Falcipain 2 and 3 as malarial drug targets: deciphering the effects of missense mutations and identification of allosteric modulators via computational approaches
- Authors: Okeke, Chiamaka Jessica
- Date: 2023-10-13
- Subjects: Antimalarials , Cysteine proteinases , Missense mutation , Allostery , Cysteine proteinase falcipain 2a , Cysteine proteinase falcipain 3
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/432170 , vital:72848 , DOI 10.21504/10962/432170
- Description: Malaria, caused by an obligate unicellular protozoan parasite of the genus Plasmodium, is a disease of global health importance that remains a major cause of morbidity and mortality in developing countries. The World Health Organization (WHO) reported nearly 247 million malaria cases in 2021, causing 619,000 deaths, the vast majority ascribed to pregnant women and young children in sub-Saharan Africa. A critical component of malaria mitigation and elimination efforts worldwide is antimalarial drugs. However, resistance to available antimalarial drugs jeopardizes the treatment, prevention, and eradication of the disease. The recent emergence and spread of resistance to artemisinin (ART), the currently recommended first-line antimalarial drug, emphasizes the need to understand the resistance mechanism and apply this knowledge in developing new drugs that are effective against malaria. An insight into ART's mechanism of action indicates that ferrous iron (Fe2+) or heme, released when hemoglobin is degraded, cleaves the endoperoxide bridge. As a result, free radicals are formed, which alkylate many intracellular targets and result in plasmodial proteopathy. Aside from the existing evidence that mutations in the Kelch 13 protein propeller domain affect ART sensitivity and clearance rate by Plasmodium falciparum (Pf) parasites, recent investigations raise the possibility that additional target loci may be involved, and these include a nonsense (S69stop) and four missense variants (K255R, N257E, T343P, and D345G) in falcipain 2 (FP-2) protein. FP-2 and falcipain 3 (FP-3) are cysteine proteases responsible for hydrolyzing hemoglobin in the host erythrocytic cycle, a key virulence factor for malaria parasite growth and metabolism. Due to the obligatory nature of the hemoglobin degradation process, both proteases have become potential antimalarial drug targets attracting attention in recent years for the development of blood-stage antimalarial drugs. The alteration of the expression profile of FP-2 and FP-3 through gene manipulation approaches (knockout) or compound inhibition assays, respectively, induced parasites with swollen food vacuoles due to the accumulation of undegraded hemoglobin. Furthermore, missense mutations in FP-2 confer parasites with decreased ART sensitivity, probably due to altered enzyme efficiency and momentary decreased hemoglobin degradation. Hence, understanding how these mutations affect FP-2 (including those implicated in ART resistance) and FP-3 is imperative to finding potentially effective inhibitors. The first aim of this thesis is to characterize the effects of missense mutations on the partial zymogen complex and the catalytic domain of FP-2 and FP-3 using a range of computational approaches and tools such as homology modeling, molecular dynamics (MD) simulations, comparative essential dynamics, dynamic residue network (DRN) analysis, weighted residue contact map analysis, amongst others. The Pf genomic resource database (PlasmoDB) identified 41 missense mutations located in the partial zymogen and catalytic domains of FP-2 and FP-3. Using structure-based tools, six putative allosteric pockets were identified in FP-2 and FP-3. The effect of mutations on the whole protein, the central core, binding pocket residues and allosteric pockets was evaluated. The accurate 3D homology models of the WT and mutants were calculated. MD simulations were performed on the various systems as a quick starting point. MD simulations have provided a cornerstone for establishing numerous computational tools for describing changes arising from mutations, ligand binding, and environmental changes such as pH and temperature. Post-MD analysis was performed in two stages viz global and local analysis. Global analysis via radius of gyration (Rg) and comparative essential dynamic analysis revealed the conformational variability associated with all mutations. In the catalytic domain of FP-2, the presence of M245I mutation triggered the formation of a cryptic pocket via an exclusive mechanism involving the fusion of pockets 2 and 6. This striking observation was also detected in the partial zymogen complex of FP-2 and induced by A159V, M245I and E249A mutations. A similar observation was uncovered in the presence of A422T mutation in the catalytic domain of FP-3. Local DRN and contact map analyses identified conserved inter-residue interaction changes on important communication networks. This study brings a novel understanding of the effects of missense mutations in FP-2 and FP-3 and provides important insight which may help discover new anti-hemoglobinase drugs. The second aim is the identification of potential allosteric ligands against the WT and mutant systems of FP-2 and FP-3 using various computational tools. Of the six potential allosteric pockets identified in FP-2 and FP-3, pocket 1 was evaluated by SiteMap as the most druggable in both proteins. This pipeline was implemented to screen pocket 1 of FP-2 and FP-3 against 2089 repositionable compounds obtained from the DrugBank database. In order to ensure selectivity and specificity to the Plasmodium protein, the human homologs (Cat K and Cat L) were screened, and compounds binding to these proteins were exempted from further analysis. Subsequently, eight compounds (DB00128, DB00312, DB00766, DB00951, DB02893, DB03754, DB13972, and DB14159) were identified as potential allosteric hits for FP-2 and five (DB00853, DB00951, DB01613, DB04173 and DB09419) for FP-3. These compounds were subjected to MD simulation and post-MD trajectory analysis to ascertain their stability in their respective protein structures. The effects of the stable compounds on the WT and mutant systems of FP-2 and FP-3 were then evaluated using DRN analysis. Attention has recently been drawn towards identifying novel allosteric compounds targeting FP-2 and FP-3; hence this study explores the potential allosteric inhibitory mechanisms in the presence and absence of mutations in FP-2 and FP-3. Overall, the results presented in this thesis provide (i) an understanding of the role mutations in the partial zymogen complex play in the activation of the active enzyme, (ii) an insight into the possible allosteric mechanisms induced by mutations on the active enzymes, and (iii) a computational pipeline for the development of novel allosteric modulators for malaria inhibition studies. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Authors: Okeke, Chiamaka Jessica
- Date: 2023-10-13
- Subjects: Antimalarials , Cysteine proteinases , Missense mutation , Allostery , Cysteine proteinase falcipain 2a , Cysteine proteinase falcipain 3
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/432170 , vital:72848 , DOI 10.21504/10962/432170
- Description: Malaria, caused by an obligate unicellular protozoan parasite of the genus Plasmodium, is a disease of global health importance that remains a major cause of morbidity and mortality in developing countries. The World Health Organization (WHO) reported nearly 247 million malaria cases in 2021, causing 619,000 deaths, the vast majority ascribed to pregnant women and young children in sub-Saharan Africa. A critical component of malaria mitigation and elimination efforts worldwide is antimalarial drugs. However, resistance to available antimalarial drugs jeopardizes the treatment, prevention, and eradication of the disease. The recent emergence and spread of resistance to artemisinin (ART), the currently recommended first-line antimalarial drug, emphasizes the need to understand the resistance mechanism and apply this knowledge in developing new drugs that are effective against malaria. An insight into ART's mechanism of action indicates that ferrous iron (Fe2+) or heme, released when hemoglobin is degraded, cleaves the endoperoxide bridge. As a result, free radicals are formed, which alkylate many intracellular targets and result in plasmodial proteopathy. Aside from the existing evidence that mutations in the Kelch 13 protein propeller domain affect ART sensitivity and clearance rate by Plasmodium falciparum (Pf) parasites, recent investigations raise the possibility that additional target loci may be involved, and these include a nonsense (S69stop) and four missense variants (K255R, N257E, T343P, and D345G) in falcipain 2 (FP-2) protein. FP-2 and falcipain 3 (FP-3) are cysteine proteases responsible for hydrolyzing hemoglobin in the host erythrocytic cycle, a key virulence factor for malaria parasite growth and metabolism. Due to the obligatory nature of the hemoglobin degradation process, both proteases have become potential antimalarial drug targets attracting attention in recent years for the development of blood-stage antimalarial drugs. The alteration of the expression profile of FP-2 and FP-3 through gene manipulation approaches (knockout) or compound inhibition assays, respectively, induced parasites with swollen food vacuoles due to the accumulation of undegraded hemoglobin. Furthermore, missense mutations in FP-2 confer parasites with decreased ART sensitivity, probably due to altered enzyme efficiency and momentary decreased hemoglobin degradation. Hence, understanding how these mutations affect FP-2 (including those implicated in ART resistance) and FP-3 is imperative to finding potentially effective inhibitors. The first aim of this thesis is to characterize the effects of missense mutations on the partial zymogen complex and the catalytic domain of FP-2 and FP-3 using a range of computational approaches and tools such as homology modeling, molecular dynamics (MD) simulations, comparative essential dynamics, dynamic residue network (DRN) analysis, weighted residue contact map analysis, amongst others. The Pf genomic resource database (PlasmoDB) identified 41 missense mutations located in the partial zymogen and catalytic domains of FP-2 and FP-3. Using structure-based tools, six putative allosteric pockets were identified in FP-2 and FP-3. The effect of mutations on the whole protein, the central core, binding pocket residues and allosteric pockets was evaluated. The accurate 3D homology models of the WT and mutants were calculated. MD simulations were performed on the various systems as a quick starting point. MD simulations have provided a cornerstone for establishing numerous computational tools for describing changes arising from mutations, ligand binding, and environmental changes such as pH and temperature. Post-MD analysis was performed in two stages viz global and local analysis. Global analysis via radius of gyration (Rg) and comparative essential dynamic analysis revealed the conformational variability associated with all mutations. In the catalytic domain of FP-2, the presence of M245I mutation triggered the formation of a cryptic pocket via an exclusive mechanism involving the fusion of pockets 2 and 6. This striking observation was also detected in the partial zymogen complex of FP-2 and induced by A159V, M245I and E249A mutations. A similar observation was uncovered in the presence of A422T mutation in the catalytic domain of FP-3. Local DRN and contact map analyses identified conserved inter-residue interaction changes on important communication networks. This study brings a novel understanding of the effects of missense mutations in FP-2 and