Analysis of bacterial Mur amide ligase enzymes for the identification of inhibitory compounds by in silico methods
- Chamboko, Chiratidzo Respina
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Pathogenic microorganisms -- Analysis , Drug resistance in microorganisms , Microorganisms -- Effect of drugs on , Antibiotics -- Effectiveness , Pathogenic bacteria , Drug tolerance , Enzymes -- Analysis , Peptide antibiotics
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/161911 , vital:40690
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Pathogenic microorganisms -- Analysis , Drug resistance in microorganisms , Microorganisms -- Effect of drugs on , Antibiotics -- Effectiveness , Pathogenic bacteria , Drug tolerance , Enzymes -- Analysis , Peptide antibiotics
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/161911 , vital:40690
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
Antibacterial activity of liposome encapsulated cyclo(TYR-PRO)
- Authors: Tshanga, Siphokazi Sisanda
- Date: 2011
- Subjects: Peptide antibiotics , Antibacterial agents -- Therapeutic use -- Testing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10132 , http://hdl.handle.net/10948/1450 , Peptide antibiotics , Antibacterial agents -- Therapeutic use -- Testing
- Description: Cyclic dipeptides (CDPs) are amino acid-based compounds, some of which possess antibacterial activity. The encapsulation of certain drugs into liposomes has been found to improve their activity in terms of bioavailability and duration of action. Liposomes are small vesicles that are under investigation as drug carriers for the delivery of therapeutic agents. A number of liposome formulations are currently under clinical trial review, whilst some have already been approved for clinical use. The aim of this study was to optimize a liposomal cyclo(Tyr-Pro) formulation and to assess its antibacterial activity against various Gram-positive and Gram-negative bacteria. Response surface methodology (RSM) using the central composite design (CCD) model was used to optimize liposomal formulations of cyclo(Tyr-Pro) for each of the four bacteria, namely Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Percent drug encapsulated and bacterial inhibition were investigated with respect to two independent variables, i.e. lipid composition and cholesterol content. Design Expert 8 was used for the purpose of finding the combination of independent variables that would yield an optimal formulation for each bacterium. The model selected by the software failed to adequately correlate the predicted models to the experimental data. The in vitro experiments showed that the antibacterial activity of liposome-encapsulated cyclo(Tyr-Pro) was superior to that of its free counterpart. Binding maximum or Bmax for the encapsulated compound at concentrations as low as 0.412 mg/ml, was significantly higher than that obtained for free cyclo(Tyr-Pro) which was tested at a concentration of 20 mg/ml. Furthermore, encapsulation of cyclo(Tyr-Pro) into a liposome formulation enhanced its potency. This was evident in the lower IC50 values for the liposomal compound when compared to free cyclo(Tyr-Pro).
- Full Text:
- Date Issued: 2011
- Authors: Tshanga, Siphokazi Sisanda
- Date: 2011
- Subjects: Peptide antibiotics , Antibacterial agents -- Therapeutic use -- Testing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10132 , http://hdl.handle.net/10948/1450 , Peptide antibiotics , Antibacterial agents -- Therapeutic use -- Testing
- Description: Cyclic dipeptides (CDPs) are amino acid-based compounds, some of which possess antibacterial activity. The encapsulation of certain drugs into liposomes has been found to improve their activity in terms of bioavailability and duration of action. Liposomes are small vesicles that are under investigation as drug carriers for the delivery of therapeutic agents. A number of liposome formulations are currently under clinical trial review, whilst some have already been approved for clinical use. The aim of this study was to optimize a liposomal cyclo(Tyr-Pro) formulation and to assess its antibacterial activity against various Gram-positive and Gram-negative bacteria. Response surface methodology (RSM) using the central composite design (CCD) model was used to optimize liposomal formulations of cyclo(Tyr-Pro) for each of the four bacteria, namely Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Percent drug encapsulated and bacterial inhibition were investigated with respect to two independent variables, i.e. lipid composition and cholesterol content. Design Expert 8 was used for the purpose of finding the combination of independent variables that would yield an optimal formulation for each bacterium. The model selected by the software failed to adequately correlate the predicted models to the experimental data. The in vitro experiments showed that the antibacterial activity of liposome-encapsulated cyclo(Tyr-Pro) was superior to that of its free counterpart. Binding maximum or Bmax for the encapsulated compound at concentrations as low as 0.412 mg/ml, was significantly higher than that obtained for free cyclo(Tyr-Pro) which was tested at a concentration of 20 mg/ml. Furthermore, encapsulation of cyclo(Tyr-Pro) into a liposome formulation enhanced its potency. This was evident in the lower IC50 values for the liposomal compound when compared to free cyclo(Tyr-Pro).
