An in-silico investigation of Morita-Baylis-Hillman accessible heterocyclic analogues for applications as novel HIV-1 C protease inhibitors
- Authors: Sigauke, Lester Takunda
- Date: 2015
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , HIV infections , Drug resistance , Cheminformatics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4152 , http://hdl.handle.net/10962/d1017913
- Description: Cheminformatic approaches have been employed to optimize the bis-coumarin scaffold identified by Onywera et al. (2012) as a potential hit against the protease HIV-1 protein. The Open Babel library of commands was used to access functions that were incorporated into a markov chain recursive program that generated 17750 analogues of the bis-coumarin scaffold. The Morita-Baylis-Hillman accessible heterocycles were used to introduce structural diversity within the virtual library. In silico high through-put virtual screening using AutoDock Vina was used to rapidly screen the virtual library ligand set against 61 protease models built by Onywera et al. (2012). CheS-Mapper computed a principle component analysis of the compounds based on 13 selected chemical descriptors. The compounds were plotted against the principle component analysis within a 3 dimensional chemical space in order to inspect the diversity of the virtual library. The physicochemical properties and binding affinities were used to identify the top 3 performing ligands. ACPYPE was used to inspect the constitutional properties and eliminated virtual compounds that possessed open valences. Chromene based ligand 805 and ligand 6610 were selected as the lead candidates from the high-throughput virtual screening procedure we employed. Molecular dynamic simulations of the lead candidates performed for 5 ns allowed the stability of the ligand protein complexes with protease model 305152. The free energy of binding of the leads with protease model 305152 was computed over the first 50 ps of simulation using the molecular mechanics Poisson-Boltzmann method. Analysis structural features and energy profiles from molecular dynamic simulations of the protein–ligand complexes indicated that although ligand 805 had a weaker binding affinity in terms of docking, it outperformed ligand 6610 in terms of complex stability and free energy of binding. Medicinal chemistry approaches will be used to optimize the lead candidates before their analogues will be synthesized and assayed for in vivo protease activity.
- Full Text:
- Date Issued: 2015
- Authors: Sigauke, Lester Takunda
- Date: 2015
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , HIV infections , Drug resistance , Cheminformatics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4152 , http://hdl.handle.net/10962/d1017913
- Description: Cheminformatic approaches have been employed to optimize the bis-coumarin scaffold identified by Onywera et al. (2012) as a potential hit against the protease HIV-1 protein. The Open Babel library of commands was used to access functions that were incorporated into a markov chain recursive program that generated 17750 analogues of the bis-coumarin scaffold. The Morita-Baylis-Hillman accessible heterocycles were used to introduce structural diversity within the virtual library. In silico high through-put virtual screening using AutoDock Vina was used to rapidly screen the virtual library ligand set against 61 protease models built by Onywera et al. (2012). CheS-Mapper computed a principle component analysis of the compounds based on 13 selected chemical descriptors. The compounds were plotted against the principle component analysis within a 3 dimensional chemical space in order to inspect the diversity of the virtual library. The physicochemical properties and binding affinities were used to identify the top 3 performing ligands. ACPYPE was used to inspect the constitutional properties and eliminated virtual compounds that possessed open valences. Chromene based ligand 805 and ligand 6610 were selected as the lead candidates from the high-throughput virtual screening procedure we employed. Molecular dynamic simulations of the lead candidates performed for 5 ns allowed the stability of the ligand protein complexes with protease model 305152. The free energy of binding of the leads with protease model 305152 was computed over the first 50 ps of simulation using the molecular mechanics Poisson-Boltzmann method. Analysis structural features and energy profiles from molecular dynamic simulations of the protein–ligand complexes indicated that although ligand 805 had a weaker binding affinity in terms of docking, it outperformed ligand 6610 in terms of complex stability and free energy of binding. Medicinal chemistry approaches will be used to optimize the lead candidates before their analogues will be synthesized and assayed for in vivo protease activity.