FP-3 and provides important insight which may help discover new anti-hemoglobinase drugs. The second aim is the identification of potential allosteric ligands against the WT and mutant systems of FP-2 and FP-3 using various computational tools. Of the six potential allosteric pockets identified in FP-2 and FP-3, pocket 1 was evaluated by SiteMap as the most druggable in both proteins. This pipeline was implemented to screen pocket 1 of FP-2 and FP-3 against 2089 repositionable compounds obtained from the DrugBank database. In order to ensure selectivity and specificity to the Plasmodium protein, the human homologs (Cat K and Cat L) were screened, and compounds binding to these proteins were exempted from further analysis. Subsequently, eight compounds (DB00128, DB00312, DB00766, DB00951, DB02893, DB03754, DB13972, and DB14159) were identified as potential allosteric hits for FP-2 and five (DB00853, DB00951, DB01613, DB04173 and DB09419) for FP-3. These compounds were subjected to MD simulation and post-MD trajectory analysis to ascertain their stability in their respective protein structures. The effects of the stable compounds on the WT and mutant systems of FP-2 and FP-3 were then evaluated using DRN analysis. Attention has recently been drawn towards identifying novel allosteric compounds targeting FP-2 and FP-3; hence this study explores the potential allosteric inhibitory mechanisms in the presence and absence of mutations in FP-2 and FP-3. Overall, the results presented in this thesis provide (i) an understanding of the role mutations in the partial zymogen complex play in the activation of the active enzyme, (ii) an insight into the possible allosteric mechanisms induced by mutations on the active enzymes, and (iii) a computational pipeline for the development of novel allosteric modulators for malaria inhibition studies. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
An in-silico study of the type II NADH: Quinone Oxidoreductase (ndh2). A new anti-malaria drug target
- Authors: Baye, Bertha Cinthia
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium , Molecular dynamics , Computer simulation , Quinone , Antimalarials , Molecules Models , Docking , Drugs Computer-aided design
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365633 , vital:65767 , DOI https://doi.org/10.21504/10962/365633
- Description: Malaria is caused by Plasmodium parasites, spread to people through the bites of infected female Anopheles mosquitoes. This study focuses on all 5 (Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax) parasites that cause malaria in humans. Africa is a developing continent, and it is the most affected with an estimation of 90% of more than 400 000 malaria-related deaths reported by the World Health Organization (WHO) report in 2020, in which 61% of that number are children under the ages of five. Malaria resistance was initially observed in early 1986 and with the progression of time anti-malarial drug resistance has only increased. As a result, there is a need to study the malarial proteins mechanism of action and identify alternative treatment strategies for this disease. Type II NADH: quinone oxidoreductase (NDH2) is a monotopic protein that catalyses the electron transfer from NADH to quinone via FAD without a proton-pumping activity, and functions as an initial enzyme, either in addition to or as an alternative to proton-pumping NADH dehydrogenase (complex I) in the respiratory chain of bacteria, archaea, and fungal and plant mitochondrial. The structures for the Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax were modelled from the crystal structure of Plasmodium falciparum (5JWA). Compounds from the South African natural compounds database (SANCDB) were docked against both the NDH2 crystal structure and modelled structures. By performing in silico screening the study aimed to find potential compounds that might interrupt the electron transfer to quinone therefore disturbing the enzyme‟s function and thereby possibly eliminating the plasmodium parasite. CHARMM-GUI was used to create the membrane (since this work is with membrane-bound proteins) and to orient the protein on the membrane using OPM server guidelines, the interface produced GROMACS topology files that were used in molecular dynamics simulations. Molecular dynamics simulations were performed in the Centre for high performance computing (CHPC) cluster under the CHEM0802 project and the trajectories produced were further analysed. In this work not only were hit compounds from SANCDB identified, but also differences in behaviour across species and in the presence or absence of the membrane were described. This highlights the need to include the correct protein environment when studying these systems. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Authors: Baye, Bertha Cinthia
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium , Molecular dynamics , Computer simulation , Quinone , Antimalarials , Molecules Models , Docking , Drugs Computer-aided design
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365633 , vital:65767 , DOI https://doi.org/10.21504/10962/365633
- Description: Malaria is caused by Plasmodium parasites, spread to people through the bites of infected female Anopheles mosquitoes. This study focuses on all 5 (Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax) parasites that cause malaria in humans. Africa is a developing continent, and it is the most affected with an estimation of 90% of more than 400 000 malaria-related deaths reported by the World Health Organization (WHO) report in 2020, in which 61% of that number are children under the ages of five. Malaria resistance was initially observed in early 1986 and with the progression of time anti-malarial drug resistance has only increased. As a result, there is a need to study the malarial proteins mechanism of action and identify alternative treatment strategies for this disease. Type II NADH: quinone oxidoreductase (NDH2) is a monotopic protein that catalyses the electron transfer from NADH to quinone via FAD without a proton-pumping activity, and functions as an initial enzyme, either in addition to or as an alternative to proton-pumping NADH dehydrogenase (complex I) in the respiratory chain of bacteria, archaea, and fungal and plant mitochondrial. The structures for the Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax were modelled from the crystal structure of Plasmodium falciparum (5JWA). Compounds from the South African natural compounds database (SANCDB) were docked against both the NDH2 crystal structure and modelled structures. By performing in silico screening the study aimed to find potential compounds that might interrupt the electron transfer to quinone therefore disturbing the enzyme‟s function and thereby possibly eliminating the plasmodium parasite. CHARMM-GUI was used to create the membrane (since this work is with membrane-bound proteins) and to orient the protein on the membrane using OPM server guidelines, the interface produced GROMACS topology files that were used in molecular dynamics simulations. Molecular dynamics simulations were performed in the Centre for high performance computing (CHPC) cluster under the CHEM0802 project and the trajectories produced were further analysed. In this work not only were hit compounds from SANCDB identified, but also differences in behaviour across species and in the presence or absence of the membrane were described. This highlights the need to include the correct protein environment when studying these systems. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
Identification of novel compounds against Plasmodium falciparum Cytochrome bc1 Complex inhibiting the trans-membrane electron transfer pathway: an In Silico study
- Authors: Chebon, Lorna Jemosop
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium falciparum , Molecular dynamics , Antimalarials , Molecules Models , Docking , Cytochromes , Drug resistance , Computer simulation , Drugs Computer-aided design , System analysis
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365666 , vital:65774 , DOI https://doi.org/10.21504/10962/365666
- Description: Malaria continues to be a burden globally with a myriad of challenges deterring eradication efforts. With most antimalarials facing drug resistance, such as atovaquone (ATQ), alternative compounds that can withstand resistance are warranted. The Plasmodium falciparum cytochrome b (PfCytb), a subunit of P. falciparum cytochrome bc1 complex, is a validated drug target. Structurally, cytochrome b, cytochrome c1, and iron sulphur protein (ISP) subunits form the catalytic domain of the protein complex having heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors. These cofactos have redox centres to aid in the electron transfer (ET) process. These subunits promote ET mainly through the enzyme’s ubiquinol oxidation (Qo) and ubiquinone reduction (Qi) processes in the catalytic domain. ATQ drug has been used in the prevention and treatment of uncomplicated malaria by targeting PfCytb protein. Once the mitochondrial transmembrane ET pathway is inhibited, it causes a collapse in its membrane potential. Previously reported ATQ drug resistance has been associated with the point mutations Y268C, Y268N and Y268S. Thus, in finding alternatives to the ATQ drug, this research aimed to: i) employ in silico approaches incorporating protein into phospholipid bilayer for the first time to understand the parasites’ resistance mechanism; ii) determine any sequence and structural differences that could be explored in drug design studies; and iii) screen for PfCytb-iron sulphur protein (Cytb-ISP) hit compounds from South African natural compound database (SANCDB) and Medicines for Malaria Venture (MMV) that can withstand the identified mutations. Using computational tools, comparative sequence and structural analyses were performed on the cytochrome b protein, where the ultimate focus was on P. falciparum cytochrome b and its human homolog. Through multiple sequence alignment, motif discovery and phylogeny, differences between P. falciparum and H. sapiens cytochrome b were identified. Protein modelling of both P. falciparum and H. sapiens cytochrome b - iron sulphur protein (PfCytb-ISP and HsCytb-ISP) was performed. Results showed that at the sequence level, there were few amino acid residue differences because the protein is highly conserved. Important to note is the four-residue deletion in Plasmodium spp. absent in the human homolog. Motif analysis discovered five unique motifs in P. falciparum cytochrome b protein which were mapped onto the predicted protein model. These motifs were not in regions of functional importance; hence their function is still unknown. At a structural level, the four-residue deletion was observed to alter the Qo substrate binding pocket as reported in previous studies and confirmed in this study. This deletion resulted in a 0.83 Å structural displacement. Also, there are currently no in silico studies that have performed experiments with P. falciparum cytochrome b protein incorporated into a phospholipid bilayer. Using 350 ns molecular dynamics (MD) simulations of the holo and ATQ-bound systems, the study highlighted the resistance mechanism of the parasite protein where the loss of active site residue-residue interactions was identified, all linked to the three mutations. The identified compromised interactions are likely to destabilise the protein’s function, specifically in the Qo substrate binding site. This showed the possible effect of mutations on ATQ drug activity, where all three mutations were reported to share a similar resistance mechanism. Thereafter, this research work utilised in silico approaches where both