Synthesis and evaluation of novel inhibitors of 1-Deoxy-D-xylolose-5-phosphate reductoisomerase as potential antimalarials
- Authors: Conibear, Anne Claire
- Date: 2013-07-19
- Subjects: Antimalarials -- Development , Malaria -- Chemotherapy , Drug development , Enzyme kinetics , Phosphate esters
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4451 , http://hdl.handle.net/10962/d1008282 , Antimalarials -- Development , Malaria -- Chemotherapy , Drug development , Enzyme kinetics , Phosphate esters
- Description: Malaria continues to be an enormous health-threat in the developing world and the emergence of drug resistance has further compounded the problem. The parasite-specific enzyme, 1-deoxY-D-xylulose-S-phosphate reductoisomerase (DXR), has recently been validated as a promising antimalarial drug target. The present study comprises a combination of synthetic, physical organic, computer modelling and bioassay techniques directed towards the development of novel DXR inhibitors. A range of 2-heteroarylamino-2-oxoethyl- and 2- heteroarylamino-2-oxopropyl phosphonate esters and their corresponding phosphonic acid salts have been synthesised as analogues of the highly active DXR inhibitor, fosmidomycin. Treatment of the heteroarylamino precursors with chloroacetyl chloride or chloropropionyl chloride afforded chloroamide intermediates, Arbuzov reactions of which led to the corresponding diethyl phosphonate esters. Hydrolysis of the esters has been effected using bromotrimethylsilane. Twenty-four new compounds have been prepared and fully characterised using elemental (HRMS or combustion) and spectroscopic (1- and 2-D NMR and IR) analysis. A 31p NMR kinetic study has been carried out on the two-step silylation reaction involved in the hydrolysis of the phosphonate esters and has provided activation parameters for the reaction. The kinetic analysis was refined using a computational method to give an improved fit with the experimental data. Saturation transfer difference (STD) NMR analysis, computer-simulated docking and enzyme inhibition assays have been used to evaluate the enzyme-binding and -inhibition potential of the synthesised ligands. Minimal to moderate inhibitory activity has been observed and several structure-activity relationships have been identified. In silica exploration of the DXR active site has revealed an additional binding pocket and information on the topology of the active site has led to the de novo design of a new series of potential ligands. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Authors: Conibear, Anne Claire
- Date: 2013-07-19
- Subjects: Antimalarials -- Development , Malaria -- Chemotherapy , Drug development , Enzyme kinetics , Phosphate esters
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4451 , http://hdl.handle.net/10962/d1008282 , Antimalarials -- Development , Malaria -- Chemotherapy , Drug development , Enzyme kinetics , Phosphate esters
- Description: Malaria continues to be an enormous health-threat in the developing world and the emergence of drug resistance has further compounded the problem. The parasite-specific enzyme, 1-deoxY-D-xylulose-S-phosphate reductoisomerase (DXR), has recently been validated as a promising antimalarial drug target. The present study comprises a combination of synthetic, physical organic, computer modelling and bioassay techniques directed towards the development of novel DXR inhibitors. A range of 2-heteroarylamino-2-oxoethyl- and 2- heteroarylamino-2-oxopropyl phosphonate esters and their corresponding phosphonic acid salts have been synthesised as analogues of the highly active DXR inhibitor, fosmidomycin. Treatment of the heteroarylamino precursors with chloroacetyl chloride or chloropropionyl chloride afforded chloroamide intermediates, Arbuzov reactions of which led to the corresponding diethyl phosphonate esters. Hydrolysis of the esters has been effected using bromotrimethylsilane. Twenty-four new compounds have been prepared and fully characterised using elemental (HRMS or combustion) and spectroscopic (1- and 2-D NMR and IR) analysis. A 31p NMR kinetic study has been carried out on the two-step silylation reaction involved in the hydrolysis of the phosphonate esters and has provided activation parameters for the reaction. The kinetic analysis was refined using a computational method to give an improved fit with the experimental data. Saturation transfer difference (STD) NMR analysis, computer-simulated docking and enzyme inhibition assays have been used to evaluate the enzyme-binding and -inhibition potential of the synthesised ligands. Minimal to moderate inhibitory activity has been observed and several structure-activity relationships have been identified. In silica exploration of the DXR active site has revealed an additional binding pocket and information on the topology of the active site has led to the de novo design of a new series of potential ligands. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
