Dr Sanjib Bhakta - Academia.edu (original) (raw)
Papers by Dr Sanjib Bhakta
Molecules
Amaryllidaceae is a significant source of bioactive phytochemicals with a strong propensity to de... more Amaryllidaceae is a significant source of bioactive phytochemicals with a strong propensity to develop new drugs. The genera Allium, Tulbaghia, Cyrtanthus and Crinum biosynthesize novel alkaloids and other phytochemicals with traditional and pharmacological uses. Amaryllidaceae biomolecules exhibit multiple pharmacological activities such as antioxidant, antimicrobial, and immunomodulatory effects. Traditionally, natural products from Amaryllidaceae are utilized to treat non-communicable and infectious human diseases. Galanthamine, a drug from this family, is clinically relevant in treating the neurocognitive disorder, Alzheimer’s disease, which underscores the importance of the Amaryllidaceae alkaloids. Although Amaryllidaceae provide a plethora of biologically active compounds, there is tardiness in their development into clinically pliable medicines. Other genera, including Cyrtanthus and Tulbaghia, have received little attention as potential sources of promising drug candidates....
Antibiotics
The bulbs of Allium species are a known source of antibacterial phytochemicals. Anti-infective, e... more The bulbs of Allium species are a known source of antibacterial phytochemicals. Anti-infective, efflux pump and biofilm inhibitory activities of bulb extracts of selected Ghanaian shallots Allium cepa var aggregatum were evaluated using the HT-SPOTi assay and other whole-cell phenotypic screening techniques to determine their possible mechanisms of action. Ethanol and aqueous extracts of white A. cepa inhibited the growth of Mycobacterium smegmatis mc2 155 and Escherichia coli, respectively. The majority of the Allium extracts significantly (p < 0.05) exhibited efflux pump inhibitory activity against all the acid-fast, Gram-positive and Gram-negative strains used. Hexane and chloroform extract of the pink A. cepa and the aqueous extract of the white A. cepa significantly inhibited M. smegmatis biofilm formation. For Pseudomonas aeruginosa, the inhibition was observed at 250 µg/mL for the aqueous extract (~77.34%) and 125 µg/mL for the hexane extract (~76.51%). The results suggest...
Arylamine N-acetyltransferase (NAT) was first identified in humans for its role in metabolising t... more Arylamine N-acetyltransferase (NAT) was first identified in humans for its role in metabolising the anti-tubercular drug isoniazid. Later functional NAT proteins were identified in bacteria including Mycobacterium tuberculosis where they were found to N-acetylate a range of arylamines, hydrazines and hydralazines both in vitro and in vivo. From gene deletion and genomic studies in mycobacteria, it became clear that NAT in Mycobacterium bovis Bacillus Calmette–Guérin (BCG) had an endogenous role in cell-wall metabolism, cholesterol degradation and the nat gene was part of an essential gene cluster for intra-cellular survival of the pathogen M. tuberculosis; this has paved the way for validation of NAT and other proteins encoded in the gene cluster as novel therapeutic targets for anti-tubercular drug design and discovery. This chapter reviews the genomic organisation of the nat gene and the function of the NAT protein in mycobacteria, including the location of the nat gene in the genome. The role of nat and its neighbouring genes, their endogenous substrates and the regulation of their expression in fast-growing environmental and slow-growing pathogenic intra-cellular mycobacteria is also covered
Fast-growing, non-infectious and intracellularly surviving drug-
Pharmaceuticals, 2021
The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB)... more The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has reinforced the need for the development of new anti-TB drugs. The first line drug isoniazid inhibits InhA. This is a prodrug requiring activation by the enzyme KatG. Mutations in KatG have largely contributed to clinical isoniazid resistance. We aimed to design new ‘direct’ InhA inhibitors that obviate the need for activation by KatG, circumventing pre-existing resistance. In silico molecular modelling was used as part of a rational structure-based drug-design approach involving inspection of protein crystal structures of InhA:inhibitor complexes, including the broad spectrum antibiotic triclosan (TCS). One crystal structure exhibited the unusual presence of two triclosan molecules within the Mycobacterium tuberculosis InhA binding site. This became the basis of a strategy for the synthesis of novel inhibitors. A series of new, flexible ligands were designed and synthesised, expandi...