Qo active site and interface pocket were targeted by screening the South African natural compounds database (SANCDB) and Medicines for Malaria Venture (MMV) compounds to identify novel selective hits. SANCDB compounds are known for their structural complexity that preserves the potency of the drug molecule. Both SANCDB and MMV compounds have not been explored as inhibitors against the PfCytb drug target. Molecular docking, molecular dynamics (MD) simulations, principal component, and dynamic residue network (DRN; global and local) analyses were utilised to identify and confirm the potential selective inhibitors. Docking results identified compounds that bound selectively onto PfCytb-ISP with a binding energy ≤ -8.7 kcal/mol-1. Further, this work validated a total of eight potential selective compounds to inhibit PfCytb-ISP protein (Qo active site) not only in the wild-type but also in the presence of the point mutations Y268C, Y268N and Y268S. The selective binding of these hit compounds could be linked to the differences reported at sequence/residue level in chapter 3. DRN and residue contact map analyses of the eight compounds in holo and ligand-bound systems revealed reduced residue interactions and decreased protein communication. This suggests that the eight compounds show the possibility of inhibiting the parasite and disrupting important residue-residue interactions. Additionally, 13 selective compounds were identified to bind at the protein’s heterodimer interface, where global and local analysis confirmed their effect on active site residues (distal location) as well as on the communication network. Based on the sequence differences between PfCytb and the human homolog, these findings suggest these selective compounds as potential allosteric modulators of the parasite enzyme, which may serve as possible replacements of the already resistant ATQ drug. Therefore, these findings pave the way for further in vitro studies to establish their anti-plasmodial inhibition levels. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Authors: Chebon, Lorna Jemosop
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium falciparum , Molecular dynamics , Antimalarials , Molecules Models , Docking , Cytochromes , Drug resistance , Computer simulation , Drugs Computer-aided design , System analysis
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365666 , vital:65774 , DOI https://doi.org/10.21504/10962/365666
- Description: Malaria continues to be a burden globally with a myriad of challenges deterring eradication efforts. With most antimalarials facing drug resistance, such as atovaquone (ATQ), alternative compounds that can withstand resistance are warranted. The Plasmodium falciparum cytochrome b (PfCytb), a subunit of P. falciparum cytochrome bc1 complex, is a validated drug target. Structurally, cytochrome b, cytochrome c1, and iron sulphur protein (ISP) subunits form the catalytic domain of the protein complex having heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors. These cofactos have redox centres to aid in the electron transfer (ET) process. These subunits promote ET mainly through the enzyme’s ubiquinol oxidation (Qo) and ubiquinone reduction (Qi) processes in the catalytic domain. ATQ drug has been used in the prevention and treatment of uncomplicated malaria by targeting PfCytb protein. Once the mitochondrial transmembrane ET pathway is inhibited, it causes a collapse in its membrane potential. Previously reported ATQ drug resistance has been associated with the point mutations Y268C, Y268N and Y268S. Thus, in finding alternatives to the ATQ drug, this research aimed to: i) employ in silico approaches incorporating protein into phospholipid bilayer for the first time to understand the parasites’ resistance mechanism; ii) determine any sequence and structural differences that could be explored in drug design studies; and iii) screen for PfCytb-iron sulphur protein (Cytb-ISP) hit compounds from South African natural compound database (SANCDB) and Medicines for Malaria Venture (MMV) that can withstand the identified mutations. Using computational tools, comparative sequence and structural analyses were performed on the cytochrome b protein, where the ultimate focus was on P. falciparum cytochrome b and its human homolog. Through multiple sequence alignment, motif discovery and phylogeny, differences between P. falciparum and H. sapiens cytochrome b were identified. Protein modelling of both P. falciparum and H. sapiens cytochrome b - iron sulphur protein (PfCytb-ISP and HsCytb-ISP) was performed. Results showed that at the sequence level, there were few amino acid residue differences because the protein is highly conserved. Important to note is the four-residue deletion in Plasmodium spp. absent in the human homolog. Motif analysis discovered five unique motifs in P. falciparum cytochrome b protein which were mapped onto the predicted protein model. These motifs were not in regions of functional importance; hence their function is still unknown. At a structural level, the four-residue deletion was observed to alter the Qo substrate binding pocket as reported in previous studies and confirmed in this study. This deletion resulted in a 0.83 Å structural displacement. Also, there are currently no in silico studies that have performed experiments with P. falciparum cytochrome b protein incorporated into a phospholipid bilayer. Using 350 ns molecular dynamics (MD) simulations of the holo and ATQ-bound systems, the study highlighted the resistance mechanism of the parasite protein where the loss of active site residue-residue interactions was identified, all linked to the three mutations. The identified compromised interactions are likely to destabilise the protein’s function, specifically in the Qo substrate binding site. This showed the possible effect of mutations on ATQ drug activity, where all three mutations were reported to share a similar resistance mechanism. Thereafter, this research work utilised in silico approaches where both Qo active site and interface pocket were targeted by screening the South African natural compounds database (SANCDB) and Medicines for Malaria Venture (MMV) compounds to identify novel selective hits. SANCDB compounds are known for their structural complexity that preserves the potency of the drug molecule. Both SANCDB and MMV compounds have not been explored as inhibitors against the PfCytb drug target. Molecular docking, molecular dynamics (MD) simulations, principal component, and dynamic residue network (DRN; global and local) analyses were utilised to identify and confirm the potential selective inhibitors. Docking results identified compounds that bound selectively onto PfCytb-ISP with a binding energy ≤ -8.7 kcal/mol-1. Further, this work validated a total of eight potential selective compounds to inhibit PfCytb-ISP protein (Qo active site) not only in the wild-type but also in the presence of the point mutations Y268C, Y268N and Y268S. The selective binding of these hit compounds could be linked to the differences reported at sequence/residue level in chapter 3. DRN and residue contact map analyses of the eight compounds in holo and ligand-bound systems revealed reduced residue interactions and decreased protein communication. This suggests that the eight compounds show the possibility of inhibiting the parasite and disrupting important residue-residue interactions. Additionally, 13 selective compounds were identified to bind at the protein’s heterodimer interface, where global and local analysis confirmed their effect on active site residues (distal location) as well as on the communication network. Based on the sequence differences between PfCytb and the human homolog, these findings suggest these selective compounds as potential allosteric modulators of the parasite enzyme, which may serve as possible replacements of the already resistant ATQ drug. Therefore, these findings pave the way for further in vitro studies to establish their anti-plasmodial inhibition levels. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
The characterization of GTP Cyclohydrolase I and 6-Pyruvoyl Tetrahydropterin Synthase enzymes as potential anti-malarial drug targets
- Khairallah, Afrah Yousif Huseein
- Authors: Khairallah, Afrah Yousif Huseein
- Date: 2022-04-08
- Subjects: Antimalarials , Plasmodium falciparum , Malaria Chemotherapy , Malaria Africa , Drug resistance , Drug development , Molecular dynamics
- Language: English
- Type: Doctoral thesis , text
- Identifier: http://hdl.handle.net/10962/233784 , vital:50127 , DOI 10.21504/10962/233784
- Description: Malaria remains a public health problem and a high burden of disease, especially in developing countries. The unicellular protozoan malaria parasite of the genus Plasmodium infects about a quarter of a billion people annually, with an estimated 409 000 death cases. The majority of malaria cases occurred in Africa; hence, the region is regarded as endemic for malaria. Global efforts to eradicate the disease led to a decrease in morbidity and mortality rates. However, an enormous burden of malaria infection remains, and it cannot go unnoticed. Countries with limited resources are more affected by the disease, mainly on its public health and socio-economic development, due to many factors besides malaria itself, such as lack of access to adequate, affordable treatments and preventative regimes. Furthermore, the current antimalarial drugs are losing their efficacy because of parasite drug resistance. The emerged drug resistance has reduced the drug efficacy in clearing the parasite from the host system, causing prolonged illness and a higher risk of death. Therefore, the emerged antimalarial drug resistance has hindered the global efforts for malaria control and elimination and established an urgent need for new treatment strategies. When the resistance against classical antimalarial drugs emerged, the class of antifolate antimalarial medicines became the most common alternative. The antifolate antimalarial drugs target the malaria parasite de novo folate biosynthesis pathway by limiting folate derivates, which are essential for the parasite cell growth and survival. Yet again, the malaria parasite developed resistance against the available antifolate drugs, rendering the drugs ineffective in many cases. Given the previous success in targeting the malaria parasite de novo folate biosynthesis pathway, alternative enzymes within this pathway stand as good targets and can be explored to develop new antifolate drugs with novel mechanisms of action. The primary focus of this thesis is to contribute to the existing and growing knowledge of antimalarial drug discovery. The study aims to characterise the malaria parasite de novo folate synthesis pathway enzymes guanosine-5'-triphosphate (GTP) cyclohydrolase I (GCH1) and 6-pyruvoyl tetrahydropterin synthase (PTPS) as alternative drug targets for malaria treatment by using computational approaches. Further, discover new allosteric drug targeting sites within the two enzymes' 3D structures for future drug design and discovery. Sequence and structural analysis were carried out to characterise and pinpoint the two enzymes' unique sequence and structure-based features. From the analyses, key sequence and structure differences were identified between the malaria parasite enzymes relative to their human homolog; the identified sites can aid significantly in designing and developing new antimalarial antifolate drugs with good selectivity toward the parasites’ enzymes. GCH1 and PTPS contain a catalytically essential metal ion in their active site; therefore, force field parameters were needed to study their active sites accurately during all-atom molecular dynamic simulations (MD). The force field parameters were derived through quantum mechanics potential energy surface scans of the metals bonded terms and evaluated via all-atom MD simulations. Proteins structural dynamics is imperative for many biological processes; thus, it is essential to consider the structural dynamics of proteins whilst understanding their function. In this regard, the normal mode analysis (NMA) approach based on the elastic network model (ENM) was employed to study the intrinsic dynamics and conformations changes of GCH1 and PTPS enzymes. The NMA disclosed essential structural information about the protein’s intrinsic dynamics and mechanism of allosteric modulation of their binding properties, further highlighting regions that govern their conformational changes. The analysis also disclosed hotspot residues that are crucial for the proteins' fold stability and function. The NMA was further combined with sequence motif results and showed that conserved residues of GCH1 and PTPS were located within the identified key structural sites modulating the proteins' conformational rearrangement. The characterized structural features and hotspot residues were regarded as potential allosteric sites of important value for the design and development of allosteric drugs. Both GCH1 and PTPS enzymes have never been targeted before and can provide an excellent opportunity to overcome the antimalarial antifolate drug resistance problem. The data presented in this thesis contribute to the understanding of the sequence, structure, and global dynamics of both GCH1 and PTPS, further disclose potential allosteric drug targeting sites and unique structural features of both enzymes that can establish a solid starting point for drug design and development of new antimalarial drugs of a novel mechanism of actions. Lastly, the reported force field parameters will be of value for MD simulations for future in-silico drug discovery studies involving the two enzymes and other enzymes with the same Zn2+ binding motifs and coordination environments. The impact of this research can facilitate the discovery of new effective antimalarial medicines with novel mechanisms of action. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Authors: Khairallah, Afrah Yousif Huseein
- Date: 2022-04-08
- Subjects: Antimalarials , Plasmodium falciparum , Malaria Chemotherapy , Malaria Africa , Drug resistance , Drug development , Molecular dynamics
- Language: English
- Type: Doctoral thesis , text
- Identifier: http://hdl.handle.net/10962/233784 , vital:50127 , DOI 10.21504/10962/233784
- Description: Malaria remains a public health problem and a high burden of disease, especially in developing countries. The unicellular protozoan malaria parasite of the genus Plasmodium infects about a quarter of a billion people annually, with an estimated 409 000 death cases. The majority of malaria cases occurred in Africa; hence, the region is regarded as endemic for malaria. Global efforts to eradicate the disease led to a decrease in morbidity and mortality rates. However, an enormous burden of malaria infection remains, and it cannot go unnoticed. Countries with limited resources are more affected by the disease, mainly on its public health and socio-economic development, due to many factors besides malaria itself, such as lack of access to adequate, affordable treatments and preventative regimes. Furthermore, the current antimalarial drugs are losing their efficacy because of parasite drug resistance. The emerged drug resistance has reduced the drug efficacy in clearing the parasite from the host system, causing prolonged illness and a higher risk of death. Therefore, the emerged antimalarial drug resistance has hindered the global efforts for malaria control and elimination and established an urgent need for new treatment strategies. When the resistance against classical antimalarial drugs emerged, the class of antifolate antimalarial medicines became the most common alternative. The antifolate antimalarial drugs target the malaria parasite de novo folate biosynthesis pathway by limiting folate derivates, which are essential for the parasite cell growth and survival. Yet again, the malaria parasite developed resistance against the available antifolate drugs, rendering the drugs ineffective in many cases. Given the previous success in targeting the malaria parasite de novo folate biosynthesis pathway, alternative enzymes within this pathway stand as good targets and can be explored to develop new antifolate drugs with novel mechanisms of action. The primary focus of this thesis is to contribute to the existing and growing knowledge of antimalarial drug discovery. The study aims to characterise the malaria parasite de novo folate synthesis pathway enzymes guanosine-5'-triphosphate (GTP) cyclohydrolase I (GCH1) and 6-pyruvoyl tetrahydropterin synthase (PTPS) as alternative drug targets for malaria treatment by using computational approaches. Further, discover new allosteric drug targeting sites within the two enzymes' 3D structures for future drug design and discovery. Sequence and structural analysis were carried out to characterise and pinpoint the two enzymes' unique sequence and structure-based features. From the analyses, key sequence and structure differences were identified between the malaria parasite enzymes relative to their human homolog; the identified sites can aid significantly in designing and developing new antimalarial antifolate drugs with good selectivity toward the parasites’ enzymes. GCH1 and PTPS contain a catalytically essential metal ion in their active site; therefore, force field parameters were needed to study their active sites accurately during all-atom molecular dynamic simulations (MD). The force field parameters were derived through quantum mechanics potential energy surface scans of the metals bonded terms and evaluated via all-atom MD simulations. Proteins structural dynamics is imperative for many biological processes; thus, it is essential to consider the structural dynamics of proteins whilst understanding their function. In this regard, the normal mode analysis (NMA) approach based on the elastic network model (ENM) was employed to study the intrinsic dynamics and conformations changes of GCH1 and PTPS enzymes. The NMA disclosed essential structural information about the protein’s intrinsic dynamics and mechanism of allosteric modulation of their binding properties, further highlighting regions that govern their conformational changes. The analysis also disclosed hotspot residues that are crucial for the proteins' fold stability and function. The NMA was further combined with sequence motif results and showed that conserved residues of GCH1 and PTPS were located within the identified key structural sites modulating the proteins' conformational rearrangement. The characterized structural features and hotspot residues were regarded as potential allosteric sites of important value for the design and development of allosteric drugs. Both GCH1 and PTPS enzymes have never been targeted before and can provide an excellent opportunity to overcome the antimalarial antifolate drug resistance problem. The data presented in this thesis contribute to the understanding of the sequence, structure, and global dynamics of both GCH1 and PTPS, further disclose potential allosteric drug targeting sites and unique structural features of both enzymes that can establish a solid starting point for drug design and development of new antimalarial drugs of a novel mechanism of actions. Lastly, the reported force field parameters will be of value for MD simulations for future in-silico drug discovery studies involving the two enzymes and other enzymes with the same Zn2+ binding motifs and coordination environments. The impact of this research can facilitate the discovery of new effective antimalarial medicines with novel mechanisms of action. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
Antimalarial activity of quinoline thiosemicarbazones: synthesis and antiplasmodial evaluation
- Nqeno, Lukhanyiso Khanyisile
- Authors: Nqeno, Lukhanyiso Khanyisile
- Date: 2022-04-06
- Subjects: Antimalarials , Quinoline , Thiosemicarbazones , Malaria Chemotherapy , Plasmodium falciparum , Malaria Africa, Sub-Saharan , Iron chelates Therapeutic use
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/291292 , vital:56841
- Description: Africa is one of the regions that is most affected by malaria, as 90% of all malaria deaths occur in sub-saharan Africa. Malaria is a life threatening disease responsible for an estimated 800000 deaths each year, the majority of these deaths occurred in children under the age of five. The disease is a mosquito-borne, and it is transmitted to humans by the female Anopheles mosquito. The parasite responsible for this disease belong to the Plasmodium genus with Plasmodium falciparum causing the most severe cases of the disease in humans. The most widely available anti-malarials were designed to specifically target the pathogenic blood stage in humans, however, in order to completely eradicate malaria there is a need for the development of medicines that not only target the pathogenic blood stage of the parasite but also block parasite transmission and eliminate asymptomatic and cryptic hepatic forms of the parasite. Iron chelators have recently gained importance as potent antimalarials, to cause infection nearly all protozoa obtain growth essential iron from their hosts. Iron is required for the development of the parasite. Deprivation of utilizable iron by chelation is a proficient approach to arrest parasite growth and associated infection. Thiosemicarbazones are known iron chelating agents by bonding through the sulfur and azomethine nitrogen atoms. This study is aimed at the identification of thiosemicarbazone based derivatives as possible antimalarial agents. Due to their iron chelation abilities there has been increasing interest in the investigation of thiosemicarbazones as possible antimalarials. During the course of this project, several thiosemicarbazone derivatives were synthesized and their structure confirmed using routine analytical techniques (NMR, FTIR, and HRMS). The synthesized compounds were evaluated in vitro against the chloroquine sensitive strain (3D7) of P. falciparum for antimarial activity. The compounds were also evaluated agsinst Hela cells for overt cytotoxicity. The compounds generally showed poor antimalarial activity. One compound (LKN11) was identified to possess intrinsic and moderate antimalarial activity of 6.6 μM. The compounds were generally not cytotoxic against Hela cell at concentrations of up to 20 μM, with only compound LKN10 showing modest cytotoxic activity of 9.5 μM. This research went on to identify two thiosemicarbazone based derivatives which had a significant effect on HeLa and pLDH cells. , Thesis (MSc) -- Faculty of Science, Chemistry, 2022
- Full Text:
- Authors: Nqeno, Lukhanyiso Khanyisile
- Date: 2022-04-06
- Subjects: Antimalarials , Quinoline , Thiosemicarbazones , Malaria Chemotherapy , Plasmodium falciparum , Malaria Africa, Sub-Saharan , Iron chelates Therapeutic use
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/291292 , vital:56841
