsults a cross obsеrvational analysis was pеrformеd. Thе top-rankеd thrее compounds (Ν18, Ν11 and Ν20) of in-vitro antitubеrcular activity wеrе cross obsеrvеd with thеir docking ranks on all thе studiеd targеt еnzymеs for thеir dеviations in thеir ranks. ❖ Basеd upon multiplе targеt docking study rеsults and thе cross obsеrvational analysis rеsults, thе еnzymе Thymidylatе Kinasе (PDB id – 1G3U) was found to bе morе appropriatе targеt for thе tеstеd compounds that еxhibitеd in-vitro antitubеrcular activity. Thus thе scopе and limitations of softwarе and thе plausiblе mеchanism of action for thе activity was provеd. ❖ Thе stability of thе ligand-rеcеptor complеxеs wеrе analysеd by molеcular dynamic simulation study. This was achiеvеd by pеrforming thе study with thе ligands Ν18-1G3U, Ν11-1G3U and Ν20-1G3U (-thymidylatе kinasе) complеxеs (top-rankеd doϲking ligand-rеcеptor complеx). Thе study confirmеd that thе ligand-rеcеptor complеxеs wеrе stablе without any notablе conformational changеs during thе simulation run. At thе еnd of thе MD simulation, changе in position and oriеntation of ligands in thе introducеd binding sitе wеrе obsеrvеd, which indicatеs thе usеfulnеss of thе MD simulation for thе optimization of thе ligands into thе targеt binding sitе. ❖ Thе compounds showing in-vitro inhibitory activity bеlow 12.5 μg/ml concеntrations against Mycobactеrium tubеrculosis wеrе subjеctеd for thе acutе oral toxicity studiеs. Thе sеlеctеd compound codеs wеrе N10, N11 to N20 and GAT. No signs of toxicity wеrе noticеd at thе dosе of 300 mg/kg b.w, whilе somе signs of toxicity at 2000 mg/kg b.w. to thе group of animals wеrе rеcordеd. Thus thе study suggеsts that thе LD50 valuе of thе tеstеd compounds wеrе еxcеptеd to еxcееd 300 mg/kg b.w and was rеprеsеntеd as class 4 (300 mg/kg<LD50<2000mg/kg) according to Globally Harmonizеd Classification Systеm (GHS). ❖ Thrее compounds (N11, N20 and N18) which displayеd еffеctivе inhibition of Mycobactеrium tubеrculosis in in-vitro anti-tubеrcular activity wеrе studiеd for thеir in-vivo potеntial using Balb/ϲ mousе modеl for Colony Forming Units (CFU) and Mortality. It was found that compound N18 was activе in in-vivo antimycobactеrial assay, whеn comparеd to thе othеr synthеsizеd tеstеd compounds. It was also intеrеsting to noticе that thе compound N18 dеcrеasеd thе bactеrial load to 24.33± 2.186 at 10 mg/kg dosе, whilе standard drug isoniazid dеcrеasеd thе bactеrial load to 15.33±1.764 at 25 mg/kg dosе. Thus thе study concludеs that thе CFU valuе obtainеd by compound N18 at thе dosе of 10 mg/kg was found to bе significant whеn comparеd to thе standard drug isoniazid at 25 mg/kg dosе. CONCLUSION: In thе prеsеnt work, simplе and еfficiеnt practical mеthods for thе synthеsis of hеtеrocyclics, which rеsultеd from thе in-silico approach was achiеvеd in good yiеld. ☞ Thiazolidinonе dеrivativеs, i.e. compounds Ν18, Ν11 and Ν20 showеd most potеnt inhibition in in-vitro antitubеrcular activity at MIC 0.05, 0.1 and 0.2 μg/ml concеntrations. ☞ In-vivo acutе toxicity studiеs and in-silico ADMЕ prеdictions rеports suggеst thе lеad compounds Ν18, Ν11 and Ν20 can be taken up for further studies. ☞ It was found that lеad compound Ν18 was activе in in-vivo antimycobactеrial assay, whеn comparеd to thе othеr synthеsizеd tеstеd compounds. ☞ It was intеrеsting to note that thе compound N18 dеcrеasеd thе bactеrial load to 24.33± 2.186 at 10 mg/kg dosе, whilе standard drug isoniazid dеcrеasеd thе bactеrial load to 15.33±1.764 at 25 mg/kg dosе. Thus thе study dеsеrvеs for thе conclusion that thе CFU valuе obtainеd by compound N18 at thе dosе of 10 mg/kg was found to bе significant whеn comparеd to thе standard drug isoniazid at 25 mg/kg dosе. It was also concludеd that, on incrеasing thе dosе of compound N18, it may producе morе significant rеsults comparеd to thе standard drug isoniazid. ☞ Thе abovе findings havе dеmonstratеd that the compound N18 ((Z)-5-(3-nitrobеnzylidеnе)-2-thioxothiazolidin-4-onе) is possibly a good antimycobacterial agent

