Kinetic and thermodynamic characterization of the South African subtype C HIV-1 protease : implications for drug resistance

ABSTRACT The magnitude of the AIDS epidemic is well documented. It has been shown that Africa constitutes about 70 % of people infected with HIV worldwide. Efforts to control the AIDS epidemic have focused heavily on studies pertaining to the biology, biochemistry and structural biology of HIV and on the interactions between HIV proteins and new drugs. One of the most challenging problems in AIDS therapy is that HIV develops drug-resistant variants rapidly. Extensive research has been dedicated to designing resistance-evading drugs for HIV-1 protease (predominantly subtype B), which is crucial for the maturation of viral, structural and enzymatic proteins. There are 10 subtypes of HIV-1 within the major group of the virus, with subtype C accounting for about 95 % of infections in South Africa. Since HIV-1 antiretroviral treatment has been developed and tested against the B subtype, which is prevalent in North America, Western Europe and Australia, an important question relates to the effectiveness of these drugs against the C subtype. At this point, however, little is known about inhibitor-resistant mutations in the subtype C. The study, therefore, looked at the two active site mutations (V82A and V82F/I84V) in the South African HIV-1 subtype C protease (C-SA) emerging from the viral population circulating in patients. These mutations are well-characterized within the framework of the subtype B and are known to cause cross-resistance to most of inhibitors currently in clinical use. Protein engineering techniques were used to generate the V82A and the V82F/I84V variants. Comparative studies with the wild-type HIV-1 C-SA protease were performed. The spectral properties of the V82A and the V82F/I84V variants indicated no changes in the secondary structure in the respective variant proteins. Tryptophan and tyrosine fluorescence indicated a major difference in the intensities at the emission maxima for all three proteins. The fluorescence intensity of the V82F/I84V variant, in particular, was significantly enhanced indicating the occurrence of tertiary structural changes at/near the flap region. Both mutations did not impact significantly upon catalytic function. Both variants also had the same Km values comparable to that of the wild-type enzyme. The catalytic efficiencies and the kinetic constants were lowered 3.6-fold for the V82A mutation and 6-fold for the V82F/I84V mutation relative to the wild-type C-SA protease. Inhibition studies were performed using four inhibitors in clinical use (saquinavir, ritonavir, indinavir and nelfinavir). For the V82A variant, IC50 and Ki values for saquinavir and nelfinavir iv were not affected, whilst those for ritonavir and indinavir were 5- and 9-fold higher than the wild-type C-SA protease, respectively. Against the V82F/I84V variant, however, the inhibition constants were drastically weaker and characterized by IC50 and Ki ratios ranging from 50 to 450. Isothermal titration calorimetry (ITC) was also used to determine the binding energetics of saquinavir, ritonavir, indinavir and nelfinavir to the wild-type C-SA, V82A and V82F/I84V HIV-1 protease. The V82A mutation lowered the Gibbs energy of binding for the respective four clinical inhibitors by 0.4 kcal/mol, 1.3 kcal/mol, 1.5 kcal/mol and 0.6 kcal/mol, respectively, relative to the wild-type C-SA HIV-1 protease. The affinity of V82A HIV-1 protease for saquinavir, ritonavir, indinavir and nelfinavir (Kd = 1.85 nM, 2.00 nM, 12.70 nM and 0.66 nM, respectively, at 25 °C) was in the range of 2- to 13-fold of magnitude weaker than that of the wild-type C-SA protein. The clinical inhibitors exhibited the highest binding affinity to both the wild-type and the V82A enzymes, but were extremely sensitive to the V82F/I84V mutation. The V82F/I84V mutant reduced the binding of saquinavir, ritonavir, indinavir and nelfinavir 117-, 1095-, 474- and 367- fold, respectively. A drop in Kd values obtained for the V82F/I84V in association with saquinavir, ritonavir, indinavir and nelfinavir was consistent with a decrease of between 2.8 - 4.2 kcal/mol in ΔG, which is equivalent to at least 2 to 3 orders of magnitude in binding affinity. Taken together, thermodynamic data indicated that the V82A and V82F/I84V active site mutations in the C-SA subtype lower the affinity of the first-generation inhibitors by making the binding entropy less positive (unfavorable) and making the enthalpy change slightly less favorable