- Description: Africa is one of the regions that is most affected by malaria, as 90% of all malaria deaths occur in sub-saharan Africa. Malaria is a life threatening disease responsible for an estimated 800000 deaths each year, the majority of these deaths occurred in children under the age of five. The disease is a mosquito-borne, and it is transmitted to humans by the female Anopheles mosquito. The parasite responsible for this disease belong to the Plasmodium genus with Plasmodium falciparum causing the most severe cases of the disease in humans. The most widely available anti-malarials were designed to specifically target the pathogenic blood stage in humans, however, in order to completely eradicate malaria there is a need for the development of medicines that not only target the pathogenic blood stage of the parasite but also block parasite transmission and eliminate asymptomatic and cryptic hepatic forms of the parasite. Iron chelators have recently gained importance as potent antimalarials, to cause infection nearly all protozoa obtain growth essential iron from their hosts. Iron is required for the development of the parasite. Deprivation of utilizable iron by chelation is a proficient approach to arrest parasite growth and associated infection. Thiosemicarbazones are known iron chelating agents by bonding through the sulfur and azomethine nitrogen atoms. This study is aimed at the identification of thiosemicarbazone based derivatives as possible antimalarial agents. Due to their iron chelation abilities there has been increasing interest in the investigation of thiosemicarbazones as possible antimalarials. During the course of this project, several thiosemicarbazone derivatives were synthesized and their structure confirmed using routine analytical techniques (NMR, FTIR, and HRMS). The synthesized compounds were evaluated in vitro against the chloroquine sensitive strain (3D7) of P. falciparum for antimarial activity. The compounds were also evaluated agsinst Hela cells for overt cytotoxicity. The compounds generally showed poor antimalarial activity. One compound (LKN11) was identified to possess intrinsic and moderate antimalarial activity of 6.6 μM. The compounds were generally not cytotoxic against Hela cell at concentrations of up to 20 μM, with only compound LKN10 showing modest cytotoxic activity of 9.5 μM. This research went on to identify two thiosemicarbazone based derivatives which had a significant effect on HeLa and pLDH cells. , Thesis (MSc) -- Faculty of Science, Chemistry, 2022
- Full Text:
Application of computer-aided drug design for identification of P. falciparum inhibitors
- Authors: Diallo, Bakary N’tji
- Date: 2021-10-29
- Subjects: Plasmodium falciparum , Malaria -- Chemotherapy , Molecular dynamics , Antimalarials , Cheminformatics , Drug development , Ligand binding (Biochemistry) , Plasmodium falciparum1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) , South African Natural Compounds Database
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/192798 , vital:45265 , 10.21504/10962/192798
- Description: Malaria is a millennia-old disease with the first recorded cases dating back to 2700 BC found in Chinese medical records, and later in other civilizations. It has claimed human lives to such an extent that there are a notable associated socio-economic consequences. Currently, according to the World Health Organization (WHO), Africa holds the highest disease burden with 94% of deaths and 82% of cases with P. falciparum having ~100% prevalence. Chemotherapy, such as artemisinin combination therapy, has been and continues to be the work horse in the fight against the disease, together with seasonal malaria chemoprevention and the use of insecticides. Natural products such as quinine and artemisinin are particularly important in terms of their antimalarial activity. The emphasis in current chemotherapy research is the need for time and cost-effective workflows focussed on new mechanisms of action (MoAs) covering the target candidate profiles (TCPs). Despite a decline in cases over the past decades with, countries increasingly becoming certified malaria free, a stalling trend has been observed in the past five years resulting in missing the 2020 Global Technical Strategy (GTS) milestones. With no effective vaccine, a reduction in funding, slower drug approval than resistance emergence from resistant and invasive vectors, and threats in diagnosis with the pfhrp2/3 gene deletion, malaria remains a major health concern. Motivated by these reasons, the primary aim of this work was a contribution to the antimalarial pipeline through in silico approaches focusing on P. falciparum. We first intended an exploration of malarial targets through a proteome scale screening on 36 targets using multiple metrics to account for the multi-objective nature of drug discovery. The continuous growth of structural data offers the ideal scenario for mining new MoAs covering antimalarials TCPs. This was combined with a repurposing strategy using a set of orally available FDA approved drugs. Further, use was made of time- and cost-effective strategies combining QVina-W efficiency metrics that integrate molecular properties, GRIM rescoring for molecular interactions and a hydrogen mass repartitioning (HMR) molecular dynamics (MD) scheme for accelerated development of antimalarials in the context of resistance. This pipeline further integrates a complex ranking for better drug-target selectivity, and normalization strategies to overcome docking scoring function bias. The different metrics, ranking, normalization strategies and their combinations were first assessed using their mean ranking error (MRE). A version combining all metrics was used to select 36 unique protein-ligand complexes, assessed in MD, with the final retention of 25. From the 16 in vitro tested hits of the 25, fingolimod, abiraterone, prazosin, and terazosin showed antiplasmodial activity with IC50 2.21, 3.37, 16.67 and 34.72 μM respectively and of these, only fingolimod was found to be not safe with respect to human cell viability. These compounds were predicted active on different molecular targets, abiraterone was predicted to interact with a putative liver-stage essential target, hence promising as a transmission-blocking agent. The pipeline had a promising 25% hit rate considering the proteome-scale and use of cost-effective approaches. Secondly, we focused on Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) using a more extensive screening pipeline to overcome some of the current in silico screening limitations. Starting from the ZINC lead-like library of ~3M, hierarchical ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches with molecular docking and re-scoring using eleven scoring functions (SFs) were used. Later ranking with an exponential consensus strategy was included. Selected hits were further assessed through Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA), advanced MD sampling in a ligand pulling simulations and (Weighted Histogram Analysis Method) WHAM analysis for umbrella sampling (US) to derive binding free energies. Four leads had better predicted affinities in US than LC5, a 280 nM potent PfDXR inhibitor with ZINC000050633276 showing a promising binding of -20.43 kcal/mol. As shown with fosmidomycin, DXR inhibition offers fast acting compounds fulfilling antimalarials TCP1. Yet, fosmidomycin has a high polarity causing its short half-life and hampering its clinical use. These leads scaffolds are different from fosmidomycin and hence may offer better pharmacokinetic and pharmacodynamic properties and may also be promising for lead optimization. A combined analysis of residues’ contributions to the free energy of binding in MM-PBSA and to steered molecular dynamics (SMD) Fmax indicated GLU233, CYS268, SER270, TRP296, and HIS341 as exploitable for compound optimization. Finally, we updated the SANCDB library with new NPs and their commercially available analogs as a solution to NP availability. The library is extended to 1005 compounds from its initial 600 compounds and the database is integrated to Mcule and Molport APIs for analogs automatic update. The new set may contribute to virtual screening and to antimalarials as the most effective ones have NP origin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Authors: Diallo, Bakary N’tji
- Date: 2021-10-29
- Subjects: Plasmodium falciparum , Malaria -- Chemotherapy , Molecular dynamics , Antimalarials , Cheminformatics , Drug development , Ligand binding (Biochemistry) , Plasmodium falciparum1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) , South African Natural Compounds Database
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/192798 , vital:45265 , 10.21504/10962/192798
- Description: Malaria is a millennia-old disease with the first recorded cases dating back to 2700 BC found in Chinese medical records, and later in other civilizations. It has claimed human lives to such an extent that there are a notable associated socio-economic consequences. Currently, according to the World Health Organization (WHO), Africa holds the highest disease burden with 94% of deaths and 82% of cases with P. falciparum having ~100% prevalence. Chemotherapy, such as artemisinin combination therapy, has been and continues to be the work horse in the fight against the disease, together with seasonal malaria chemoprevention and the use of insecticides. Natural products such as quinine and artemisinin are particularly important in terms of their antimalarial activity. The emphasis in current chemotherapy research is the need for time and cost-effective workflows focussed on new mechanisms of action (MoAs) covering the target candidate profiles (TCPs). Despite a decline in cases over the past decades with, countries increasingly becoming certified malaria free, a stalling trend has been observed in the past five years resulting in missing the 2020 Global Technical Strategy (GTS) milestones. With no effective vaccine, a reduction in funding, slower drug approval than resistance emergence from resistant and invasive vectors, and threats in diagnosis with the pfhrp2/3 gene deletion, malaria remains a major health concern. Motivated by these reasons, the primary aim of this work was a contribution to the antimalarial pipeline through in silico approaches focusing on P. falciparum. We first intended an exploration of malarial targets through a proteome scale screening on 36 targets using multiple metrics to account for the multi-objective nature of drug discovery. The continuous growth of structural data offers the ideal scenario for mining new MoAs covering antimalarials TCPs. This was combined with a repurposing strategy using a set of orally available FDA approved drugs. Further, use was made of time- and cost-effective strategies combining QVina-W efficiency metrics that integrate molecular properties, GRIM rescoring for molecular interactions and a hydrogen mass repartitioning (HMR) molecular dynamics (MD) scheme for accelerated development of antimalarials in the context of resistance. This pipeline further integrates a complex ranking for better drug-target selectivity, and normalization strategies to overcome docking scoring function bias. The different metrics, ranking, normalization strategies and their combinations were first assessed using their mean ranking error (MRE). A version combining all metrics was used to select 36 unique protein-ligand complexes, assessed in MD, with the final retention of 25. From the 16 in vitro tested hits of the 25, fingolimod, abiraterone, prazosin, and terazosin showed antiplasmodial activity with IC50 2.21, 3.37, 16.67 and 34.72 μM respectively and of these, only fingolimod was found to be not safe with respect to human cell viability. These compounds were predicted active on different molecular targets, abiraterone was predicted to interact with a putative liver-stage essential target, hence promising as a transmission-blocking agent. The pipeline had a promising 25% hit rate considering the proteome-scale and use of cost-effective approaches. Secondly, we focused on Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) using a more extensive screening pipeline to overcome some of the current in silico screening limitations. Starting from the ZINC lead-like library of ~3M, hierarchical ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches with molecular docking and re-scoring using eleven scoring functions (SFs) were used. Later ranking with an exponential consensus strategy was included. Selected hits were further assessed through Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA), advanced MD sampling in a ligand pulling simulations and (Weighted Histogram Analysis Method) WHAM analysis for umbrella sampling (US) to derive binding free energies. Four leads had better predicted affinities in US than LC5, a 280 nM potent PfDXR inhibitor with ZINC000050633276 showing a promising binding of -20.43 kcal/mol. As shown with fosmidomycin, DXR inhibition offers fast acting compounds fulfilling antimalarials TCP1. Yet, fosmidomycin has a high polarity causing its short half-life and hampering its clinical use. These leads scaffolds are different from fosmidomycin and hence may offer better pharmacokinetic and pharmacodynamic properties and may also be promising for lead optimization. A combined analysis of residues’ contributions to the free energy of binding in MM-PBSA and to steered molecular dynamics (SMD) Fmax indicated GLU233, CYS268, SER270, TRP296, and HIS341 as exploitable for compound optimization. Finally, we updated the SANCDB library with new NPs and their commercially available analogs as a solution to NP availability. The library is extended to 1005 compounds from its initial 600 compounds and the database is integrated to Mcule and Molport APIs for analogs automatic update. The new set may contribute to virtual screening and to antimalarials as the most effective ones have NP origin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