The Journal of antimicrobial chemotherapy, Jan 4, 2015
Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñ an,... more Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñ an, was found to inhibit the growth of Mycobacterium tuberculosis H 37 Rv. A biomimetic approach for the chemical synthesis of a wide array of 1-substituted tetrahydroisoquinolines was undertaken with the aim of elucidating a common pharmacophore for these compounds with novel mode(s) of anti-TB action. Methods: Biomimetic Pictet-Spengler or Bischler-Napieralski synthetic routes were employed followed by an evaluation of the biological activity of the synthesized compounds. Results: In this work, the synthesized tetrahydroisoquinolines were found to inhibit the growth of M. tuberculosis H 37 Rv and affect its whole-cell phenotype as well as the activity of the ATP-dependent MurE ligase, a key enzyme involved in the early stage of cell wall peptidoglycan biosynthesis. Conclusions: As the correlation between the MIC and the half-inhibitory enzymatic concentration was not particularly strong, there is a credible possibility that these compounds have pleiotropic mechanism(s) of action in M. tuberculosis.
Tuberculosis, 2010
New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan bio... more New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan biosynthesis is a potential therapeutic target. UDP-MurNAc-tripeptide ligase (MurE) is a member of the ATP-dependent ligase family, which incorporate amino acids including meso-diaminopimelic acid (m-DAP) into peptidoglycan during synthesis in a species-specific manner. In the present study, we have cloned, over-expressed, and characterised MurE from M. tuberculosis (Mtb-MurE). The crystal structure has been determined at 3.0A resolution in the presence of the substrate UDP-MurNAc-l-Ala-d-Glu (UAG). The activity of the enzyme was measured through estimating inorganic phosphate released in a non-radioactive high-throughput colourimetric assay. UDP-MurNAc-l-Ala-d-Glu-m-DAP (UMT) formation coupled to inorganic phosphate release was confirmed by HPLC and mass spectrometric analyses. Kinetic constants were determined for a range of natural substrates using optimised conditions. From our findings, it is evident that Mtb-MurE is highly specific in adding m-DAP to UDP-MurNAc-dipeptide and ATP-hydrolysis is an absolute requirement for its activity.
Journal of Antimicrobial Chemotherapy, 2010
New anti-mycobacterial entities with novel mechanisms of action are clinically needed for treatin... more New anti-mycobacterial entities with novel mechanisms of action are clinically needed for treating resistant forms of tuberculosis. The purpose of this study was to evaluate anti-tubercular activity and selectivity of seven recently isolated natural products from Colombian plants. Methods: MICs were determined using a liquid medium growth inhibition assay for Mycobacterium tuberculosis H 37 Rv and both solid and liquid media growth inhibition assays for Mycobacterium bovis BCG. Escherichia coli growth inhibition and mammalian macrophage cell toxicity were evaluated to establish the degree of selectivity of the natural product against whole cell organisms. Enzymatic inhibition of ATP-dependent MurE ligase from M. tuberculosis was assayed using a colorimetric phosphate detection method. The most active compound, 3-methoxynordomesticine hydrochloride, was further investigated on M. bovis BCG for its inhibition of sigmoidal growth, acid-fast staining and viability counting analysis. Results: Aporphine alkaloids were found to be potent inhibitors of slow-growing mycobacterial pathogens showing favourable selectivity and cytotoxicity. In terms of their endogenous action, the aporphine alkaloids were found inhibitory to M. tuberculosis ATP-dependent MurE ligase at micromolar concentrations. A significantly low MIC was detected for 3-methoxynordomesticine hydrochloride against both M. bovis BCG and M. tuberculosis H 37 Rv. Conclusions: Considering all the data, 3-methoxynordomesticine hydrochloride was found to be a potent antitubercular compound with a favourable specificity profile. The alkaloid showed MurE inhibition and is considered an initial hit for exploring related chemical space.
Current Eye Research, 1995
Nitroreductase is a 24 KDa flavoprotein from E.coli. It has potential therapeutic use as an anti-... more Nitroreductase is a 24 KDa flavoprotein from E.coli. It has potential therapeutic use as an anti-cancer enzyme in VDEPT (Virus Directed Enzyme Prodrug Therapy). Nitroreductase is very unusual in being able to use both N A D H and NADPH in the reduction of a broad range of substrates. The enzyme has been purified to near homogeniety and subjected to a series of crystallization trials. Crystals have been obtained. Preliminary characterisation shows them to be tetragonal (P41212, unit cell 57,57,262 Angstroms, 90" 90" 90"). Recent progress will be reported.
Journal of Antimicrobial Chemotherapy, 2012
The intracellularly surviving and slow-growing pathogen, Mycobacterium tuberculosis, adapts the h... more The intracellularly surviving and slow-growing pathogen, Mycobacterium tuberculosis, adapts the host cell environment for its active and dormant life cycle. It is evident that the lack of appropriate highthroughput screening of inhibitors within host cells is an impediment for the early stages of anti-tubercular drug discovery. We aimed to develop an integrated surrogate model that enhances the screening of large inhibitor libraries. Methods: Different mycobacterial species were compared for their growth, drug susceptibility and intracellular uptake. A 6-well plate solid agar-based spot culture growth inhibition (SPOTi) assay was developed into a higher throughput format. The uptake and intracellular survival of Mycobacterium aurum within mouse macrophage cells (RAW 264.7) were optimized using 24/96-well plate formats. Results: Fast-growing, non-pathogenic M. aurum was found to have an antibiotic-susceptibility profile similar to that of M. tuberculosis. The sensitivity to an acidic pH environment and the ability to multiply inside RAW 264.7 macrophages provided additional advantages for employing M. aurum in intracellular drug screening methods. A selection of anti-tubercular drugs inhibited the growth and viability of M. aurum inside the macrophages at different levels. Conclusions: We present a rapid, convenient, high-throughput surrogate model, which provides a comprehensive evaluation platform for new chemical scaffolds against different physiological stages of mycobacteria within the primary cell environment of the host. The results using anti-tubercular drugs validate this model for screening libraries of existing and novel chemical entities.
Novel drugs to treat tuberculosis are required and the identification of potential targets is imp... more Novel drugs to treat tuberculosis are required and the identification of potential targets is important. Piperidinols have been identified as potential antimycobacterial agents (MIC < 5 μg/mL), which also inhibit mycobacterial arylamine N-acetyltransferase (NAT), an enzyme essential for mycobacterial survival inside macrophages. The NAT inhibition involves a prodrug-like mechanism in which activation leads to the formation of bioactive phenyl vinyl ketone (PVK). The PVK fragment selectively forms an adduct with the cysteine residue in the active site. Time dependent inhibition of the NAT enzyme from Mycobacterium marinum (M. marinum) demonstrates a covalent binding mechanism for all inhibitory piperidinol analogues. The structure activity relationship highlights the
This paper examines the courses, which are called "Metropolitan Planning in Istanbul" in Marmara ... more This paper examines the courses, which are called "Metropolitan Planning in Istanbul" in Marmara University, using a Bourdieu's participant objectivation method. These courses are in Global Cities and Istanbul Studies Master's Program in which there is a protocol between Marmara University and Turkish Association of Municipalities. Most of students in this program have been working at different level management in several municipalities. They got a scholarship according to the protocol. The courses are about planning theory and practices. The center of discussion in these courses is the planning agenda of Istanbul. This text discusses the possibilities and the hidden hardships of teaching planning, using the Istanbul Metropolitan Planning course in 2016-2017 academic year, as a case study.
The authors wish to make the following corrections to this paper [...].
Objectives: The aim of this study was to comprehensively evaluate the antibacterial activity and ... more Objectives: The aim of this study was to comprehensively evaluate the antibacterial activity and MurE inhibition of a set of N-methyl-2-alkenyl-4-quinolones found to inhibit the growth of fast-growing mycobacteria. Methods: Using the spot culture growth inhibition assay, MICs were determined for Mycobacterium tuberculosis H37Rv, Mycobacterium bovis BCG and Mycobacterium smegmatis mc 2155. MICs were determined for Mycobac-terium fortuitum, Mycobacterium phlei, methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudo-monas aeruginosa using microplate dilution assays. Inhibition of M. tuberculosis MurE ligase activity was determined both by colorimetric and HPLC methods. Computational modelling and binding prediction of the quinolones in the MurE structure was performed using Glide. Kinetic experiments were conducted for under-standing possible competitive relations of the quinolones with the endogenous substrates of MurE ligase.
ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall... more ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthe...
Antibiotics
Tuberculosis (TB) is still a leading cause of death worldwide. Treatments remain unsatisfactory d... more Tuberculosis (TB) is still a leading cause of death worldwide. Treatments remain unsatisfactory due to an incomplete understanding of the underlying host–pathogen interactions during infection. In the present study, weighted gene co-expression network analysis (WGCNA) was conducted to identify key macrophage modules and hub genes associated with mycobacterial infection. WGCNA was performed combining our own transcriptomic results using Mycobacterium aurum-infected human monocytic macrophages (THP1) with publicly accessible datasets obtained from three types of macrophages infected with seven different mycobacterial strains in various one-to-one combinations. A hierarchical clustering tree of 11,533 genes was built from 198 samples, and 47 distinct modules were revealed. We identified a module, consisting of 226 genes, which represented the common response of host macrophages to different mycobacterial infections that showed significant enrichment in innate immune stimulation, bacter...
Journal of Antimicrobial Chemotherapy, 2020
Background The rise of antimicrobial drug resistance in Mycobacterium tuberculosis coupled with t... more Background The rise of antimicrobial drug resistance in Mycobacterium tuberculosis coupled with the shortage of new antibiotics has elevated TB to a major global health priority. Repurposing drugs developed or used for other conditions has gained special attention in the current scenario of accelerated drug development for several global infectious diseases. In a similar effort, previous studies revealed that carprofen, a non-steroidal anti-inflammatory drug, selectively inhibited the growth of replicating, non-replicating and MDR clinical isolates of M. tuberculosis. Objectives We aimed to reveal the whole-cell phenotypic and transcriptomic effects of carprofen in mycobacteria. Methods Integrative molecular and microbiological approaches such as resazurin microtitre plate assay, high-throughput spot-culture growth inhibition assay, whole-cell efflux inhibition, biofilm inhibition and microarray analyses were performed. Analogues of carprofen were also synthesized and assessed for t...
Molecules
Amaryllidaceae is a significant source of bioactive phytochemicals with a strong propensity to de... more Amaryllidaceae is a significant source of bioactive phytochemicals with a strong propensity to develop new drugs. The genera Allium, Tulbaghia, Cyrtanthus and Crinum biosynthesize novel alkaloids and other phytochemicals with traditional and pharmacological uses. Amaryllidaceae biomolecules exhibit multiple pharmacological activities such as antioxidant, antimicrobial, and immunomodulatory effects. Traditionally, natural products from Amaryllidaceae are utilized to treat non-communicable and infectious human diseases. Galanthamine, a drug from this family, is clinically relevant in treating the neurocognitive disorder, Alzheimer’s disease, which underscores the importance of the Amaryllidaceae alkaloids. Although Amaryllidaceae provide a plethora of biologically active compounds, there is tardiness in their development into clinically pliable medicines. Other genera, including Cyrtanthus and Tulbaghia, have received little attention as potential sources of promising drug candidates....
Antibiotics
The bulbs of Allium species are a known source of antibacterial phytochemicals. Anti-infective, e... more The bulbs of Allium species are a known source of antibacterial phytochemicals. Anti-infective, efflux pump and biofilm inhibitory activities of bulb extracts of selected Ghanaian shallots Allium cepa var aggregatum were evaluated using the HT-SPOTi assay and other whole-cell phenotypic screening techniques to determine their possible mechanisms of action. Ethanol and aqueous extracts of white A. cepa inhibited the growth of Mycobacterium smegmatis mc2 155 and Escherichia coli, respectively. The majority of the Allium extracts significantly (p < 0.05) exhibited efflux pump inhibitory activity against all the acid-fast, Gram-positive and Gram-negative strains used. Hexane and chloroform extract of the pink A. cepa and the aqueous extract of the white A. cepa significantly inhibited M. smegmatis biofilm formation. For Pseudomonas aeruginosa, the inhibition was observed at 250 µg/mL for the aqueous extract (~77.34%) and 125 µg/mL for the hexane extract (~76.51%). The results suggest...