- Full Text:
- Date Issued: 2011
Development and testing of liposome encapsulated cyclic dipeptides
- Authors: Kilian, Gareth
- Date: 2011
- Subjects: Peptide antibiotics , Peptide drugs -- Therapeutic use , Peptides -- Synthesis , Antibacterial agents -- Therapeutic use -- Testing , Cyclic peptides , Liposomes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10136 , http://hdl.handle.net/10948/1397 , Peptide antibiotics , Peptide drugs -- Therapeutic use , Peptides -- Synthesis , Antibacterial agents -- Therapeutic use -- Testing , Cyclic peptides , Liposomes
- Description: Cyclic dipeptides have been well characterized for their multitude of biological activities, including antimicrobial and anticancer activities. Cyclo(His-Gly) and cyclo(His-Ala) have also recently been shown to possess significant anticancer activity against a range of cell lines, despite the limitations of these two molecules with respect to their physicochemical properties. Low Log P results in poor cell permeability which can often be problematic for drugs with intracellular mechanisms of action. It can also results in poor biodistribution, and theoretical Log P values for cyclo(His-Gly) and cyclo(His-Ala) were extremely low making them ideal candidates for inclusion into a nanoparticulate drug delivery system. The aim of this study was therefore to formulate and evaluate liposome-encapsulated cyclic dipeptides that increase the tumour-suppressive actions of the cyclic dipeptides, while showing a high degree of specificity for tumour cells. While liposomes are relatively simple to prepare, inter batch variation, low encapsulation and poor stability are often problematic in their production and this has lead to very few liposomal products on the market. This study aimed at using a comprehensive statistical methodology in optimizing liposome formulations encapsulating cyclo(His-Gly) and cyclo(His-Ala). Initial screening of potential factors was conducted using a 25-1 fractional factorial design. This design made use of two levels for each of the five factors and abbreviated the design to minimize runs. Although not much information is provided by these types of designs, the design was sufficient in identifying two critical factors that would be studies further in a more robust design. The two factors selected, based on the screening study, were cholesterol and stearylamine content. These two factors were then used in designing a response surface methodology (RSM) design making use of a central composite rotatable vii design (CCRD) at five levels (-1.5, -1, 0, 1, 1.5) for each factor in order to better understand the design space. Various factors influenced the measured responses of encapsulation efficiency, zeta potential, polydispersity index, cellular uptake and leakage, but most notable were the adverse effects of increasing stearylamine levels on encapsulations efficiency and cholesterol levels on leakage for both cyclo(His-Gly) and cyclo(His-Ala) liposomes. Optimized formulations were derived from the data and prepared. Fair correlation between the predicted and measured responses was obtained. The cytotoxic activity of the encapsulated cyclic dipeptides were assessed against HeLa and MCF-7 cells and found to have limited improvement in activity. However, modification of the polyethylene glycol (PEG) grafted to the liposome surface in order to target folate receptors showed good benefit in significantly decreasing the IC50 values recorded in all cells lines tested, particularly low folate HeLa cells with the lowest IC50 being recorded as 0.0962 mM for folate targeted cyclo(His-Ala). The results therefore indicate that hydrophilic cyclic dipeptides are ideal candidates for inclusion into targeted drug delivery systems such as liposomes. Key words: Liposomes, cyclo(His-Gly), cyclo(His-Ala), cyclic dipeptides, HeLa, MCF-7, folate receptors, factorial design, response surface methodology (RSM), central composite rotatable design (CCRD).
- Full Text:
- Date Issued: 2011
- Authors: Kilian, Gareth
- Date: 2011
- Subjects: Peptide antibiotics , Peptide drugs -- Therapeutic use , Peptides -- Synthesis , Antibacterial agents -- Therapeutic use -- Testing , Cyclic peptides , Liposomes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10136 , http://hdl.handle.net/10948/1397 , Peptide antibiotics , Peptide drugs -- Therapeutic use , Peptides -- Synthesis , Antibacterial agents -- Therapeutic use -- Testing , Cyclic peptides , Liposomes
- Description: Cyclic dipeptides have been well characterized for their multitude of biological activities, including antimicrobial and anticancer activities. Cyclo(His-Gly) and cyclo(His-Ala) have also recently been shown to possess significant anticancer activity against a range of cell lines, despite the limitations of these two molecules with respect to their physicochemical properties. Low Log P results in poor cell permeability which can often be problematic for drugs with intracellular mechanisms of action. It can also results in poor biodistribution, and theoretical Log P values for cyclo(His-Gly) and cyclo(His-Ala) were extremely low making them ideal candidates for inclusion into a nanoparticulate drug delivery system. The aim of this study was therefore to formulate and evaluate liposome-encapsulated cyclic dipeptides that increase the tumour-suppressive actions of the cyclic dipeptides, while showing a high degree of specificity for tumour cells. While liposomes are relatively simple to prepare, inter batch variation, low encapsulation and poor stability are often problematic in their production and this has lead to very few liposomal products on the market. This study aimed at using a comprehensive statistical methodology in optimizing liposome formulations encapsulating cyclo(His-Gly) and cyclo(His-Ala). Initial screening of potential factors was conducted using a 25-1 fractional factorial design. This design made use of two levels for each of the five factors and abbreviated the design to minimize runs. Although not much information is provided by these types of designs, the design was sufficient in identifying two critical factors that would be studies further in a more robust design. The two factors selected, based on the screening study, were cholesterol and stearylamine content. These two factors were then used in designing a response surface methodology (RSM) design making use of a central composite rotatable vii design (CCRD) at five levels (-1.5, -1, 0, 1, 1.5) for each factor in order to better understand the design space. Various factors influenced the measured responses of encapsulation efficiency, zeta potential, polydispersity index, cellular uptake and leakage, but most notable were the adverse effects of increasing stearylamine levels on encapsulations efficiency and cholesterol levels on leakage for both cyclo(His-Gly) and cyclo(His-Ala) liposomes. Optimized formulations were derived from the data and prepared. Fair correlation between the predicted and measured responses was obtained. The cytotoxic activity of the encapsulated cyclic dipeptides were assessed against HeLa and MCF-7 cells and found to have limited improvement in activity. However, modification of the polyethylene glycol (PEG) grafted to the liposome surface in order to target folate receptors showed good benefit in significantly decreasing the IC50 values recorded in all cells lines tested, particularly low folate HeLa cells with the lowest IC50 being recorded as 0.0962 mM for folate targeted cyclo(His-Ala). The results therefore indicate that hydrophilic cyclic dipeptides are ideal candidates for inclusion into targeted drug delivery systems such as liposomes. Key words: Liposomes, cyclo(His-Gly), cyclo(His-Ala), cyclic dipeptides, HeLa, MCF-7, folate receptors, factorial design, response surface methodology (RSM), central composite rotatable design (CCRD).
- Full Text:
- Date Issued: 2011
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