A medicinal chemistry study in nitrogen containing heterocycles
- Authors: Lunga, Mayibongwe Junior
- Date: 2018
- Subjects: Indole , Tetrazoles , Antimalarials , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63521 , vital:28430
- Description: Heterocyclic structures have found extensive utility in the field of medicinal chemistry, as prominent regions of pharmacophores resulting in numerous drug treatments for many diseases. Accordingly, in this project we explored the respective antimalarial and anticancer activity exhibited by compounds featuring nitrogen containing indole and tetrazole heterocycles respectively. This thesis therefore comprises of two distinct parts. Part 1. Following the development of resistance towards traditional antimalarial therapy such as chloroquine and emerging resistance towards artemisinin combination therapies, the WHO reported the urgent need for new, effective drugs and identification of new drug targets to combat the Plasmodium falciparum parasite. In 2015 the parasite was the cause of 429 000 deaths, the majority occurring in the sub-Saharan region of Africa. This highlights the failing effectiveness of vector control strategies, reiterating the need to develop alternative control and treatment strategies. In response to this need we wanted to expand and further describe the SAR of the indole based series, indolyl-3-ethanone-α- thioethers, previously synthesized in our laboratory. These compounds were found to exhibit antimalarial activity with compounds 2.26 and 2.27 exhibiting activity against P. falciparum 3D7 in the nanomolar range. Based on these compounds we synthesized compounds 3.21 and 3.24 – 3.32 following a three step reaction pathway. Our results in this study, indicate that compound 3.28, a pnitrothiophenol analogue of 2.27 was the most active of the compounds we synthesized and furthermore was superior in activity against Plasmodium compared to 2.27. This result indicated that the presence of p-NO2 is important in enhancing anti-plasmodial activity. Comparing compounds 3.25 and 3.26 with an oxygen on the ether bridge to compounds 3.29 and 3.30 with a sulfur, we observed an increase in hydrophilicity coupled to a decrease in anti-plasmodial activity in the compounds, thus, highlighting the importance of sulfur for enhanced activity. Furthermore, we investigated bioisosteric replacement of the 5-chloro substituent present in hit compounds 2.27 and 3.28, with an electron withdrawing nitrile (3.27) and electron donating methyl (3.29) and methoxy (3.31) substituents. These substituents decreased anti-plasmodial activity, confirming that a chlorine substituent is optimal for biological activity. This study furthered our understanding of the SAR of indolyl-3-ethanone-α- thioethers for the development of potent anti-plasmodial lead compounds. Part 2. Triple negative breast cancer (TNBC), which disproportionately affects women of sub-Saharan Africa, is unresponsive to hormone-based therapies. This emergence presents a population of patients devoid of effective drug treatment, signaling the urgent need to develop new effective therapies with novel drug targets. Therefore, we identified our target in TNBC cells as the protein-protein interaction between the co-chaperones HOP and HSP90. We reasoned that a disruption of this interaction would ultimately result in cancer cell death via the degradation of essential oncogenic client proteins. Following a fragment screening campaign, which identified several acid and tetrazole containing hits (4.56 – 4.58) which bound to HOP, with low anticancer activity, we sought to develop synthetic methodology to elaborate our fragment hits synthesizing tetrazole containing fragments to target TNBC cell lines. We therefore proceeded to synthesize a range of multi substituted fragments (4.59 – 4.63), utilizing a nitrile (4.66) to access tetrazoles via 1,3-cycloaddition and an acid by nitrile hydrolysis. We successfully synthesized the tetrazole and acid fragments which are currently undergoing characterization for activity against TNBC. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Authors: Lunga, Mayibongwe Junior
- Date: 2018
- Subjects: Indole , Tetrazoles , Antimalarials , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63521 , vital:28430
- Description: Heterocyclic structures have found extensive utility in the field of medicinal chemistry, as prominent regions of pharmacophores resulting in numerous drug treatments for many diseases. Accordingly, in this project we explored the respective antimalarial and anticancer activity exhibited by compounds featuring nitrogen containing indole and tetrazole heterocycles respectively. This thesis therefore comprises of two distinct parts. Part 1. Following the development of resistance towards traditional antimalarial therapy such as chloroquine and emerging resistance towards artemisinin combination therapies, the WHO reported the urgent need for new, effective drugs and identification of new drug targets to combat the Plasmodium falciparum parasite. In 2015 the parasite was the cause of 429 000 deaths, the majority occurring in the sub-Saharan region of Africa. This highlights the failing effectiveness of vector control strategies, reiterating the need to develop alternative control and treatment strategies. In response to this need we wanted to expand and further describe the SAR of the indole based series, indolyl-3-ethanone-α- thioethers, previously synthesized in our laboratory. These compounds were found to exhibit antimalarial activity with compounds 2.26 and 2.27 exhibiting activity against P. falciparum 3D7 in the nanomolar range. Based on these compounds we synthesized compounds 3.21 and 3.24 – 3.32 following a three step reaction pathway. Our results in this study, indicate that compound 3.28, a pnitrothiophenol analogue of 2.27 was the most active of the compounds we synthesized and furthermore was superior in activity against Plasmodium compared to 2.27. This result indicated that the presence of p-NO2 is important in enhancing anti-plasmodial activity. Comparing compounds 3.25 and 3.26 with an oxygen on the ether bridge to compounds 3.29 and 3.30 with a sulfur, we observed an increase in hydrophilicity coupled to a decrease in anti-plasmodial activity in the compounds, thus, highlighting the importance of sulfur for enhanced activity. Furthermore, we investigated bioisosteric replacement of the 5-chloro substituent present in hit compounds 2.27 and 3.28, with an electron withdrawing nitrile (3.27) and electron donating methyl (3.29) and methoxy (3.31) substituents. These substituents decreased anti-plasmodial activity, confirming that a chlorine substituent is optimal for biological activity. This study furthered our understanding of the SAR of indolyl-3-ethanone-α- thioethers for the development of potent anti-plasmodial lead compounds. Part 2. Triple negative breast cancer (TNBC), which disproportionately affects women of sub-Saharan Africa, is unresponsive to hormone-based therapies. This emergence presents a population of patients devoid of effective drug treatment, signaling the urgent need to develop new effective therapies with novel drug targets. Therefore, we identified our target in TNBC cells as the protein-protein interaction between the co-chaperones HOP and HSP90. We reasoned that a disruption of this interaction would ultimately result in cancer cell death via the degradation of essential oncogenic client proteins. Following a fragment screening campaign, which identified several acid and tetrazole containing hits (4.56 – 4.58) which bound to HOP, with low anticancer activity, we sought to develop synthetic methodology to elaborate our fragment hits synthesizing tetrazole containing fragments to target TNBC cell lines. We therefore proceeded to synthesize a range of multi substituted fragments (4.59 – 4.63), utilizing a nitrile (4.66) to access tetrazoles via 1,3-cycloaddition and an acid by nitrile hydrolysis. We successfully synthesized the tetrazole and acid fragments which are currently undergoing characterization for activity against TNBC. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
Exploring para-thiophenols to expand the SAR of antimalarial 3-indolylethanones
- Authors: Chisango, Ruramai Lissa
- Date: 2018
- Subjects: Antimalarials , Malaria Chemotherapy , Thiols , Plasmodium falciparum , Blood-brain barrier
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63515 , vital:28428
- Description: According to the WHO, malaria is responsible for over half a million deaths annually especially in populations from disadvantaged settings. Although there has been a documented improvement in the mortality rates, malaria has proved to be a global emergency. Mostly affecting the poor population, this disease is perpetuating a vicious cycle of poverty in the developing world as current preventive measures are not adequate unless adopted in addition to effective treatment. However, there has been a worldwide increase in resistance to available treatment which presents a need for novel, affordable treatment. A study conducted in our laboratory identified two hit thiophenol containing compounds 2.24 and 2.25. These molecules provided initial insight into the SAR and potential pharmacophore of this class of compounds. We decided to further explore these compounds by making bioisosteric replacements to optimize the structure as we monitor the effect of these modifications on the anti-plasmodial activity. The synthetic pathway to form the target compounds of our study comprised of three steps which were initiated by the Friedel-Crafts acetylation of the indoles resulting in compounds 3.5 - 3.7. A bromination step followed which yielded the -bromo ketones (3.8 - 3.11). Some of the thiophenols (3.14 and 3.16) were not readily available in our laboratory and so were synthesized for the final synthetic step. This step involved the nucleophilic displacement of the -bromine to generate the -aryl substituted 3-indolylethanones (3.17 - 3.27). The thioethers displayed improved antimalarial activity from 2.24 and 2.25 against the chloroquine sensitive 3D7 Plasmodium falciparum strain. In addition, these compounds were non-toxic against HeLa cells which indicated this potential novel class of antimalarials is selective for the malaria parasite as hypothesized in the previous study conducted in our laboratory. In an attempt to predict the bioavailability of some of our compounds, in silico studies were conducted revealing that these compounds could be passively absorbed by the gastrointestinal tract, a positive result for bioavailability purposes. However, results from these studies indicate that modifications of these compounds would be necessary to allow for permeation through the blood brain barrier (BBB) for instances when the patient has cerebral malaria. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Authors: Chisango, Ruramai Lissa