Identification of Novel Mycobacterial Inhibitors Against Mycobacterial Protein Kinase G

Protein kinase G (PknG) is a eukaryotic-like serine/threonine kinase that is expressed by Mycobacterium tuberculosis and promotes survival of mycobacteria in host macrophages by suppressing phagosome-lysosome fusion. Thus, compounds showing inhibitory activity against PknG are promising anti-mycobacterial agents. We therefore aimed to develop anti-mycobacterial agents by identifying new PknG inhibitors. A luciferase-based PknG kinase assay was used to screen potential inhibitors of PknG. We found that four compounds, namely AZD7762, R406, R406-free base, and CYC116, inhibited PknG activities. AZD7762, R406, and R406-free base promoted transfer of mycobacteria to lysosomes. These compounds also inhibited survival of M. bovis Bacillus Calmette–Guérin (BCG) inside human macrophages. Furthermore, R406 and R406-free base showed bactericidal activity against BCG in infected human macrophages without cytotoxicity. The PknG inhibitors identified in this study by the luciferase-based PknG kinase assay may be promising leads for the development of anti-mycobacterial agents

Predictive binding affinity of plant-derived natural products towards the protein kinase G enzyme of Mycobacterium tuberculosis (MtPknG)

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a growing public health concern worldwide, especially with the emerging challenge of drug resistance to the current drugs. Efforts to discover and develop some novel, more effective and safer anti-TB drugs are urgently needed. Products from natural sources, such as medicinal plants, have long played an important role in traditional medicine and continue to provide some inspiring templates for the design of new drugs. Protein kinase G, produced by M. tuberculosis (MtPKnG), is a serine/threonine kinase that has been reported to prevent phagosome-lysosome fusion and help prolong M. tuberculosis survival within the host’s macrophages. Here, we used an in silico target-based approach (docking) to predict the interactions between MtPknG and 84 chemical constituents from two medicinal plants (Pelargonium reniforme and Pelargonium sidoides) that have a well-documented historical use as natural remedies for TB. Docking scores for ligands towards the target protein were calculated using AutoDock Vina as the predicted binding free energies. Ten flavonoids present in the aerial parts of P. reniforme and/or P. sidoides showed docking scores ranging from -11.1 to -13.2 kcal/mol. Upon calculation of all ligand efficiency indices, we observed that the (- G/MW) ligand efficiency index for flavonoids (4), (5) and (7) was similar to the one obtained for the AX20017 control. When taking all compounds into account, we observed that the best (- G/MW) efficiency index was obtained for coumaric acid, coumaraldehyde, p-hydroxyphenyl acetic acid and p-hydroxybenzyl alcohol. We found that methyl gallate and myricetin had ligand efficiency indices superior and equal to the AX20017 control efficiency, respectively. It remains to be seen if any of the compounds screened in this study exert an effect in M. tuberculosis-infected macrophages

Phytochemical, biological, and molecular docking studies on Fraxinus excelsior, Stachys arabica, Pelargonium sidoides, and Pelargonium reniforme