Thiazole derivatives as potential hiv-1 protease inhibitors
- Authors: Hlongwe, Zola
- Date: 2021-04
- Subjects: Gqeberha (South Africa) , Eastern Cape (South Africa) , Enzyme kinetics
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/52116 , vital:43427
- Description: Series of compounds were screened using Schrodinger suite (Maestro). The DFT calculations were used for geometry optimization of the ligands using the B3YLYP functional and 6-31G basis set, and these structures were used for docking studies. Maestro was used to predict the activity of thiazole derivatives against HIV-1 protease. The range of estimated inhibition constants for these thiazole derivatives (65 nM-5 μM) indicate moderate to weak activity against HIV-1 protease, given that the activity of current protease inhibitors is typically found have experimental inhibition constants around 0.1-2.0 nM. Twenty compounds were selected based on the docking results and they were synthesized and characterized by NMR, FT-IR and elemental analysis. The cytotoxicity studies were done at two different concentrations (100 μM and 10 μM), using the brine shrimp bioassay. All compounds were highly toxic at 100 μM, with the percentage mortality between 20 to 75%. Eight compounds were selected for the enzyme bioassay based on the results obtained from lower concentration (10 μM). In the enzyme inhibition studies, the profile of HIV-1 activity was done at different inhibitor concentrations (800 μM – 10 μM) by measuring the cleavage of the synthetic substrate (Abz-Thr-lle-PNO2Phe=Gln-Arg-NH2) at excitation wavelength of 345/490 nm using fluorescence. Ligands 5 (unsubstituted derivative), 7 (4-nitro derivative) and 16 (4-methoxy derivative) gave percentage inhibition of 39, 45 and 42%, and this activity was very low compared to the activity of the positive control ritonavir (85% enzyme inhibition). Ligands 8 (4-methoxy derivative) and 12 (4-methoxy derivative) gave enzyme inhibition of 70% and 75%. These results suggest that the presence of the methoxy substituents ii increases activity of these compounds against HIV-1 protease. Most of the compounds gave good IC50 values between 12.5-42.7 nM. The bromo-substituted ligand 7 gave the lowest IC50 (12.5 nM). Ligand 11 also gave a good IC50 value of 14.86 nM. The bromo-substituted derivatives showed to be very active compared to other types of thiazole derivatives. Enzyme kinetics were carried out to compare the inhibition constants obtained via computational modelling. Ligand 7 (4-methoxy derivatives) binds better in the active site of HIV-1 protease than other compounds in Class B, with Ki = 50 nM, Km = 23.8 Nm and Vmax = 83.3 nM/min. The unsubstituted (L5), 4-bromo (L7) and 4-nitro (L8) substituted compounds gave inhibition constants of 100 to 112 nM. The in vitro testing yielded higher activity than that determined in silico. , Thesis (MSc) -- Faculty of Science, School of Biomolecular and Chemical Sciences, 2021
- Full Text: false
- Date Issued: 2021-04
- Authors: Hlongwe, Zola
- Date: 2021-04
- Subjects: Gqeberha (South Africa) , Eastern Cape (South Africa) , Enzyme kinetics
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/52116 , vital:43427
- Description: Series of compounds were screened using Schrodinger suite (Maestro). The DFT calculations were used for geometry optimization of the ligands using the B3YLYP functional and 6-31G basis set, and these structures were used for docking studies. Maestro was used to predict the activity of thiazole derivatives against HIV-1 protease. The range of estimated inhibition constants for these thiazole derivatives (65 nM-5 μM) indicate moderate to weak activity against HIV-1 protease, given that the activity of current protease inhibitors is typically found have experimental inhibition constants around 0.1-2.0 nM. Twenty compounds were selected based on the docking results and they were synthesized and characterized by NMR, FT-IR and elemental analysis. The cytotoxicity studies were done at two different concentrations (100 μM and 10 μM), using the brine shrimp bioassay. All compounds were highly toxic at 100 μM, with the percentage mortality between 20 to 75%. Eight compounds were selected for the enzyme bioassay based on the results obtained from lower concentration (10 μM). In the enzyme inhibition studies, the profile of HIV-1 activity was done at different inhibitor concentrations (800 μM – 10 μM) by measuring the cleavage of the synthetic substrate (Abz-Thr-lle-PNO2Phe=Gln-Arg-NH2) at excitation wavelength of 345/490 nm using fluorescence. Ligands 5 (unsubstituted derivative), 7 (4-nitro derivative) and 16 (4-methoxy derivative) gave percentage inhibition of 39, 45 and 42%, and