Enumeration, conformation sampling and population of libraries of peptide macrocycles for the search of chemotherapeutic cardioprotection agents
- Authors: Sigauke, Lester Takunda
- Date: 2019
- Subjects: Peptides -- Synthesis , Macrocyclic compounds , Drug development , Drug discovery , Cardiovascular system -- Diseases -- Prevention , Proteins -- Synthesis
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/116056 , vital:34293
- Description: Peptides are uniquely endowed with features that allow them to perturb previously difficult to drug biomolecular targets. Peptide macrocycles in particular have seen a flurry of recent interest due to their enhanced bioavailability, tunability and specificity. Although these properties make them attractive hit-candidates in early stage drug discovery, knowing which peptides to pursue is non‐trivial due to the magnitude of the peptide sequence space. Computational screening approaches show promise in their ability to address the size of this search space but suffer from their inability to accurately interrogate the conformational landscape of peptide macrocycles. We developed an in‐silico compound enumerator that was tasked with populating a conformationally laden peptide virtual library. This library was then used in the search for cardio‐protective agents (that may be administered, reducing tissue damage during reperfusion after ischemia (heart attacks)). Our enumerator successfully generated a library of 15.2 billion compounds, requiring the use of compression algorithms, conformational sampling protocols and management of aggregated compute resources in the context of a local cluster. In the absence of experimental biophysical data, we performed biased sampling during alchemical molecular dynamics simulations in order to observe cyclophilin‐D perturbation by cyclosporine A and its mitochondrial targeted analogue. Reliable intermediate state averaging through a WHAM analysis of the biased dynamic pulling simulations confirmed that the cardio‐protective activity of cyclosporine A was due to its mitochondrial targeting. Paralleltempered solution molecular dynamics in combination with efficient clustering isolated the essential dynamics of a cyclic peptide scaffold. The rapid enumeration of skeletons from these essential dynamics gave rise to a conformation laden virtual library of all the 15.2 Billion unique cyclic peptides (given the limits on peptide sequence imposed). Analysis of this library showed the exact extent of physicochemical properties covered, relative to the bare scaffold precursor. Molecular docking of a subset of the virtual library against cyclophilin‐D showed significant improvements in affinity to the target (relative to cyclosporine A). The conformation laden virtual library, accessed by our methodology, provided derivatives that were able to make many interactions per peptide with the cyclophilin‐D target. Machine learning methods showed promise in the training of Support Vector Machines for synthetic feasibility prediction for this library. The synergy between enumeration and conformational sampling greatly improves the performance of this library during virtual screening, even when only a subset is used.
- Full Text:
- Authors: Sigauke, Lester Takunda
- Date: 2019
- Subjects: Peptides -- Synthesis , Macrocyclic compounds , Drug development , Drug discovery , Cardiovascular system -- Diseases -- Prevention , Proteins -- Synthesis
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/116056 , vital:34293
- Description: Peptides are uniquely endowed with features that allow them to perturb previously difficult to drug biomolecular targets. Peptide macrocycles in particular have seen a flurry of recent interest due to their enhanced bioavailability, tunability and specificity. Although these properties make them attractive hit-candidates in early stage drug discovery, knowing which peptides to pursue is non‐trivial due to the magnitude of the peptide sequence space. Computational screening approaches show promise in their ability to address the size of this search space but suffer from their inability to accurately interrogate the conformational landscape of peptide macrocycles. We developed an in‐silico compound enumerator that was tasked with populating a conformationally laden peptide virtual library. This library was then used in the search for cardio‐protective agents (that may be administered, reducing tissue damage during reperfusion after ischemia (heart attacks)). Our enumerator successfully generated a library of 15.2 billion compounds, requiring the use of compression algorithms, conformational sampling protocols and management of aggregated compute resources in the context of a local cluster. In the absence of experimental biophysical data, we performed biased sampling during alchemical molecular dynamics simulations in order to observe cyclophilin‐D perturbation by cyclosporine A and its mitochondrial targeted analogue. Reliable intermediate state averaging through a WHAM analysis of the biased dynamic pulling simulations confirmed that the cardio‐protective activity of cyclosporine A was due to its mitochondrial targeting. Paralleltempered solution molecular dynamics in combination with efficient clustering isolated the essential dynamics of a cyclic peptide scaffold. The rapid enumeration of skeletons from these essential dynamics gave rise to a conformation laden virtual library of all the 15.2 Billion unique cyclic peptides (given the limits on peptide sequence imposed). Analysis of this library showed the exact extent of physicochemical properties covered, relative to the bare scaffold precursor. Molecular docking of a subset of the virtual library against cyclophilin‐D showed significant improvements in affinity to the target (relative to cyclosporine A). The conformation laden virtual library, accessed by our methodology, provided derivatives that were able to make many interactions per peptide with the cyclophilin‐D target. Machine learning methods showed promise in the training of Support Vector Machines for synthetic feasibility prediction for this library. The synergy between enumeration and conformational sampling greatly improves the performance of this library during virtual screening, even when only a subset is used.
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Studies on an autolysin produced by clostridium acetobutylicum
- Authors: Webster, Jocelyn Rowena
- Date: 1981
- Subjects: Clostridium acetobutylicum , Autolysis , Bacteriocins , Proteins -- Synthesis , DNA -- Synthesis , RNA -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3893 , http://hdl.handle.net/10962/d1003724
- Description: An extracellular bacteriocin-like substance produced by Clostridium acetobutylicum was detected during studies on an industrial fermentation process. The bacteriocin-like substance was not inducible by either ultraviolet light or mitomycin C, and its production was not associated with the induction of a protease. Studies on the mode of action of the bacteriocin-like substance indicated that it had no significant effect on DNA, RNA, or protein synthesis, and it did not cause the loss of intracellular ATP. However, the bacteriocin-like substance was able to lyse SDS-treated cells and cell walls of C. acetobutylicum and was identified as an autolysin. Some of the characteristics of this extracellular autolysin were determined, and after purification it was shown to be a glycoprotein with a molecular weight of 28 000.