Plants have a long history of use in traditional medicine and several plant species are natural cures for tuberculosis (TB) or TB-related symptoms. Such natural sources can be a worthy starting point in the search for new drugs since they are rich in diverse phytochemicals which may possess antimicrobial and/or immunomodulatory activity. TB is still a growing public health concern worldwide, especially with the emerging challenge of drug resistance to current anti-TB drugs. There is an urgent need for effective and safe therapeutic interventions to tackle TB globally. One approach to achieve this goal is to combine host directed therapies (HDT) with current anti-TB therapies. Pro-inflammatory cytokines (TNF-α and IL-12) are essential for an effective immune response against bacterial infections and play a crucial role in controlling TB. Another promising approach is to identify compounds that could target key virulence enzymes of Mtb such as protein kinase G (MtPKnG). This study describes the phytochemical investigation of two medicinal plants (Fraxinus excelsior and Stachys arabica) selected based on their traditional and/or chemo-taxonomical use in the treatment for TB or TB-related symptoms. The work also focused on the evaluation of these plant extracts and selected isolated phytochemicals for their potential immunomodulatory effect in LPS-stimulated THP-1 cells. A total of 28 phytochemicals were isolated and characterised from both plants. The phytochemical investigation of the n-hexane and ethyl acetate extracts of F. excelsior leaves revealed the presence of 20 phytochemicals. The isolated phytochemicals included two pentacyclic triterpenoids (oleanolic acid and ursolic acid), squalene, one flavonoid (pinocembrin), five phenylethanoid esters (4-hydroxyphenethyl dotriacontanoate, 4-hydroxyphenethyl triacontanoate, 4-hydroxyphenethyl octacosanoate, 4-hydroxyphenethyl hexacosanoate, and 4-hydroxyphenethyl tetracosanoate), steryl fatty acid esters (β-sitosterol oleate), phytyl fatty acid esters (phytyl palmitate, phytyl oleate, and phytyl linolenate), and a mixture of acyclic alkanes (heptacosane, octacosane, nonacosane, triacontane, hentriacontane, dotriacontane and tritriacontane). Although ursolic acid, phytyl linolenate, nonacosane, and hentriacontane have already been reported in F. excelsior leaves, all other phytochemicals are reported here for the first time A total of 17 phytochemicals were isolated from the n-hexane and ethyl acetate extracts of S. arabica leaves. This included pheophytins (pheophytin a, 132 (R,S)-hydroxy pheophytin a, and pheophytin b), a mixture of phytosterols (stigmasterol, β-sitosterol and campesterol), a mixture of steryl fatty acid esters (β-sitosterol oleate & campesterol oleate), a mixture of phytyl fatty acid esters (phytyl palmitate, phytyl oleate), and a mixture of acyclic alkanes (heptacosane, octacosane, nonacosane, triacontane, hentriacontane and tritriacontane). Although all are known compounds, they are reported from S. arabica for the first time. When screened for potential Immunomodulatory effect in LPS-stimulated THP-1 cells, all the plant extracts and selected purified phytochemicals (oleanolic acid, ursolic acid, pinocembrin, and pheophytins) were able to reduce the production of the pro-inflammatory cytokines (TNF- α and IL-12). The mixture of phenylethanoid esters did not appear to influence the production of either of the cytokines studied. Our results indicate that the two plant extracts and their selected phytochemicals (oleanolic acid, ursolic acid, pinocembrin, and pheophytins) exhibited anti-inflammatory effect that could validate to a certain extent their potential use for complications associated with inflammation including TB as adjunct host therapy. Molecular docking using AutoDock Vina was conducted to predict the interactions between MtPknG and eighty-four phytochemicals from Pelargonium sidoides and Pelargonium reniforme as well as six selected phytochemicals identified through the phytochemical work. The flavonoids present in the aerial parts of Pelargonium plants displayed the best predicted binding energy towards MtPknG. The highest binding affinity towards MtPknG was recorded for isoorientin 2”-O-gallate (79), isovitexin 2”-O-gallate (77), nicotiflorin (61), orientin (74) and populnin (60) (−13.2, −12.6, −12.2, −11.8, and −11.6 kcal/mol, respectively) accompanied with SILE values (4.27, 4.11, 3.98, 4.17 and 4.10, respectively) that were superior to the control inhibitor AX20017 (−7.9 kcal/mol and SILE value of 3.32, respectively). Medicinal plants and their phytochemicals, owing to their abundance and diversity, could provide valuable alternatives for design of new generation of anti-TB drugs by serving as adjunct host-directed therapy and/or pathogen-directed therapy.Plants