- Full Text:
- Date Issued: 2015
Ostrich calpastatin purification and partial characterization of the liver inhibitor
- Authors: Roman, Henry James
- Date: 2000
- Subjects: Calpastatin , Protease inhibitors , Ion exchange chromatography , Ostriches
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11090 , http://hdl.handle.net/10948/d1015522
- Description: The isolation and purification of calpastatin from ostrich liver is presented, along with its physicochemical and kinetic properties. By using extraction from liver, ion-exchange chromatography on DEAE-Toyopearl, heating to 90 °C for 10 min and rechromatography on Toyopearl Super-Q 650 S, ostrich calpastatin was isolated and purified from ostrich liver. The purified intact calpastatin showed homogeneity on SDS-PAGE (Mr of 105.6 K). Amino acid analysis showed that ostrich calpastatin resembled that of rabbit liver and human erythrocyte calpastatin. An N-terminal sequence could not be obtained because the N-terminus was found to be blocked by an as yet unknown amino acid residue. The Mr values of degradative forms of ostrich liver calpastatin were determined to be 56 K and 90 K. By using PAG-IEF the pI of the intact form was determined to be 5.1. Ostrich liver calpastatin behaved characteristically like other calpastatins during kinetic analysis. Calpastatin inhibited calpain from pH 6 to 9 and was found to be unaffected by temperatures as high as 100 °C. Calpastatin also inhibited calpain activity at Ca2+ concentrations ranging from 1 to 10 mM. The inhibitor was shown to be phosphorylated because after incubation with alkaline phosphatase there was a decrease in inhibitory activity. No inhibitory effects were detected against other proteases such as chymotrypsin and trypsin, with both proteases inactivating calpastatin completely. Ostrich liver calpain was shown to have a pH optimum of 7.5 and a temperature optimum of 30 °C. In terms of its thermodynamic properties it resembled that of other ostrich proteases; DH, DS and DG being 47.07 kJ/mol, -91.1 J/mol/K and 74.237 kJ/mol, respectively. Ostrich liver calpain showed a Km of 0.14 % (w/v). The enzyme was active at both milli- and micro-molar concentrations of Ca2+. Ostrich liver calpastatin showed many physical, chemical and kinetic properties similar to those of other known calpastatins.
- Full Text: false
- Date Issued: 2000
- Authors: Roman, Henry James
- Date: 2000
- Subjects: Calpastatin , Protease inhibitors , Ion exchange chromatography , Ostriches
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11090 , http://hdl.handle.net/10948/d1015522
- Description: The isolation and purification of calpastatin from ostrich liver is presented, along with its physicochemical and kinetic properties. By using extraction from liver, ion-exchange chromatography on DEAE-Toyopearl, heating to 90 °C for 10 min and rechromatography on Toyopearl Super-Q 650 S, ostrich calpastatin was isolated and purified from ostrich liver. The purified intact calpastatin showed homogeneity on SDS-PAGE (Mr of 105.6 K). Amino acid analysis showed that ostrich calpastatin resembled that of rabbit liver and human erythrocyte calpastatin. An N-terminal sequence could not be obtained because the N-terminus was found to be blocked by an as yet unknown amino acid residue. The Mr values of degradative forms of ostrich liver calpastatin were determined to be 56 K and 90 K. By using PAG-IEF the pI of the intact form was determined to be 5.1. Ostrich liver calpastatin behaved characteristically like other calpastatins during kinetic analysis. Calpastatin inhibited calpain from pH 6 to 9 and was found to be unaffected by temperatures as high as 100 °C. Calpastatin also inhibited calpain activity at Ca2+ concentrations ranging from 1 to 10 mM. The inhibitor was shown to be phosphorylated because after incubation with alkaline phosphatase there was a decrease in inhibitory activity. No inhibitory effects were detected against other proteases such as chymotrypsin and trypsin, with both proteases inactivating calpastatin completely. Ostrich liver calpain was shown to have a pH optimum of 7.5 and a temperature optimum of 30 °C. In terms of its thermodynamic properties it resembled that of other ostrich proteases; DH, DS and DG being 47.07 kJ/mol, -91.1 J/mol/K and 74.237 kJ/mol, respectively. Ostrich liver calpain showed a Km of 0.14 % (w/v). The enzyme was active at both milli- and micro-molar concentrations of Ca2+. Ostrich liver calpastatin showed many physical, chemical and kinetic properties similar to those of other known calpastatins.
- Full Text: false
- Date Issued: 2000
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