this activity was very low compared to the activity of the positive control ritonavir (85% enzyme inhibition). Ligands 8 (4-methoxy derivative) and 12 (4-methoxy derivative) gave enzyme inhibition of 70% and 75%. These results suggest that the presence of the methoxy substituents ii increases activity of these compounds against HIV-1 protease. Most of the compounds gave good IC50 values between 12.5-42.7 nM. The bromo-substituted ligand 7 gave the lowest IC50 (12.5 nM). Ligand 11 also gave a good IC50 value of 14.86 nM. The bromo-substituted derivatives showed to be very active compared to other types of thiazole derivatives. Enzyme kinetics were carried out to compare the inhibition constants obtained via computational modelling. Ligand 7 (4-methoxy derivatives) binds better in the active site of HIV-1 protease than other compounds in Class B, with Ki = 50 nM, Km = 23.8 Nm and Vmax = 83.3 nM/min. The unsubstituted (L5), 4-bromo (L7) and 4-nitro (L8) substituted compounds gave inhibition constants of 100 to 112 nM. The in vitro testing yielded higher activity than that determined in silico. , Thesis (MSc) -- Faculty of Science, School of Biomolecular and Chemical Sciences, 2021
- Full Text: false
- Date Issued: 2021-04
Synthesis and interaction of secondary N-nitrosamines with acetylcholinesterase
- Authors: Mmutle, Tsietso Bernard
- Date: 1991
- Subjects: Chemistry, Physical and theoretical , Enzyme kinetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4058 , http://hdl.handle.net/10962/d1004119 , Chemistry, Physical and theoretical , Enzyme kinetics
- Description: Secondary N-nitrosamines: diphenylnitrosamine (DPhNA), dimethylnitrosamine (DMNA), diethylnitrosamine (DENA), dipropylnitrosamine (DPNA), dibutylnitrosamine (DBNA), diethanolnitrosamine (DEtNA), methylnitrosoglycine (MNGly), nitrosopyrrolidine (NPyr), nitrosomorpholine (NMor) and nitrosopiperidine (NPip) were synthesised and their interaction with acetylcholinesterase (AChE) was investigated. Analyses of kinetic results show that DMNA (Ki=34.78 μM); DENA (Ki=54.24 μM); DPNA(Ki=60.36 μM); DBNA(Ki=95.54 μM); DEtNA(Ki=43.68 μM)MNGly (Ki=30.18 μM); NPip (Ki=123 μM); NPyr (Ki=66.07 μM), NMor (Ki=73.93 μM) and DPhNA (Ki=20.32 μM) are competitive and reversible inhibitors of acetylcholinesterase, with respect to the substrate, acetylthiocholine chloride, ATChCl. With time they act as irreversible covalent inhibitors with dipropy1nitrosamine producing 72% inactivation after 60 minutes. Scatchard analyses of f1uorometric titrations, (Kd=0.75mM-4.09mM); gel chromatography (Kd=O. 80mM-4. 60mM) and equilibrium dia1ysis (Kd=O. 71mM- 4.21mM) for MNG1y, DMNA, DEtNA, DENA, DPNA, NPyr, DSNA, NMor and NPip show that these compounds have weaker affinity for the enzyme, as compared to the much tightly binding aromatic DPhNA, Kd values (0.65mM, 0.68mM and 0.68mM) for fluorometric experiments, gel chromatography and equilibrium dialysis respectively. In all cases, the number of binding sites of acetylcholinesterase averaged to four.
- Full Text:
- Date Issued: 1991
- Authors: Mmutle, Tsietso Bernard
- Date: 1991
- Subjects: Chemistry, Physical and theoretical , Enzyme kinetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4058 , http://hdl.handle.net/10962/d1004119 , Chemistry, Physical and theoretical , Enzyme kinetics
- Description: Secondary N-nitrosamines: diphenylnitrosamine (DPhNA), dimethylnitrosamine (DMNA), diethylnitrosamine (DENA), dipropylnitrosamine (DPNA), dibutylnitrosamine (DBNA), diethanolnitrosamine (DEtNA), methylnitrosoglycine (MNGly), nitrosopyrrolidine (NPyr), nitrosomorpholine (NMor) and nitrosopiperidine (NPip) were synthesised and their interaction with acetylcholinesterase (AChE) was investigated. Analyses of kinetic results show that DMNA (Ki=34.78 μM); DENA (Ki=54.24 μM); DPNA(Ki=60.36 μM); DBNA(Ki=95.54 μM); DEtNA(Ki=43.68 μM)MNGly (Ki=30.18 μM); NPip (Ki=123 μM); NPyr (Ki=66.07 μM), NMor (Ki=73.93 μM) and DPhNA (Ki=20.32 μM) are competitive and reversible inhibitors of acetylcholinesterase, with respect to the substrate, acetylthiocholine chloride, ATChCl. With time they act as irreversible covalent inhibitors with dipropy1nitrosamine producing 72% inactivation after 60 minutes. Scatchard analyses of f1uorometric titrations, (Kd=0.75mM-4.09mM); gel chromatography (Kd=O. 80mM-4. 60mM) and equilibrium dia1ysis (Kd=O. 71mM- 4.21mM) for MNG1y, DMNA, DEtNA, DENA, DPNA, NPyr, DSNA, NMor and NPip show that these compounds have weaker affinity for the enzyme, as compared to the much tightly binding aromatic DPhNA, Kd values (0.65mM, 0.68mM and 0.68mM) for fluorometric experiments, gel chromatography and equilibrium dialysis respectively. In all cases, the number of binding sites of acetylcholinesterase averaged to four.
- Full Text:
- Date Issued: 1991
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