have a long history of use in traditional medicine and several plant species are natural cures for tuberculosis (TB) or TB-related symptoms. Such natural sources can be a worthy starting point in the search for new drugs since they are rich in diverse phytochemicals which may possess antimicrobial and/or immunomodulatory activity. TB is still a growing public health concern worldwide, especially with the emerging challenge of drug resistance to current anti-TB drugs. There is an urgent need for effective and safe therapeutic interventions to tackle TB globally. One approach to achieve this goal is to combine host directed therapies (HDT) with current anti-TB therapies. Pro-inflammatory cytokines (TNF-α and IL-12) are essential for an effective immune response against bacterial infections and play a crucial role in controlling TB. Another promising approach is to identify compounds that could target key virulence enzymes of Mtb such as protein kinase G (MtPKnG). This study describes the phytochemical investigation of two medicinal plants (Fraxinus excelsior and Stachys arabica) selected based on their traditional and/or chemo-taxonomical use in the treatment for TB or TB-related symptoms. The work also focused on the evaluation of these plant extracts and selected isolated phytochemicals for their potential immunomodulatory effect in LPS-stimulated THP-1 cells. A total of 28 phytochemicals were isolated and characterised from both plants. The phytochemical investigation of the n-hexane and ethyl acetate extracts of F. excelsior leaves revealed the presence of 20 phytochemicals. The isolated phytochemicals included two pentacyclic triterpenoids (oleanolic acid and ursolic acid), squalene, one flavonoid (pinocembrin), five phenylethanoid esters (4-hydroxyphenethyl dotriacontanoate, 4-hydroxyphenethyl triacontanoate, 4-hydroxyphenethyl octacosanoate, 4-hydroxyphenethyl hexacosanoate, and 4-hydroxyphenethyl tetracosanoate), steryl fatty acid esters (β-sitosterol oleate), phytyl fatty acid esters (phytyl palmitate, phytyl oleate, and phytyl linolenate), and a mixture of acyclic alkanes (heptacosane, octacosane, nonacosane, triacontane, hentriacontane, dotriacontane and tritriacontane). Although ursolic acid, phytyl linolenate, nonacosane, and hentriacontane have already been reported in F. excelsior leaves, all other phytochemicals are reported here for the first time A total of 17 phytochemicals were isolated from the n-hexane and ethyl acetate extracts of S. arabica leaves. This included pheophytins (pheophytin a, 132 (R,S)-hydroxy pheophytin a, and pheophytin b), a mixture of phytosterols (stigmasterol, β-sitosterol and campesterol), a mixture of steryl fatty acid esters (β-sitosterol oleate & campesterol oleate), a mixture of phytyl fatty acid esters (phytyl palmitate, phytyl oleate), and a mixture of acyclic alkanes (heptacosane, octacosane, nonacosane, triacontane, hentriacontane and tritriacontane). Although all are known compounds, they are reported from S. arabica for the first time. When screened for potential Immunomodulatory effect in LPS-stimulated THP-1 cells, all the plant extracts and selected purified phytochemicals (oleanolic acid, ursolic acid, pinocembrin, and pheophytins) were able to reduce the production of the pro-inflammatory cytokines (TNF- α and IL-12). The mixture of phenylethanoid esters did not appear to influence the production of either of the cytokines studied. Our results indicate that the two plant extracts and their selected phytochemicals (oleanolic acid, ursolic acid, pinocembrin, and pheophytins) exhibited anti-inflammatory effect that could validate to a certain extent their potential use for complications associated with inflammation including TB as adjunct host therapy. Molecular docking using AutoDock Vina was conducted to predict the interactions between MtPknG and eighty-four phytochemicals from Pelargonium sidoides and Pelargonium reniforme as well as six selected phytochemicals identified through the phytochemical work. The flavonoids present in the aerial parts of Pelargonium plants displayed the best predicted binding energy towards MtPknG. The highest binding affinity towards MtPknG was recorded for isoorientin 2”-O-gallate (79), isovitexin 2”-O-gallate (77), nicotiflorin (61), orientin (74) and populnin (60) (−13.2, −12.6, −12.2, −11.8, and −11.6 kcal/mol, respectively) accompanied with SILE values (4.27, 4.11, 3.98, 4.17 and 4.10, respectively) that were superior to the control inhibitor AX20017 (−7.9 kcal/mol and SILE value of 3.32, respectively). Medicinal plants and their phytochemicals, owing to their abundance and diversity, could provide valuable alternatives for design of new generation of anti-TB drugs by serving as adjunct host-directed therapy and/or pathogen-directed therapy