- Date: 2018
- Subjects: Antimalarials , Malaria Chemotherapy , Thiols , Plasmodium falciparum , Blood-brain barrier
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63515 , vital:28428
- Description: According to the WHO, malaria is responsible for over half a million deaths annually especially in populations from disadvantaged settings. Although there has been a documented improvement in the mortality rates, malaria has proved to be a global emergency. Mostly affecting the poor population, this disease is perpetuating a vicious cycle of poverty in the developing world as current preventive measures are not adequate unless adopted in addition to effective treatment. However, there has been a worldwide increase in resistance to available treatment which presents a need for novel, affordable treatment. A study conducted in our laboratory identified two hit thiophenol containing compounds 2.24 and 2.25. These molecules provided initial insight into the SAR and potential pharmacophore of this class of compounds. We decided to further explore these compounds by making bioisosteric replacements to optimize the structure as we monitor the effect of these modifications on the anti-plasmodial activity. The synthetic pathway to form the target compounds of our study comprised of three steps which were initiated by the Friedel-Crafts acetylation of the indoles resulting in compounds 3.5 - 3.7. A bromination step followed which yielded the -bromo ketones (3.8 - 3.11). Some of the thiophenols (3.14 and 3.16) were not readily available in our laboratory and so were synthesized for the final synthetic step. This step involved the nucleophilic displacement of the -bromine to generate the -aryl substituted 3-indolylethanones (3.17 - 3.27). The thioethers displayed improved antimalarial activity from 2.24 and 2.25 against the chloroquine sensitive 3D7 Plasmodium falciparum strain. In addition, these compounds were non-toxic against HeLa cells which indicated this potential novel class of antimalarials is selective for the malaria parasite as hypothesized in the previous study conducted in our laboratory. In an attempt to predict the bioavailability of some of our compounds, in silico studies were conducted revealing that these compounds could be passively absorbed by the gastrointestinal tract, a positive result for bioavailability purposes. However, results from these studies indicate that modifications of these compounds would be necessary to allow for permeation through the blood brain barrier (BBB) for instances when the patient has cerebral malaria. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
Synthesis and biolgical screening of potential plasmodium falciparum DXR inhibitors
- Authors: Adeyemi, Christiana Modupe
- Date: 2017-04
- Subjects: Plasmodium falciparum , Enzyme inhibitors , Malaria , Antimalarials , Drug development , Malaria -- Chemotherapy , Isopentenoids -- Synthesis , Fosmidomycin , 1-Deoxy-D-xylulose 5-phosphate
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61790 , vital:28060
- Description: The non-mevalonate isoprenoid pathway, also known as the 1-deoxy-D-xylulose-5- phosphate DXP pathway, is absent in humans, but present in the anopheles mosquito responsible for the transmission of malaria. DXP reductoisomerase - a key enzyme in the DXP pathway in Plasmodium falciparum (PfDXR) has been identified as a target for the design of novel anti-malarial drugs. Fosmidomycin and its acetyl analogue (FR900098) are known to be inhibitors of PfDXR and, in this study, synthetic variations of the fosmidomycin scaffold have led to four series of novel analogues. Particular attention has been centred on the introduction of various substituted benzyl groups in each of these series in order to occupy a recently discovered vacant pocket in the PfDXR active-site and thus enhance ligand-enzyme binding. In the process 160 ligands and precursors have been prepared, no less than 119 of them novel. Fistly, a series of C-benzylated phosphonate esters and phosphonic acids were synthesised, in which the fosmidomycin hydroxamate Mg2+- coordinating moiety was replaced by an amide funtionality and the number of methylene groups in the “hydrophobic patch” between the phosphonate and the hydroxamate moiety was decreased from two to one. Several approaches were explored for this series, the most successful involving reaction of 3- substituted anilines with a-bromo propanoic acid in the presence of the coupling agent 1,1'- carbonyldiimidazole (CDI), followed by Michaelis-Arbuzov phosphonation using triethyl phosphite. Reaction of the resulting chiral phosphonate esters with bromotrimethylsilane gave the corresponding phosphonic acids in good yields. In order to obviate chirality issues, a second series of potential “reverse” fosmidomycin analogues was synthesised by replacing the methylene group adjacent to the the phosphonate moiety with a nitrogen atom. Deprotonation, alkylation and phosphorylation of various amines gave diethyl #-benzylphosphoramidate ester intermediate. Aza-Michael addition of these intermediates, followed by hydrolysis gave the corresponding carboxylic acids which could be reacted with different hydroxylamine hydrochloride derivatives to afford the novel hydroxamic acid derivatives in good yields. Thirdly, a series of a novel #-benzylated phosphoramidate derivatives were prepared as aza- FR900098 analogues. Alkylation of different amines using bromoacetalde-hyde diethylacetal gave a series of N-benzyl-2,2-diethoxyethylamine compounds, which were then elaborated via a futher six steps to afford novel #-benzylated phosphoramidate derivatives. Finally, in order to ensure syn-orientation of the donor atoms in the Mg - coordinating group and, at the same time, introduce conformational constraints in the ligand, the hydrophobic patch and the hydroxamate moiety were replaced by cyclic systems. Several approaches towards the synthesis of such conformationally constrained phosphoramidate analogues from maleic anhydride led to the unexpected isolation of an unprecedented acyclic furfuryl compound, and 1H NMR and DFT-level theoretical studies have been initiated to explore the reaction sequence. A series of #-benzylated phosphoramidate derivatives containing dihydroxy aromatic rings (as the conformationally constrained groups) to replace the hydroxamate moiety, were successfully obtained in six steps from the starting material, 3,4-dihydroxylbenzaldehyde. While in vitro assays have been conducted on all of the synthesised compounds, and some of the ligands show promising anti-malarial inhibitory activity - most especially the conformationally constrained cyclic #-benzylated phosphoramidate series. Interestingly, a number of these compounds has also shown activity against T.brucei - the causative agent of sleeping sickness. In silico docking studies of selected compounds has revealed the capacity of some of the ligands to bind effectively in the PfDXR active-site with the newly introduced benzyl group occupying the adjacent vacant pocket. The physico-chemical properties of these ligands were also explored in order to predict the oral-bioavailability. Most of the ligands obeyed the Lipinski rule of 5, while QSAR methods have been used in an attempt to correlate structural variations and calculated molecular properties with the bioassay data. , Thesis (PhD) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Authors: Adeyemi, Christiana Modupe
- Date: 2017-04
- Subjects: Plasmodium falciparum , Enzyme inhibitors , Malaria , Antimalarials , Drug development , Malaria -- Chemotherapy , Isopentenoids -- Synthesis , Fosmidomycin , 1-Deoxy-D-xylulose 5-phosphate
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61790 , vital:28060
- Description: The non-mevalonate isoprenoid pathway, also known as the 1-deoxy-D-xylulose-5- phosphate DXP pathway, is absent in humans, but present in the anopheles mosquito responsible for the transmission of malaria. DXP reductoisomerase - a key enzyme in the DXP pathway in Plasmodium falciparum (PfDXR) has been identified as a target for the design of novel anti-malarial drugs. Fosmidomycin and its acetyl analogue (FR900098) are known to be inhibitors of PfDXR and, in this study, synthetic variations of the fosmidomycin scaffold have led to four series of novel analogues. Particular attention has been centred on the introduction of various substituted benzyl groups in each of these series in order to occupy a recently discovered vacant pocket in the PfDXR active-site and thus enhance ligand-enzyme binding. In the process 160 ligands and precursors have been prepared, no less than 119 of them novel. Fistly, a series of C-benzylated phosphonate esters and phosphonic acids were synthesised, in which the fosmidomycin hydroxamate Mg2+- coordinating moiety was replaced by an amide funtionality and the number of methylene groups in the “hydrophobic patch” between the phosphonate and the hydroxamate moiety was decreased from two to one. Several approaches were explored for this series, the most successful involving reaction of 3- substituted anilines with a-bromo propanoic acid in the presence of the coupling agent 1,1'- carbonyldiimidazole (CDI), followed by Michaelis-Arbuzov phosphonation using triethyl phosphite. Reaction of the resulting chiral phosphonate esters with bromotrimethylsilane gave the corresponding phosphonic acids in good yields. In order to obviate chirality issues, a second series of potential “reverse” fosmidomycin analogues was synthesised by replacing the methylene group adjacent to the the phosphonate moiety with a nitrogen atom. Deprotonation, alkylation and phosphorylation of various amines gave diethyl #-benzylphosphoramidate ester intermediate. Aza-Michael addition of these intermediates, followed by hydrolysis gave the corresponding carboxylic acids which could be reacted with different hydroxylamine hydrochloride derivatives to afford the novel hydroxamic acid derivatives in good yields. Thirdly, a series of a novel #-benzylated phosphoramidate derivatives were prepared as aza- FR900098 analogues. Alkylation of different amines using bromoacetalde-hyde diethylacetal gave a series of N-benzyl-2,2-diethoxyethylamine compounds, which were then elaborated via a futher six steps to afford novel #-benzylated phosphoramidate derivatives. Finally, in order to ensure syn-orientation of the donor atoms in the Mg - coordinating group and, at the same time, introduce conformational constraints in the ligand, the hydrophobic patch and the hydroxamate moiety were replaced by cyclic systems. Several approaches towards the synthesis of such conformationally constrained phosphoramidate analogues from maleic anhydride led to the unexpected isolation of an unprecedented acyclic furfuryl compound, and 1H NMR and DFT-level theoretical studies have been initiated to explore the reaction sequence. A series of #-benzylated phosphoramidate derivatives containing dihydroxy aromatic rings (as the conformationally constrained groups) to replace the hydroxamate moiety, were successfully obtained in six steps from the starting material, 3,4-dihydroxylbenzaldehyde. While in vitro assays have been conducted on all of the synthesised compounds, and some of the ligands show promising anti-malarial inhibitory activity - most especially the conformationally constrained cyclic #-benzylated phosphoramidate series. Interestingly, a number of these compounds has also shown activity against T.brucei - the causative agent of sleeping sickness. In silico docking studies of selected compounds has revealed the capacity of some of the ligands to bind effectively in the PfDXR active-site with the newly introduced benzyl group occupying the adjacent vacant pocket. The physico-chemical properties of these ligands were also explored in order to predict the oral-bioavailability. Most of the ligands obeyed the Lipinski rule of 5, while QSAR methods have been used in an attempt to correlate structural variations and calculated molecular properties with the bioassay data. , Thesis (PhD) -- Faculty of Science, Chemistry, 2017