Arylamine N-acetyltransferase (NAT) was first identified in humans for its role in metabolising t... more Arylamine N-acetyltransferase (NAT) was first identified in humans for its role in metabolising the anti-tubercular drug isoniazid. Later functional NAT proteins were identified in bacteria including Mycobacterium tuberculosis where they were found to N-acetylate a range of arylamines, hydrazines and hydralazines both in vitro and in vivo. From gene deletion and genomic studies in mycobacteria, it became clear that NAT in Mycobacterium bovis Bacillus Calmette–Guérin (BCG) had an endogenous role in cell-wall metabolism, cholesterol degradation and the nat gene was part of an essential gene cluster for intra-cellular survival of the pathogen M. tuberculosis; this has paved the way for validation of NAT and other proteins encoded in the gene cluster as novel therapeutic targets for anti-tubercular drug design and discovery. This chapter reviews the genomic organisation of the nat gene and the function of the NAT protein in mycobacteria, including the location of the nat gene in the genome. The role of nat and its neighbouring genes, their endogenous substrates and the regulation of their expression in fast-growing environmental and slow-growing pathogenic intra-cellular mycobacteria is also covered
Fast-growing, non-infectious and intracellularly surviving drug-
Pharmaceuticals, 2021
The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB)... more The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has reinforced the need for the development of new anti-TB drugs. The first line drug isoniazid inhibits InhA. This is a prodrug requiring activation by the enzyme KatG. Mutations in KatG have largely contributed to clinical isoniazid resistance. We aimed to design new ‘direct’ InhA inhibitors that obviate the need for activation by KatG, circumventing pre-existing resistance. In silico molecular modelling was used as part of a rational structure-based drug-design approach involving inspection of protein crystal structures of InhA:inhibitor complexes, including the broad spectrum antibiotic triclosan (TCS). One crystal structure exhibited the unusual presence of two triclosan molecules within the Mycobacterium tuberculosis InhA binding site. This became the basis of a strategy for the synthesis of novel inhibitors. A series of new, flexible ligands were designed and synthesised, expandi...
The Journal of antimicrobial chemotherapy, Jan 4, 2015
Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñ an,... more Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñ an, was found to inhibit the growth of Mycobacterium tuberculosis H 37 Rv. A biomimetic approach for the chemical synthesis of a wide array of 1-substituted tetrahydroisoquinolines was undertaken with the aim of elucidating a common pharmacophore for these compounds with novel mode(s) of anti-TB action. Methods: Biomimetic Pictet-Spengler or Bischler-Napieralski synthetic routes were employed followed by an evaluation of the biological activity of the synthesized compounds. Results: In this work, the synthesized tetrahydroisoquinolines were found to inhibit the growth of M. tuberculosis H 37 Rv and affect its whole-cell phenotype as well as the activity of the ATP-dependent MurE ligase, a key enzyme involved in the early stage of cell wall peptidoglycan biosynthesis. Conclusions: As the correlation between the MIC and the half-inhibitory enzymatic concentration was not particularly strong, there is a credible possibility that these compounds have pleiotropic mechanism(s) of action in M. tuberculosis.
Tuberculosis, 2010
New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan bio... more New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan biosynthesis is a potential therapeutic target. UDP-MurNAc-tripeptide ligase (MurE) is a member of the ATP-dependent ligase family, which incorporate amino acids including meso-diaminopimelic acid (m-DAP) into peptidoglycan during synthesis in a species-specific manner. In the present study, we have cloned, over-expressed, and characterised MurE from M. tuberculosis (Mtb-MurE). The crystal structure has been determined at 3.0A resolution in the presence of the substrate UDP-MurNAc-l-Ala-d-Glu (UAG). The activity of the enzyme was measured through estimating inorganic phosphate released in a non-radioactive high-throughput colourimetric assay. UDP-MurNAc-l-Ala-d-Glu-m-DAP (UMT) formation coupled to inorganic phosphate release was confirmed by HPLC and mass spectrometric analyses. Kinetic constants were determined for a range of natural substrates using optimised conditions. From our findings, it is evident that Mtb-MurE is highly specific in adding m-DAP to UDP-MurNAc-dipeptide and ATP-hydrolysis is an absolute requirement for its activity.