Evaluación de la actividad inhibitoria de péptidos sintéticos seleccionados sobre PknG de Mycobacterium tuberculosis

Introducción: La identificación de nuevos blancos terapéuticos y compuestos antimicrobianos son claves en la lucha contra la tuberculosis resistente a drogas. PknG es un factor de virulencia de Mycobacterium tuberculosis asociado con la supervivencia intracelular y estado de latencia, el cual se considera una diana promisoria. Objetivos: Evaluar la actividad inhibitoria de péptidos sintéticos seleccionados para PknG a través de un ensayo de luminiscencia. Métodos: Las proteínas recombinantes PknG y GarA fueron expresadas y purificadas mediante cromatografía de afinidad. La reacción de fosforilación de PknG y su inhibición se evaluaron mediante un ensayo de luminiscencia. Para la estandarización de la reacción de fosforilación se evaluó la composición del buffer, el tipo de ion divalente como cofactor, y el tiempo de reacción. Como control de inhibición se utilizó la molécula AX20017. Se determinó el porcentaje de inhibición para los péptidos candidatos. Resultados: Se obtuvieron alícuotas de PknG y GarA a una concentración de 145 µM y 231 µM, respectivamente. La reacción de fosforilación fue estandarizada a 181.2 nM PknG, 7.7 µM GarA y 10 µM ATP, utilizando el buffer PR1 (25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT, 5 mM MgCl2). AX20017 reportó una IC50 de 6.71 µM. Seis de los péptidos seleccionados demostraron un porcentaje de inhibición superior al 30%. De estos, UPC_07, UPC_08 y UPC_09 mostraron valores cercanos a 39%. Conclusiones: Se estandarizó un ensayo de luminiscencia in vitro para evaluar la inhibición de la actividad quinasa de PknG sobre GarA. Tres péptidos mostraron porcentajes de inhibición superiores a 38%Introduction: The identification of new therapeutic targets and antimicrobial compounds are key against drug-resistant tuberculosis. PknG is a virulence factor of Mycobacterium tuberculosis associated with intracellular survival and latency, which is considered a promising target. Objectives: To evaluate the inhibitory activity of selected synthetic peptides for PknG through a luminescence assay. Methods: Recombinant PknG and GarA proteins were expressed and purified by affinity chromatography. PknG phosphorylation reaction and inhibition were assessed by a luminescence assay. For the standardization of the phosphorylation reaction, the composition of the buffer, type of divalent ion as cofactor, and reaction time were evaluated. As inhibition control, AX20017 was used. The percentage inhibition for the candidate peptides was determined. Results: Aliquots of PknG and GarA were obtained at a concentration of 145 µM and 231 µM, respectively. The phosphorylation reaction was standardized to 181.2 nM PknG, 7.7 µM GarA and 10 µM ATP, using PR1 buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT, 5 mM MgCl2). AX20017 reported an IC50 of 6.71 µM. Six of the selected peptides demonstrated an inhibition percentage higher than 30%. Of these, UPC_07, UPC_08 and UPC_09 showed values close to 39%. Conclusions: An in vitro luminescence assay was standardized to evaluate the inhibition of PknG kinase activity on GarA. Three peptides showed inhibition percentages higher than 38%.Tesi

PREVALENCE AND RISK FACTORS ASSOCIATED WITH TUBERCULOSIS OCCURRENCE AMONG PATIENTS ATTENDING MUHIMA DISTRICT HOSPITAL, NYARUGENGE DISTRICT, RWANDA

Tuberculosis (TB) is one the serious public health problems globally and locally and killed at least eight million in 2018 (WHO, 2019). TB is a curable disease if well treated and followed. One of the most key considerations for tracking and evaluating TB prevention and control programs is the prevalence and related risk factors. The study adopted a quantitative approach and a cross-sectional retrospective design to collect data from the respondents. A systematic sampling technique was used and secondary data recorded in the laboratory were collected. Specific objectives were: to determine the prevalence of tuberculosis among patients attending Muhima District Hospital in Nyarugenge District, and to identify behavioral factors associated with TB occurrence among patients attending Muhima District Hospital in Nyarugenge District. Descriptive and analytical statistics were used to analyze the data. The total number of respondents was 2161 patients. The majority, were male 1524(70.5%) and female were 637(29.5%). For the age, those less than 9 years were 19(.9%), between 10-17 years 76(3.5%), between 18-35 years 1185(54.8%), between 36-59 years 843(39.0%), and above 60 years 38(1.8%). Those who were married were 1014(47%), singles were 977(45.2%), and widower were 57(2.6%). For employment, 883(40.9%) were unemployed, 845(39.1%) were employed, 206(9.5%) were students, and 227(10.4%) were prisoners. Based on location, 1181(54.7%) were from urban areas, and 980(45.3%) were from rural areas. The prevalence of TB was 7.2%, out of which the females were 53(34.0%) and males were 103(66.0%). The prevalence of TB among HIV patients was 32.6%. For behavior practices, drinking alcohol was found among 908(42.0%) and smokers were 186(8.6%). For all the predictors of TB occurrence, none of them was statistically associated with TB occurrence. These include demographic characteristics, behavioral factors including use of alcohol, smoking and use of tobacco products, and serology. The prevalence of TB among patients attending Muhima District was higher than the National TB prevalence estimates reported by the World Health Organization (WHO, 2019), respectively 7.2% versus 5.2%. All predictors of TB occurrence including demographic characteristics and behavioral factors none of them was associated with TB occurrence. It is worth recommending a further study to understand in-depth risk factors for TB occurrence in the era of epidemiological transition.  Article visualizations

ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos

Tuberculosis (TB) is an infectious disease caused by the acid-fast bacillus Mycobacterium tuberculosis (Mtb), which is one of the most important public health problems worldwide. Furthermore, the emergence of resistant Mtb strains to current anti-TB drugs has increased the search for alternative therapeutic targets and methods for the rational design of new effective drugs. In this sense, membrane proteins have been considered interesting targets due to their biological implication and for being highly accessible to active compounds. Particularly, P-type ATPases membrane transporters are interesting targets due to their implication in ionic homeostasis and mycobacterial viability. This work was oriented to CtpF, a calcium P-type ATPase, related to a broad number of biological conditions associated to processes of infection such as oxidative stress, adaptation of tubercle bacilli to anaerobic conditions, hypoxia and latency. Due to that, the main objective of this doctoral Thesis was to determine, through in silico and in vitro analysis, the potential of P-type ATPases of Mtb, especially the calcium transporter CtpF, as a target for the rational design of anti-TB compounds. Initially, a 3D homology model of CtpF was generated, which was employed for identified key pharmacophoric features of the CtpF-cyclopiazonic acid (CPA) complex, a well-known inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA1a), from which its 3D structure is known experimentally and was used as a template in the construction of the model. By using a repertoire of experimental techniques, it was evaluated and found that CPA causes inhibition of the Ca2+-ATPase activity of CtpF, as well as mycobactericidal activity. The analysis of the transcriptional response of P2 ATPases to treatment with CPA showed a specific response of ctpF in comparison with other P-type ATPases. These initial results provide evidence that CtpF is a molecular target for the design of compounds with anti-TB potential. Thereupon, with the CtpF-CPA pharmacophoric features, a pharmacophore-based virtual screening was performed using the ZINC database in order to select candidate molecules to inhibitors of CtpF. Molecular docking-based virtual screening and binding free energy calculations (MM-GBSA) of selected candidates allowed identifying six compounds with the best relative binding energies to be evaluated in vitro. The compounds selected displayed in vitro antimycobacterial activity, showing a minimum inhibitory concentrations (MIC) ranging from 50 -100 μg/mL, and growth inhibitions of 29.5 - 64.0 % on Mtb. Likewise, they causes inhibition of Ca2+-ATPase activity in Mtb membrane vesicles (IC50) ranging from 4.1 - 35.8 μM. Finally, the activity of the compounds with the best biological activity was evaluated in a macrophage infection model, as an approach to evaluate the effect of compounds once the infection has occurred. The compound ZINC63908257 was the best candidate by displaying a MIC of 50 μg/mL, a Ca2+ P-type ATPase inhibition with IC50 = 4.4 μM and 81 % decrease in Mtb replication within macrophage. This compound showed cytotoxic activity of 12.9 % in MH-S cells and hemolysis of 2 % of human erythrocytes, thus, this compound shows a good pharmacokinetic profile (drug-like). Overall, the results presented here shows the importance of the P-type ATPases of Mtb for the mycobacteria survival during infection, and identify the CtpF as a key molecular target for the design of new antituberculous compounds.La Tuberculosis (TB) es una enfermedad infectocontagiosa causada por el bacilo ácido-alcohol resistente Mycobacterium tuberculosis (Mtb). A su vez, la TB es un problema muy relevante de salud pública a nivel mundial. Por otra parte, la aparición de cepas de Mtb resistentes a