- Full Text:
- Authors: Webster, Jocelyn Rowena
- Date: 1981
- Subjects: Clostridium acetobutylicum , Autolysis , Bacteriocins , Proteins -- Synthesis , DNA -- Synthesis , RNA -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3893 , http://hdl.handle.net/10962/d1003724
- Description: An extracellular bacteriocin-like substance produced by Clostridium acetobutylicum was detected during studies on an industrial fermentation process. The bacteriocin-like substance was not inducible by either ultraviolet light or mitomycin C, and its production was not associated with the induction of a protease. Studies on the mode of action of the bacteriocin-like substance indicated that it had no significant effect on DNA, RNA, or protein synthesis, and it did not cause the loss of intracellular ATP. However, the bacteriocin-like substance was able to lyse SDS-treated cells and cell walls of C. acetobutylicum and was identified as an autolysin. Some of the characteristics of this extracellular autolysin were determined, and after purification it was shown to be a glycoprotein with a molecular weight of 28 000.
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Characterization and mode of action of a bacteriocin produced by a Bacteroides Fragilis strain
- Mossie, Godwin Mxolisi Kevin
- Authors: Mossie, Godwin Mxolisi Kevin
- Date: 1980
- Subjects: Bacteroides , Anaerobic bacteria , Trypsin , Dinitrophenol , Proteins -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4124 , http://hdl.handle.net/10962/d1013543
- Description: Bacteroides fragilis strain Bf-1 produces an extracellular bacteriocin at the beginning of the stationary growth phase. Production is not inducible by either ultraviolet light or mitomycin C. The low molecular weight bacteriocin (MW estimates of 13 500 and 18 800 obtained from Sephadex G-100 chromatography and SDS-PAGE electrophoresis respecively) is stable between pH 7 - 9 and is inactivated on incubation with trypsin and pronase. An unusual feature of the Bf-1 bacteriocin is its apparent biphasic temperature stability: while the majority of the activity (97%) is destroyed by heating at 60ºC (t [subscript] 1/2 = 2.5 min at 60ºC), a small proportion (3%) is stable even after autoclaving at 121ºC for 15 min. The killing of sensitive cells occurs in 2 stages and the killing action is reversed by incubation with trypsin. The transition from stage I to stage II is dependent on the temperature of incubation and the growth state of sensitive cells. 2,4-Dinitrophenol prevents this transition. The Bf-1 bacteriocin has an unusual mode of action. It specifically inhibits RNA synthesis whilst having no effect on protein or DNA synthesis. No effect on intracellular ATP levels were observed. The heat-stable (3%) fraction had a similar biochemical effect. In vitro studies involving RNA polymerase indicated that the bacteriocin and the antibiotic rifampicin have similar effects on RNA synthesis. The bacteriocinogenic strain (Bf-1) is insensitive to its own bacteriocin both in vivo and in vitro, although this immunity is overcome in vitro by the addition of higher concentrations of the Bf-1 bacteriocin. The bacteriocinogenic strain (Bf-1) harbors a cryptic plasmid (or plasmids) which on a neutral sucrose gradient, sediments faster than the Col E1 marker plasmid DNA. Attempts to cure this strain of its bacteriocinogenic phenotype were unsuccessful.
- Full Text:
- Authors: Mossie, Godwin Mxolisi Kevin
- Date: 1980
- Subjects: Bacteroides , Anaerobic bacteria , Trypsin , Dinitrophenol , Proteins -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4124 , http://hdl.handle.net/10962/d1013543
- Description: Bacteroides fragilis strain Bf-1 produces an extracellular bacteriocin at the beginning of the stationary growth phase. Production is not inducible by either ultraviolet light or mitomycin C. The low molecular weight bacteriocin (MW estimates of 13 500 and 18 800 obtained from Sephadex G-100 chromatography and SDS-PAGE electrophoresis respecively) is stable between pH 7 - 9 and is inactivated on incubation with trypsin and pronase. An unusual feature of the Bf-1 bacteriocin is its apparent biphasic temperature stability: while the majority of the activity (97%) is destroyed by heating at 60ºC (t [subscript] 1/2 = 2.5 min at 60ºC), a small proportion (3%) is stable even after autoclaving at 121ºC for 15 min. The killing of sensitive cells occurs in 2 stages and the killing action is reversed by incubation with trypsin. The transition from stage I to stage II is dependent on the temperature of incubation and the growth state of sensitive cells. 2,4-Dinitrophenol prevents this transition. The Bf-1 bacteriocin has an unusual mode of action. It specifically inhibits RNA synthesis whilst having no effect on protein or DNA synthesis. No effect on intracellular ATP levels were observed. The heat-stable (3%) fraction had a similar biochemical effect. In vitro studies involving RNA polymerase indicated that the bacteriocin and the antibiotic rifampicin have similar effects on RNA synthesis. The bacteriocinogenic strain (Bf-1) is insensitive to its own bacteriocin both in vivo and in vitro, although this immunity is overcome in vitro by the addition of higher concentrations of the Bf-1 bacteriocin. The bacteriocinogenic strain (Bf-1) harbors a cryptic plasmid (or plasmids) which on a neutral sucrose gradient, sediments faster than the Col E1 marker plasmid DNA. Attempts to cure this strain of its bacteriocinogenic phenotype were unsuccessful.
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