Screening of the NIH Clinical Collection for inhibitors of HIV-1 integrase activity

Drug repurposing offers a validated approach to reduce drug attrition within the drug discovery and development pipeline through the application of known drugs and drug candidates to treat new indications. Full exploitation of this strategy necessitates the screening of a vast number of molecules against an extensive number of diseases of high burden or unmet need and the subsequent dissemination of the findings. In order to contribute to endeavours within this field, we screened the 727 compounds comprising the US National Institutes of Health (NIH) Clinical Collection through an HIV-1 (human immunodeficiency virus type 1) integrase stand transfer inhibition assay on an automated scintillation proximity assay platform. Only two compounds were identified within the initial screen, with cefixime trihydrate and epigallocatechin gallate found to reduce integrase strand transfer activity at IC50 values of 6.03±1.29 ?M and 9.57±1.62 ?M, respectively. However, both cefixime trihydrate and epigallocatechin gallate retained their low micromolar inhibitory activity when tested against a raltegravir-resistant integrase double mutant (FCIC50 values of 0.83 and 0.06, respectively), were ineffective in an orthogonal strand transfer ELISA (<30% inhibition at 100 ?M) and produced negligible selectivity index values (<1) in vitro. While no useful inhibitors of HIV-1 integrase strand transfer activity were found within the NIH Clinical Collection, the identification of two assay-disrupting molecules demonstrates the importance of consideration of non-specific inhibitors in drug repurposing screens. Significance: • This study is the first to screen the US NIH Clinical Collection for potential HIV-1 integrase inhibitors. • The pervasive nature of promiscuous inhibitors is emphasised

Calorimetry to Quantify Protein-Ligand Binding

Isothermal titration calorimetry (ITC) is the preferred method used to study biochemical reactions like protein-ligand binding due to its sensitivity, accuracy, and precision. ITC measures directly the heat absorbed or released (∆H) associated with a given binding process. A typical ITC experiment allows the dissection of the binding energy of a reaction into ligand-enzyme association constant (Ka), change in enthalpy (∆H), change in entropy (∆S), change in Gibbs-free energy (∆G), and the stoichiometry of association (N). The change in heat capacity (∆Cp) is obtained from the measurements of binding enthalpy over a range of temperatures. The magnitude and signs of the thermodynamic parameters that were obtained provide insight into the nature of interactions involved in the binding process. The strength of interaction is thermodynamically favorable is determined by the Gibbs free energy. ∆G is an important thermodynamic descriptor of a binding reaction since it dictates the binding affinity and is in turn defined by the enthalpy and entropy changes expressed in the following equation: ∆G = ∆H–T∆S. Up-close, this reflects the contradistinctions of two thermodynamic effects at a molecular level—the propensity to drop to lower energy (bond formation, negative ∆H), counterbalanced by the innate thermal Brownian motion’s destructive characteristic (bond breakage, positive ∆S)

The Role of Death-Associated Protein Kinase-1 in Cell Homeostasis-Related Processes

Tremendous amount of financial resources and manpower have been invested to understand the function of numerous genes that are deregulated during the carcinogenesis process, which can be targeted for anticancer therapeutic interventions. Death-associated protein kinase 1 (DAPK-1) is one of the genes that have shown potential as biomarkers for cancer treatment. It is a member of the kinase family, which also includes Death-associated protein kinase 2 (DAPK-2), Death-associated protein kinase 3 (DAPK-3), Death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK-1) and Death-associated protein kinase-related apoptosis-inducing kinase 2 (DRAK-2). DAPK-1 is a tumour-suppressor gene that is hypermethylated in most human cancers. Additionally, DAPK-1 regulates a number of cellular processes, including apoptosis, autophagy and the cell cycle. The molecular basis by which DAPK-1 induces these cell homeostasis-related processes for cancer prevention is less understood; hence, they need to be investigated. The purpose of this review is to discuss the current understanding of the mechanisms of DAPK-1 in cell homeostasis-related processes, especially apoptosis, autophagy and the cell cycle. It also explores how the expression of DAPK-1 affects carcinogenesis. Since deregulation of DAPK-1 is implicated in the pathogenesis of cancer, altering DAPK-1 expression or activity may be a promising therapeutic strategy against cancer

Active-Site Mutations in the South African Human Immunodeficiency Virus Type 1 Subtype C Protease Have a Significant Impact on Clinical Inhibitor Binding: Kinetic and Thermodynamic Study▿

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Study of active-site mutations (the V82A single mutation and the V82F I84V double mutation) in the less-studied South African HIV type 1 subtype C (C-SA) protease indicated that neither mutation had a significant impact on the proteolytic functioning of the protease. However, the binding affinities of, and inhibition by, saquinavir, ritonavir, indinavir, and nelfinavir were weaker for each variant than for the wild-type protease, with the double mutant exhibiting the most dramatic change. Therefore, our results show that the C-SA V82F I84V double mutation decreased the binding affinities of protease inhibitors to levels significantly lower than that required for effective inhibition

Antimicrobial and anti-inflammatory activities of selected medicinal plants against pathogens causing sexually transmitted infections

INTRODUCTION: Worldwide, more than one million sexually transmitted infections (STIs) are acquired daily. The diversity and frequency of sexual infections caused by pathogenic microorganisms have increased thus becoming a major cause of illness and mortality amongst young adults. Medicinal plants have been good remedies for the treatment of STIs since ancient times. In this study, we evaluated antimicrobial, anti- Human immunodeficiency virus (HIV) and anti-inflammatory activities of five selected medicinal plants. METHODS: We determined the antimicrobial activities of plant extracts against the bacteria causing common STIs. Then, the anti-inflammatory activities were evaluated by measuring the inhibition of the pro-inflammatory enzyme, 15-lipoxygenase (15-LOX) and we further investigated the plants extracts of anti-HIV activities against the recombinant HIV-1 enzyme, reverse transcriptase. RESULTS: Methanol extract of Terminalia sericea and dichloromethane (DCM) extract of Bidens pilosa exhibited good activities against Neisseria gonorrhoeae and Gardnerella vaginalis. Ethyl acetate, dichloromethane and methanol extracts of Bidens pilosa exhibited good activities against Candida albicans. Ethyl acetate extract of K. africana and methanol extract of B. pilosa showed good anti-inflammatory activities. Ethyl acetate, DCM and methanol extracts of T. sericea exhibited promising anti-HIV-1 activities by inhibiting the reverse transcriptase whilst methanol extracts of T. dregeana showed low anti-HIV-1 activity. CONCLUSION: These plants showed promising activity against the propagation of inflammation, displayed good antimicrobial activities against the bacteria causing STIs and could be used as potential leads and/or source for new drug candidates.University of South Africahttp://herbmedpharmacol.compm2021Plant Production and Soil Scienc

Antimicrobial and anti-inflammatory activities of selected medicinal plants against pathogens causing sexually transmitted infections