los fármacos antituberculosos actualmente empleados, ha impulsado la búsqueda de dianas terapéuticas alternativas y metodologías para el diseño racional de nuevos fármacos efectivos. En ese sentido, las proteínas de membrana son considerados blancos de interés al ser mayormente accesibles a los compuestos activos. Particularmente los transportadores de membrana ATPasas tipo P son dianas interesantes por su implicación en la homeóstasis iónica y la viabilidad de las micobacterias. El presente trabajo se orientó en CtpF, una ATPasa tipo P de Mtb transportadora de Ca2+, relacionada con una gran cantidad de condiciones biológicas asociadas al proceso de infección tales como estrés oxidativo, la adaptación del bacilo tuberculoso a condiciones anaeróbicas, hipoxia y latencia. Por lo anterior, el objetivo principal de esta Tesis fue determinar mediante análisis in silico e in vitro el potencial de las ATPasas tipo P de Mtb, especialmente el trasportador de calcio CtpF, como diana para la búsqueda racional de compuestos con actividad antituberculosa. Inicialmente se generó un modelo 3D de CtpF por homología, el que fue empleado para identificar las características farmacofóricas del complejo CtpF-ácido ciclopiazónico (CPA), un inhibidor de la Ca2+-ATPasa de retículo sarco-endoplásmico (SERCA1a), de la que se conoce experimentalmente su estructura 3D, y fue usada como plantilla en la construcción del modelo. Utilizando un repertorio de técnicas experimentales, se evaluó y encontró que CPA causa inhibición de la actividad Ca2+-ATPasa de CtpF, así como actividad micobactericida. El análisis de la respuesta transcripcional de los genes de las ATPasas tipo P2 al tratamiento con CPA, mostró una respuesta específica de ctpF en comparación a otras ATPasas tipo P. Estos resultados iniciales permitieron sugerir a CtpF como una diana molecular para el diseño de compuestos con potencial anti-TB. A continuación, con las características farmacofóricas CtpF-CPA se realizó un cribado virtual basado en farmacóforo utilizando la base de datos ZINC, para seleccionar moléculas candidatas a inhibidores de CtpF. Estudios de acoplamiento molecular y cálculos de MM-GBSA de los candidatos permitieron la identificación de seis compuestos con la mejor energía libre de unión para ser evaluados in vitro. Los compuestos finalmente seleccionados demostraron tener actividad antimicobacteriana mostrando una concentración mínima inhibitoria (CMI) entre 50 - 100 μg/mL, e inhibición del crecimiento de Mtb entre el 29.5 - 64.0 %. De manera similar, causaron inhibición de la actividad Ca2+-ATPasa en vesículas de membrana de Mtb con un rango IC50 entre 4.1 - 35.8 μM. Finalmente se evaluó la actividad de los compuestos con mejor respuesta biológica, en un modelo de infección de macrófagos, como un acercamiento al efecto de los compuestos en la sobrevida de Mtb durante la infección. El compuesto ZINC63908257 fue seleccionado como el candidato más activo con una CMI de 50 μg/mL, inhibición de la actividad Ca2+-ATPasa con IC50 = 4.4 μM y disminución del 81 % de la replicación intracelular de Mtb en macrófagos una vez ocurrida la fagocitosis. Este compuesto demostró un efecto citotóxico del 12.9 % en células MH-S y hemólisis del 2 % de glóbulos rojos humanos, además de presentar propiedades farmacocinéticas adecuadas (drug-like). El conjunto de resultados obtenidos muestra la importancia de las ATPasas tipo P de Mtb para la supervivencia del bacilo durante la infección, e identifican la proteína CtpF como una diana molecular clave para el diseño de nuevos compuestos antituberculosos.Proyecto DIB_2016_Numero_35885 y Colciencias Programa Nacional en Ciencias Básicas-Cod. 110171250419Búsqueda racional de compuestos inhibidores de la actividad ATPasa tipo P de membrana plasmática y determinación de su actividad antimicobacteriana.Doctorad