Journal of Antimicrobial Chemotherapy, 2010
New anti-mycobacterial entities with novel mechanisms of action are clinically needed for treatin... more New anti-mycobacterial entities with novel mechanisms of action are clinically needed for treating resistant forms of tuberculosis. The purpose of this study was to evaluate anti-tubercular activity and selectivity of seven recently isolated natural products from Colombian plants. Methods: MICs were determined using a liquid medium growth inhibition assay for Mycobacterium tuberculosis H 37 Rv and both solid and liquid media growth inhibition assays for Mycobacterium bovis BCG. Escherichia coli growth inhibition and mammalian macrophage cell toxicity were evaluated to establish the degree of selectivity of the natural product against whole cell organisms. Enzymatic inhibition of ATP-dependent MurE ligase from M. tuberculosis was assayed using a colorimetric phosphate detection method. The most active compound, 3-methoxynordomesticine hydrochloride, was further investigated on M. bovis BCG for its inhibition of sigmoidal growth, acid-fast staining and viability counting analysis. Results: Aporphine alkaloids were found to be potent inhibitors of slow-growing mycobacterial pathogens showing favourable selectivity and cytotoxicity. In terms of their endogenous action, the aporphine alkaloids were found inhibitory to M. tuberculosis ATP-dependent MurE ligase at micromolar concentrations. A significantly low MIC was detected for 3-methoxynordomesticine hydrochloride against both M. bovis BCG and M. tuberculosis H 37 Rv. Conclusions: Considering all the data, 3-methoxynordomesticine hydrochloride was found to be a potent antitubercular compound with a favourable specificity profile. The alkaloid showed MurE inhibition and is considered an initial hit for exploring related chemical space.
Current Eye Research, 1995
Nitroreductase is a 24 KDa flavoprotein from E.coli. It has potential therapeutic use as an anti-... more Nitroreductase is a 24 KDa flavoprotein from E.coli. It has potential therapeutic use as an anti-cancer enzyme in VDEPT (Virus Directed Enzyme Prodrug Therapy). Nitroreductase is very unusual in being able to use both N A D H and NADPH in the reduction of a broad range of substrates. The enzyme has been purified to near homogeniety and subjected to a series of crystallization trials. Crystals have been obtained. Preliminary characterisation shows them to be tetragonal (P41212, unit cell 57,57,262 Angstroms, 90" 90" 90"). Recent progress will be reported.
Journal of Antimicrobial Chemotherapy, 2012
The intracellularly surviving and slow-growing pathogen, Mycobacterium tuberculosis, adapts the h... more The intracellularly surviving and slow-growing pathogen, Mycobacterium tuberculosis, adapts the host cell environment for its active and dormant life cycle. It is evident that the lack of appropriate highthroughput screening of inhibitors within host cells is an impediment for the early stages of anti-tubercular drug discovery. We aimed to develop an integrated surrogate model that enhances the screening of large inhibitor libraries. Methods: Different mycobacterial species were compared for their growth, drug susceptibility and intracellular uptake. A 6-well plate solid agar-based spot culture growth inhibition (SPOTi) assay was developed into a higher throughput format. The uptake and intracellular survival of Mycobacterium aurum within mouse macrophage cells (RAW 264.7) were optimized using 24/96-well plate formats. Results: Fast-growing, non-pathogenic M. aurum was found to have an antibiotic-susceptibility profile similar to that of M. tuberculosis. The sensitivity to an acidic pH environment and the ability to multiply inside RAW 264.7 macrophages provided additional advantages for employing M. aurum in intracellular drug screening methods. A selection of anti-tubercular drugs inhibited the growth and viability of M. aurum inside the macrophages at different levels. Conclusions: We present a rapid, convenient, high-throughput surrogate model, which provides a comprehensive evaluation platform for new chemical scaffolds against different physiological stages of mycobacteria within the primary cell environment of the host. The results using anti-tubercular drugs validate this model for screening libraries of existing and novel chemical entities.
Novel drugs to treat tuberculosis are required and the identification of potential targets is imp... more Novel drugs to treat tuberculosis are required and the identification of potential targets is important. Piperidinols have been identified as potential antimycobacterial agents (MIC < 5 μg/mL), which also inhibit mycobacterial arylamine N-acetyltransferase (NAT), an enzyme essential for mycobacterial survival inside macrophages. The NAT inhibition involves a prodrug-like mechanism in which activation leads to the formation of bioactive phenyl vinyl ketone (PVK). The PVK fragment selectively forms an adduct with the cysteine residue in the active site. Time dependent inhibition of the NAT enzyme from Mycobacterium marinum (M. marinum) demonstrates a covalent binding mechanism for all inhibitory piperidinol analogues. The structure activity relationship highlights the
This paper examines the courses, which are called "Metropolitan Planning in Istanbul" in Marmara ... more This paper examines the courses, which are called "Metropolitan Planning in Istanbul" in Marmara University, using a Bourdieu's participant objectivation method. These courses are in Global Cities and Istanbul Studies Master's Program in which there is a protocol between Marmara University and Turkish Association of Municipalities. Most of students in this program have been working at different level management in several municipalities. They got a scholarship according to the protocol. The courses are about planning theory and practices. The center of discussion in these courses is the planning agenda of Istanbul. This text discusses the possibilities and the hidden hardships of teaching planning, using the Istanbul Metropolitan Planning course in 2016-2017 academic year, as a case study.
The authors wish to make the following corrections to this paper [...].
Objectives: The aim of this study was to comprehensively evaluate the antibacterial activity and ... more Objectives: The aim of this study was to comprehensively evaluate the antibacterial activity and MurE inhibition of a set of N-methyl-2-alkenyl-4-quinolones found to inhibit the growth of fast-growing mycobacteria. Methods: Using the spot culture growth inhibition assay, MICs were determined for Mycobacterium tuberculosis H37Rv, Mycobacterium bovis BCG and Mycobacterium smegmatis mc 2155. MICs were determined for Mycobac-terium fortuitum, Mycobacterium phlei, methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudo-monas aeruginosa using microplate dilution assays. Inhibition of M. tuberculosis MurE ligase activity was determined both by colorimetric and HPLC methods. Computational modelling and binding prediction of the quinolones in the MurE structure was performed using Glide. Kinetic experiments were conducted for under-standing possible competitive relations of the quinolones with the endogenous substrates of MurE ligase.
ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall... more ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthe...
Antibiotics
Tuberculosis (TB) is still a leading cause of death worldwide. Treatments remain unsatisfactory d... more Tuberculosis (TB) is still a leading cause of death worldwide. Treatments remain unsatisfactory due to an incomplete understanding of the underlying host–pathogen interactions during infection. In the present study, weighted gene co-expression network analysis (WGCNA) was conducted to identify key macrophage modules and hub genes associated with mycobacterial infection. WGCNA was performed combining our own transcriptomic results using Mycobacterium aurum-infected human monocytic macrophages (THP1) with publicly accessible datasets obtained from three types of macrophages infected with seven different mycobacterial strains in various one-to-one combinations. A hierarchical clustering tree of 11,533 genes was built from 198 samples, and 47 distinct modules were revealed. We identified a module, consisting of 226 genes, which represented the common response of host macrophages to different mycobacterial infections that showed significant enrichment in innate immune stimulation, bacter...
Journal of Antimicrobial Chemotherapy, 2020
Background The rise of antimicrobial drug resistance in Mycobacterium tuberculosis coupled with t... more Background The rise of antimicrobial drug resistance in Mycobacterium tuberculosis coupled with the shortage of new antibiotics has elevated TB to a major global health priority. Repurposing drugs developed or used for other conditions has gained special attention in the current scenario of accelerated drug development for several global infectious diseases. In a similar effort, previous studies revealed that carprofen, a non-steroidal anti-inflammatory drug, selectively inhibited the growth of replicating, non-replicating and MDR clinical isolates of M. tuberculosis. Objectives We aimed to reveal the whole-cell phenotypic and transcriptomic effects of carprofen in mycobacteria. Methods Integrative molecular and microbiological approaches such as resazurin microtitre plate assay, high-throughput spot-culture growth inhibition assay, whole-cell efflux inhibition, biofilm inhibition and microarray analyses were performed. Analogues of carprofen were also synthesized and assessed for t...