- Full Text:
Synthesis, characterisation and evaluation of benzoxaborole-based hybrids as antiplasmodial agents
- Authors: Gumbo, Maureen
- Date: 2017
- Subjects: Malaria Chemotherapy , Antimalarials , Boron compounds , Drug resistance , Plasmodium falciparum , Drug development
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59193 , vital:27456
- Description: Malaria is a mosquito-borne disease, which continues to pose a threat to the entire humanity. About 40% of the world population is estimated to be at risk of infections by malaria. Despite efforts undertaken by scientific community, government entities and international organizations, malaria is still rampant. The major problem is drug resistance, where the Plasmodium spp have over the past decades developed drug resistance against available drugs. In order to counter this problem, novel antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Benzoxaborole derivatives have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported on the compounds such as 6-(2- (alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroles, which showed good antimalarial activity against both W7 and 3D7 strains without significant toxicity. On the other hand, chloroquine (CQ) and cinnamic acids have a wide variety of biological activity including antimalarial activity. Herein, a hybridisation strategy was employed to synthesise new CQ-benzoxaborole and cinnamoyl-benzoxaborole hybrids. CQ-Benzoxaborole 2.12a-c and cinnamoylbenzoxaborole 2.11a-g hydrid molecules were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (1H and 13C NMR, and mass spectrometry). CQ-benzoxaborole compounds, however, showed instability, and only 2.12b was used for in vitro biological assay and showed activity comparable to CQ. Furthermore, in vitro biological assay revealed that compounds 2.11a-g poorly inhibited the growth of P. falciparum parasites. Interestingly, these compounds, however, exhibited satisfactory activity against Trypanosoma brucei with IC50 = 0.052 μM for compound 2.11g. The cell cytotoxicity assay of all final compounds confirmed that all CQ-benzoxaborole 2.12b and cinnamoyl-benzoxaborole 2.11a-g hybrids were non-toxic against HeLa cell lines. However, efforts to further expand the structure-activity relationship (SAR) of CQbenzoxaborole by increasing the length of the linker with one extra carbon (Scheme 2.10) were not possible as an important precursor 6-formylbenzoxaborole 2.29 could not be synthesized in sufficient yields. , Thesis (MSc) -- Faculty of Faculty of Science, Chemistry, 2017
- Full Text:
- Authors: Gumbo, Maureen
- Date: 2017
- Subjects: Malaria Chemotherapy , Antimalarials , Boron compounds , Drug resistance , Plasmodium falciparum , Drug development
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59193 , vital:27456
- Description: Malaria is a mosquito-borne disease, which continues to pose a threat to the entire humanity. About 40% of the world population is estimated to be at risk of infections by malaria. Despite efforts undertaken by scientific community, government entities and international organizations, malaria is still rampant. The major problem is drug resistance, where the Plasmodium spp have over the past decades developed drug resistance against available drugs. In order to counter this problem, novel antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Benzoxaborole derivatives have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported on the compounds such as 6-(2- (alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroles, which showed good antimalarial activity against both W7 and 3D7 strains without significant toxicity. On the other hand, chloroquine (CQ) and cinnamic acids have a wide variety of biological activity including antimalarial activity. Herein, a hybridisation strategy was employed to synthesise new CQ-benzoxaborole and cinnamoyl-benzoxaborole hybrids. CQ-Benzoxaborole 2.12a-c and cinnamoylbenzoxaborole 2.11a-g hydrid molecules were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (1H and 13C NMR, and mass spectrometry). CQ-benzoxaborole compounds, however, showed instability, and only 2.12b was used for in vitro biological assay and showed activity comparable to CQ. Furthermore, in vitro biological assay revealed that compounds 2.11a-g poorly inhibited the growth of P. falciparum parasites. Interestingly, these compounds, however, exhibited satisfactory activity against Trypanosoma brucei with IC50 = 0.052 μM for compound 2.11g. The cell cytotoxicity assay of all final compounds confirmed that all CQ-benzoxaborole 2.12b and cinnamoyl-benzoxaborole 2.11a-g hybrids were non-toxic against HeLa cell lines. However, efforts to further expand the structure-activity relationship (SAR) of CQbenzoxaborole by increasing the length of the linker with one extra carbon (Scheme 2.10) were not possible as an important precursor 6-formylbenzoxaborole 2.29 could not be synthesized in sufficient yields. , Thesis (MSc) -- Faculty of Faculty of Science, Chemistry, 2017
- Full Text:
Synthesis, characterisation and evaluation of ferrocene-containing Novobiocin analogues for anticancer and antiplasmodial activity through inhibition of Hsp90
- Authors: Mbaba, Mziyanda
- Date: 2017
- Subjects: Antibiotics Synthesis , Ferrocene , Heat shock proteins , Antimalarials , Cancer Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65111 , vital:28690
- Description: Novobiocin (Nb) is a coumarin type antibiotic isolated from the bacterium species of Streptomyces and possesses modest anticancer and antimalarial activities. Nb and analogues have been extensively explored as potential anticancer agents through inhibition of the C- terminal domain of heat shock protein 90 (Hsp90), which plays a pivotal role in the proteinfolding machinery of cells. There has been little effort in the exploration of Nb and derivatives for antimalarial activity. Incorporation of organometallic units, such as ferrocene (Fc), into bioactive chemical scaffolds remains an attractive approach for developing new therapeutic agents for treatment of several ailments. The current study sought to investigate the anticancer and antiplasmodial effects of incorporating ferrocene (Fc) into Nb scaffold presumably through inhibition of Hsp90. The ferrocenyl Nb analogues containing simplified structural motifs such as phenyl, benzyl, and piperidine were synthesized in six to nine steps employing conventional synthetic organic protocols adapted from literature, and the compounds were accessed in reasonable yields. For comparison purposes, a selection of organic Nb analogues were also included in the study. The target compounds were characterized by spectroscopic techniques including 1-dimensional nuclear magnetic resonance (1D NMR) and high-resolution mass spectroscopy. The synthesized compounds were evaluated in vitro for potential anticancer and antiplasmodial activities using the breast cancer cell line (HCC38) and chloroquine-sensitive strain (3D7) of the malaria parasite, Plasmodium falciparum. The presence of the Fc unit was found to enhance both anticancer and antiplasmodial activities of the resultant ferrocenyl Nb compounds with IC50 values in the low to mid micromolar range. Hsp90 inhibitory studies of the ferrocenyl Nb analogues possessing superior activities (2.13a and 2.20c) were also conducted using different yeast strains expressing both human and malarial Hsp90 isoforms: hHsp90a/p and PfHsp90, respectively. The results of Hsp90 inhibitory studies suggested no direct correlation between the observed activities of the analogues and Hsp90 inhibition. However, since the conditions of the assay were not optimised due to time constrains of the project, these observed data remained to be confirmed. , Thesis (MSc) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Authors: Mbaba, Mziyanda
- Date: 2017
- Subjects: Antibiotics Synthesis , Ferrocene , Heat shock proteins , Antimalarials , Cancer Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65111 , vital:28690
- Description: Novobiocin (Nb) is a coumarin type antibiotic isolated from the bacterium species of Streptomyces and possesses modest anticancer and antimalarial activities. Nb and analogues have been extensively explored as potential anticancer agents through inhibition of the C- terminal domain of heat shock protein 90 (Hsp90), which plays a pivotal role in the proteinfolding machinery of cells. There has been little effort in the exploration of Nb and derivatives for antimalarial activity. Incorporation of organometallic units, such as ferrocene (Fc), into bioactive chemical scaffolds remains an attractive approach for developing new therapeutic agents for treatment of several ailments. The current study sought to investigate the anticancer and antiplasmodial effects of incorporating ferrocene (Fc) into Nb scaffold presumably through inhibition of Hsp90. The ferrocenyl Nb analogues containing simplified structural motifs such as phenyl, benzyl, and piperidine were synthesized in six to nine steps employing conventional synthetic organic protocols adapted from literature, and the compounds were accessed in reasonable yields. For comparison purposes, a selection of organic Nb analogues were also included in the study. The target compounds were characterized by spectroscopic techniques including 1-dimensional nuclear magnetic resonance (1D NMR) and high-resolution mass spectroscopy. The synthesized compounds were evaluated in vitro for potential anticancer and antiplasmodial activities using the breast cancer cell line (HCC38) and chloroquine-sensitive strain (3D7) of the malaria parasite, Plasmodium falciparum. The presence of the Fc unit was found to enhance both anticancer and antiplasmodial activities of the resultant ferrocenyl Nb compounds with IC50 values in the low to mid micromolar range. Hsp90 inhibitory studies of the ferrocenyl Nb analogues possessing superior activities (2.13a and 2.20c) were also conducted using different yeast strains expressing both human and malarial Hsp90 isoforms: hHsp90a/p and PfHsp90, respectively. The results of Hsp90 inhibitory studies suggested no direct correlation between the observed activities of the analogues and Hsp90 inhibition. However, since the conditions of the assay were not optimised due to time constrains of the project, these observed data remained to be confirmed. , Thesis (MSc) -- Faculty of Science, Chemistry, 2017
- Full Text:
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