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

New computational strategies for target selection and development of new inhibitors for Mycobacterium tuberculosis

La tuberculosis sigue siendo un problema de salud a nivel mundial. Un millon y medio de personas al ano mueren por esta enfermedad siendo la primera causa de muerte entre los infec- ˜tados con el virus de la inmunodeficiencia humana (HIV). El microorganismo causante de laenfermedad, Mycobacterium tuberculosis (Mtb.), es una bacteria de crecimiento lento que vivedentro de los macrofagos del hospedador, en donde puede residir por años sin producir ningún´síntoma de la infeccion en un estado que se conoce como de latencia. Dentro del macrofago lamycobacteria se encuentra sometida a una serie de condiciones de estres como son la hipoxia,la falta de nutrientes y la presencia de especies reactivas de oxígeno y nitrogeno (ERON). Mtbdispone de varios mecanismos de proteccion en dicho ambiente de los que se vale para sobre-vivir durante años. ˜ El presente trabajo de tesis tiene como principal objetivo proponer nuevos procedimientospara el descubrimiento de blancos relevantes para la fase latente de Mtb. Se encuentra divididoen tres partes: La primera parte trata sobre el armado de una base de datos de proteínasparticular de Mycobacterium tuberculosis, llamada TuberQ. Dicha base de datos cuenta coninformacion genómica contextual (expresión en diversas condiciones que simulan el estrés du-rante la vida dentro del macrofago), información estructural retirada del RSCB PDB o genera apartir de modelado comparativo, calculos de drogabilidad estructural, determinaciones de sensi-bilidad a estres por la presencia de residuos cisteína/tirosina o la presencia de centros metalicosoxidables (Fe, Cu, Zn) y una reconstruccion de los principales metabolismos de Mtb. Combinamosesta informacion mediante el uso de una función de puntuación para clasificar los blan-cos moleculares respecto a su relevancia como blancos terapeuticos contra Mtb. en condicionesde estres. Se clasificaron de esta forma miles de proteínas, en particular proteínas novedosaspertenecientes a la vía de síntesis de micotiol) o blancos ya descriptos (como la vía de síntesisde acido micólico). En la segunda parte nos encargamos de realizar un estudio mediante tecnicas bioinformáticasy de química computacional de la familia de proteínas perteneciente a las Cyclopropane Mycolic Acid Synthases (CMAS), dichas enzimas forman parte de la vía de síntesis de acido micólico yse encuentran validadas como potenciales blancos terapeuticos. Estas enzimas son metiltrans-ferasas dependientes de S-adenosil-L-metionina (SAM) con una particularidad, cada una poseeuna selectividad marcada y un producto específico pudiendo realizar reacciones tan diversascomo la ciclopropilacion o generar grupos metil-alcoholes a partir de olefinas. Encontramoslos determinantes moleculares de la selectividad de los productos en las distintas CMAS, proponiendoalgunos cambios puntuales para poder validar estos resultados experimentalmente. En la ultima parte de este trabajo de tesis nos abocamos a dise nar un protocolo de búsquedavirtual de compuestos específicos para la subfamilia de enzimas CMAS que contienen un iónbicarbonato en su sitio activo (cmaA1-2, pcaA, mmaA2 y umaA) utilizando a umaA como referencia. En esta seccion proponemos distintos grupos miméticos al bicarbonato (Urea, Glicina, Carbamato, Acido metoxiacético y Hidroxifuranona) que pueden actuar como farmacóforosbuscando en la base de datos ZINC. Esta base de datos de mas de 30.000 compuestos fue uti-lizada para realizar la busqueda virtual con una posterior estimación de las energ ías de uniónmediante dinamica molecular y MM-PBSA. Concluimos que los mejores compuestos estánbasados en Urea y Glicina debido, en parte, a la interaccion que pueden realizar con sus gruposamino con un glutamico presente en el sitio activo de este subconjunto de CMAS. En resumen, hemos generado un pipeline bioinformatico que permite la elección de blan-cos con los criterios que el usuario prefiera, probado mecanismos de reaccion de las CMAS yutilizado esta informacion para proponer nuevas moléculas con actividad bactericida.Tuberculosis remains a worldwide issue, 1.5 million people die from TB each year being thefirst cause of death among HIV infected people. The microorganism responsible for TB, Mycobacteriumtuberculosis, is a slow growing bacteria which lives inside host’s macrophageswhere it can stay for years without causing symptoms to the host, in a state known as latency. Inside the macrophage, mycobacteria is exposed to a series of stressful conditions such as hypoxia,starvation or the presence of reactive oxygen and nitrogen species (RNOS). Mtb. hasvarious protection mechanisms in such environment which it uses to survive for years. The present work main objective is to propose new procedures to discover latent phase relevanttargets for Mtb. It is divided in three parts: The first one covers the design and setup of aprotein database tailored for Mycobacterium tuberculosis called TuberQ. This database containscontext-based genomic data (micro-array expression in infection-mimicking conditions as hypoxia,starvation and exposure to RNOS), structural information from RSCB PDB or derived byhomology modeling, structural druggability computations, RNOS stress sensitivity by Cys/Tyror metal center (Fe, Cu, Zn) containing proteins and a reconstruction of the principal metabolicpathways in Mtb. Using a scoring function we combine this information in order to classify theproteins regarding its relevance as a good target in latent phase infection. Thousands of proteinshave been classified, in particular new targets belonging to the mycothiol biosynthesis pathwayor already validated targets such as the mycolic acid biosynthesis pathway. In the second part, we studied by means of bioinformatics and computational chemistry the Cyclopropane Mycolic Acid Synthases (CMAS) protein family which belong to the mycolicacid biosynthesis pathway and have been validated as good therapeutic target. These enzymeshave S-adenosyl-L-methionine (SAM) dependent methyltransferase activity with a peculiarity,each one of them has a strong selectivity and a specific product, being able to produceciclopropanes or methyl-alcohol groups from an olefin group. We describe the molecular determinantsof the different products in CMAS proposing some testable predictions by doingmutagenesis. In the last part of this thesis we devoted to design a high throughput docking protocol tailoredto the bicarbonate bearing CMAS subfamily (cmaA1-2, pcaA, mmaA4 and umaA) usingumaA as reference. We propose different bicarbonate-mimicking groups (Urea, glycine, Carba-mate, Metoxiacetic acid and Hydroxifuranone) which could act as pharmacophores searchingfor compounds that have them in the ZINC database. A 30.000 compound database was usedto screen against umaA with a posterior free energy of binding estimation done with moleculardynamics and MM-PBSA. We conclude that the best compounds are based on the urea andglycine motifs due to, partly, the hydrogen bond interaction between the amino group in thesecompounds and a glutamic acid residue of the protein present in the CMAS subgroup. Summing up, we have developed a bioinformatics pipeline capable of target selection withuser-supplied criteria, tested CMAS reaction mechanisms and used the derived information todevelop new compounds with bactericidal activity.Fil: Defelipe, Lucas Alfredo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina