Design, formulation and evalauation of liposomes co-loaded with human serum Albumin and Rifampicin
- Authors: Bapolisi, Alain Murhimalika
- Date: 2020
- Subjects: Liposomes , Serum albumin , Rifampin , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/161780 , vital:40670
- Description: Tuberculosis (TB) is a devastating infectious disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious agent. The high morbidity and mortality rates of TB are partly due to factors such as the lengthy regimen (of 6–24 months), the development of drug resistance, and the pathogen location within the macrophages. These, with poor physiochemical properties of existing drugs hamper the effectiveness of the treatment despite the existence of potent antibiotics such as Rifampicin (Rif). Hydrophobicity plagues many drugs, including Rif, which are then particularly affected due to inherently poor intracellular availability. Novel drug delivery approaches are therefore needed in order to optimize the cytotoxic potential of said antitubercular drugs. To improve the bioavailability of hydrophobic drugs, numerous delivery strategies have been developed. Amongst these, the coordination of cytotoxic drugs to therapeutic proteins have shown some success for improved efficacy in the management of illnesses including infectious diseases. Of therapeutic proteins, Human Serum Albumin (HSA) is an attractive drug carrier with interestingbenefits such as low immunogenicity, antioxidant properties and improving cellular uptake ofdrugs through HSA-specific binding sites which are expressed on most cells including macrophages, where M. tuberculosis often resides. Hence, combination of Rif to HSA (Rif-HSA)seems a promising approach for improved intracellular delivery of Rif. However, the in vivo stability of colloidal protein-based therapeutics is mostly challenging and an effective vehicle is needed to control the biological fate of such conjugates.Liposomes seem to be appropriate carriers for the Rif-HSA complex due to their reputable applicability for encapsulating diverse materials (i.e., hydrophobic and hydrophilic compounds or small and complex molecules) and preventing chemical and biological degradation of the cargo. Therefore, the main objective of this study was to simultaneously encapsulate Rif and HSA in liposomes, which, to the best of our knowledge, has not been done before. The dual liposomes (Rif-HSA-lip) were made by a modified “Reverse Phase Evaporation” method (REV), following a Design of Experiments (DOE) approach to determine which factors impact the formulation. In addition, liposomes were made from crude soybean lecithin (CSL), rather than expensive and highly purified lipids. The liposomes were fully characterised, and the encapsulation efficiency (î) was monitored using high-performance liquid chromatography (HPLC). The results were correlated with factors such as organic and aqueous phase composition, as well as the in vitro release profile of Rif. Transmission electron microscopy (TEM) results confirmed the formation of spherical dual liposomes nanoparticles of roughly 200 nm. Dynamic light scattering (DLS) and Zeta potential measurements showed a negative charge (<–45 mV) and with satisfactory polydispersity (PDI<0.5). HSA dramatically improved the aqueous solubility of Rif (from1.9 mg/ml in water to around 4.3 mg/ml in HSA 10% solution) mainly due to Rif-HSA hydrophobic interactions. This resulted in a good î of almost 60% for Rif, despite the presence of bulky HSA in the lipid bilayer. These details were confirmed using proton nuclear magnetic resonance (1H NMR) and Fourier-transform infrared spectroscopy (FTIR). Furthermore, energy dispersive X-ray (EDX) and DLS data suggested the presence of HSA poking out on the surface of liposomes, which is encouraging for potential targeted delivery in the future. The in vitro release studies also depicted a substantial improvement in the diffusion of Rif in dual liposomes versus free Rif, from 65% after 12 hours for free Rif to 95% after only 5 hours for Rif- HSA-lip. Finally, stability studies conducted over 30 days at room temperature, showed that the freeze-dried formulations of Rif-HSA-lip exhibited good shelf stability over liposomes with no HSA. This study represents an illustrative example of co-loading of antibiotics and proteins into liposomes, which could encourage further development of novel nanoparticulate tools for the effective management of both drug-susceptible and -resistant infectious diseases such as TB.
- Full Text:
- Authors: Bapolisi, Alain Murhimalika
- Date: 2020
- Subjects: Liposomes , Serum albumin , Rifampin , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/161780 , vital:40670
- Description: Tuberculosis (TB) is a devastating infectious disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious agent. The high morbidity and mortality rates of TB are partly due to factors such as the lengthy regimen (of 6–24 months), the development of drug resistance, and the pathogen location within the macrophages. These, with poor physiochemical properties of existing drugs hamper the effectiveness of the treatment despite the existence of potent antibiotics such as Rifampicin (Rif). Hydrophobicity plagues many drugs, including Rif, which are then particularly affected due to inherently poor intracellular availability. Novel drug delivery approaches are therefore needed in order to optimize the cytotoxic potential of said antitubercular drugs. To improve the bioavailability of hydrophobic drugs, numerous delivery strategies have been developed. Amongst these, the coordination of cytotoxic drugs to therapeutic proteins have shown some success for improved efficacy in the management of illnesses including infectious diseases. Of therapeutic proteins, Human Serum Albumin (HSA) is an attractive drug carrier with interestingbenefits such as low immunogenicity, antioxidant properties and improving cellular uptake ofdrugs through HSA-specific binding sites which are expressed on most cells including macrophages, where M. tuberculosis often resides. Hence, combination of Rif to HSA (Rif-HSA)seems a promising approach for improved intracellular delivery of Rif. However, the in vivo stability of colloidal protein-based therapeutics is mostly challenging and an effective vehicle is needed to control the biological fate of such conjugates.Liposomes seem to be appropriate carriers for the Rif-HSA complex due to their reputable applicability for encapsulating diverse materials (i.e., hydrophobic and hydrophilic compounds or small and complex molecules) and preventing chemical and biological degradation of the cargo. Therefore, the main objective of this study was to simultaneously encapsulate Rif and HSA in liposomes, which, to the best of our knowledge, has not been done before. The dual liposomes (Rif-HSA-lip) were made by a modified “Reverse Phase Evaporation” method (REV), following a Design of Experiments (DOE) approach to determine which factors impact the formulation. In addition, liposomes were made from crude soybean lecithin (CSL), rather than expensive and highly purified lipids. The liposomes were fully characterised, and the encapsulation efficiency (î) was monitored using high-performance liquid chromatography (HPLC). The results were correlated with factors such as organic and aqueous phase composition, as well as the in vitro release profile of Rif. Transmission electron microscopy (TEM) results confirmed the formation of spherical dual liposomes nanoparticles of roughly 200 nm. Dynamic light scattering (DLS) and Zeta potential measurements showed a negative charge (<–45 mV) and with satisfactory polydispersity (PDI<0.5). HSA dramatically improved the aqueous solubility of Rif (from1.9 mg/ml in water to around 4.3 mg/ml in HSA 10% solution) mainly due to Rif-HSA hydrophobic interactions. This resulted in a good î of almost 60% for Rif, despite the presence of bulky HSA in the lipid bilayer. These details were confirmed using proton nuclear magnetic resonance (1H NMR) and Fourier-transform infrared spectroscopy (FTIR). Furthermore, energy dispersive X-ray (EDX) and DLS data suggested the presence of HSA poking out on the surface of liposomes, which is encouraging for potential targeted delivery in the future. The in vitro release studies also depicted a substantial improvement in the diffusion of Rif in dual liposomes versus free Rif, from 65% after 12 hours for free Rif to 95% after only 5 hours for Rif- HSA-lip. Finally, stability studies conducted over 30 days at room temperature, showed that the freeze-dried formulations of Rif-HSA-lip exhibited good shelf stability over liposomes with no HSA. This study represents an illustrative example of co-loading of antibiotics and proteins into liposomes, which could encourage further development of novel nanoparticulate tools for the effective management of both drug-susceptible and -resistant infectious diseases such as TB.
- Full Text:
Evaluation of an NADPH-dependent assay for inhibition screening of Salmonella enterica DOXP Reguctoisomerase for identification of novel drug hit compounds
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
Exploring Quinolinyl-Thiazolidinedione hybrid compounds as potential anti-tubercular agents
- Authors: Mtshare, Thanduxolo Elihle
- Date: 2020
- Subjects: Quinoline , Mycobacterium tuberculosis , Tuberculosis -- Chemotherapy , Plasmodium falciparum , Thiazolidinedione
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/143314 , vital:38232
- Description: Tuberculosis (TB) is an infectious disease caused by the pathogen, Mycobacterium tuberculosis. According to the World Health Organization, TB is the ninth leading cause of death worldwide ranking above HIV/AIDS. This high mortality rate of TB begs the questions about the efficiency of the current therapy and raises an urgent need to create novel anti-tuberculosis agents which will aid in curbing this burden. Quinoline containing compounds have remarkable biological activities across a wide spectrum of diseases including anti-tuberculosis. On the other hand, thiazolidinedione containing compounds possess a broad spectrum of biological properties. In this study, we rationally designed compounds containing these pharmacophoric units and investigated them for their potential biological activity against Mycobacterium tuberculosis. Considering antimalarial activity of quinoline-based compounds, the compounds achieved were also cross-screened for their activity against the Plasmodium falciparum parasite, a causative agent of malaria. In all the synthesized compounds, compound 2.6a, 2.6b and 2.7b emerged as most active compounds against the H37Rv strain with MIC₉₀ values ranging in between of 1.08 – 17.1 μM. In addition, none of the compounds showed any inhibitory activities against the 3D7 strain of P. falciparum parasite. All the compounds prepared in this study showed no significant human cytotoxic effects as measured by HeLa cell line.
- Full Text:
- Authors: Mtshare, Thanduxolo Elihle
- Date: 2020
- Subjects: Quinoline , Mycobacterium tuberculosis , Tuberculosis -- Chemotherapy , Plasmodium falciparum , Thiazolidinedione
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/143314 , vital:38232
- Description: Tuberculosis (TB) is an infectious disease caused by the pathogen, Mycobacterium tuberculosis. According to the World Health Organization, TB is the ninth leading cause of death worldwide ranking above HIV/AIDS. This high mortality rate of TB begs the questions about the efficiency of the current therapy and raises an urgent need to create novel anti-tuberculosis agents which will aid in curbing this burden. Quinoline containing compounds have remarkable biological activities across a wide spectrum of diseases including anti-tuberculosis. On the other hand, thiazolidinedione containing compounds possess a broad spectrum of biological properties. In this study, we rationally designed compounds containing these pharmacophoric units and investigated them for their potential biological activity against Mycobacterium tuberculosis. Considering antimalarial activity of quinoline-based compounds, the compounds achieved were also cross-screened for their activity against the Plasmodium falciparum parasite, a causative agent of malaria. In all the synthesized compounds, compound 2.6a, 2.6b and 2.7b emerged as most active compounds against the H37Rv strain with MIC₉₀ values ranging in between of 1.08 – 17.1 μM. In addition, none of the compounds showed any inhibitory activities against the 3D7 strain of P. falciparum parasite. All the compounds prepared in this study showed no significant human cytotoxic effects as measured by HeLa cell line.
- Full Text:
Understanding of the underlying resistance mechanism of the Kat-G protein against isoniazid in Mycobacterium tuberculosis using bioinformatics approaches
- Authors: Barozi, Victor
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146592 , vital:38540
- Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
- Full Text:
- Authors: Barozi, Victor
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146592 , vital:38540
- Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
- Full Text:
Understanding the underlying resistance mechanism of Mycobacterium tuberculosis against Rifampicin by analyzing mutant DNA - directed RNA polymerase proteins via bioinformatics approaches
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
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