Introduction: Worldwide, more than one million sexually transmitted infections (STIs) are acquired daily. The diversity and frequency of sexual infections caused by pathogenic microorganisms have increased thus becoming a major cause of illness and mortality amongst young adults. Medicinal plants have been good remedies for the treatment of STIs since ancient times. In this study, we evaluated antimicrobial, anti- Human immunodeficiency virus (HIV) and anti-inflammatory activities of five selected medicinal plants. Methods: We determined the antimicrobial activities of plant extracts against the bacteria causing common STIs. Then, the anti-inflammatory activities were evaluated by measuring the inhibition of the pro-inflammatory enzyme, 15-lipoxygenase (15-LOX) and we further investigated the plants extracts of anti-HIV activities against the recombinant HIV-1 enzyme, reverse transcriptase. Results: Methanol extract of Terminalia sericea and dichloromethane (DCM) extract of Bidens pilosa exhibited good activities against Neisseria gonorrhoeae and Gardnerella vaginalis. Ethyl acetate, dichloromethane and methanol extracts of Bidens pilosa exhibited good activities against Candida albicans. Ethyl acetate extract of K. africana and methanol extract of B. pilosa showed good anti-inflammatory activities. Ethyl acetate, DCM and methanol extracts of T. sericea exhibited promising anti-HIV-1 activities by inhibiting the reverse transcriptase whilst methanol extracts of T. dregeana showed low anti-HIV-1 activity. Conclusion: These plants showed promising activity against the propagation of inflammation, displayed good antimicrobial activities against the bacteria causing STIs and could be used as potential leads and/or source for new drug candidates

Tertiary interactions stabilise the C-terminal region of human glutathione transferase A1-1: a crystallographic and calorimetric study

The C-terminal region in class Alpha glutathione transferase A1-1 (GSTA1-1), which forms an amphipathic a-helix (helix 9), is known to contribute to the catalytic and non-substrate ligand-binding functions of the enzyme. The region in the apo protein is proposed to be disordered which, upon ligand binding at the active-site, becomes structured and localised. Because Ile219 plays a pivotal role in the stability and localisation of the region, the role of tertiary interactions mediated by Ile219 in determining the conformation and dynamics of the C-terminal region were studied. Ligand-binding microcalorimetric and X-ray structural data were obtained to characterise ligand binding at the active-site and the associated localisation of the C-terminal region. In the crystal structure of the I219A hGSTA1-1$ S-hexylglutathione complex, the C-terminal region of one chain is mobile and not observed (unresolved electron density), whereas the corresponding region of the other chain is localised and structured as a result of crystal packing interactions. In solution, the mutant C-terminal region of both chains in the complex is mobile and delocalised resulting in a hydrated, less hydrophobic active-site and a reduction in the affinity of the protein for S-hexylglutathione. Complete dehydration of the active-site, important for maintaining the highly reactive thiolate form of glutathione, requires the binding of ligands and the subsequent localisation of the C-terminal region. Thermodynamic data demonstrate that the mobile C-terminal region in apo hGSTA1-1 is structured and does not undergo ligand-induced folding. Its close proximity to the surface of the wild-type protein is indicated by the concurrence between the observed heat capacity change of complex formation and the type and amount of surface area that becomes buried at the ligand–protein interface when the C-terminal region in the apo protein assumes the same localised structure as that observed in the wild-type complex

A Review of the Nucleic Acid-Based Lateral Flow Assay for Detection of Breast Cancer from Circulating Biomarkers at a Point-of-Care in Low Income Countries

The current levels of breast cancer in African women have contributed to the high mortality rates among them. In South Africa, the incidence of breast cancer is also on the rise due to changes in behavioural and biological risk factors. Such low survival rates can be attributed to the late diagnosis of the disease due to a lack of access and the high costs of the current diagnostic tools. Breast cancer is asymptomatic at early stages, which is the best time to detect it and intervene to prevent high mortality rates. Proper risk assessment, campaigns, and access to adequate healthcare need to be prioritised among patients at an early stage. Early detection of breast cancer can significantly improve the survival rate of breast cancer patients, since therapeutic strategies are more effective at this stage. Early detection of breast cancer can be achieved by developing devices that are simple, sensitive, low-cost, and employed at point-of-care (POC), especially in low-income countries (LICs). Nucleic-acid-based lateral flow assays (NABLFAs) that combine molecular detection with the immunochemical visualisation principles, have recently emerged as tools for disease diagnosis, even for low biomarker concentrations. Detection of circulating genetic biomarkers in non-invasively collected biological fluids with NABLFAs presents an appealing and suitable method for POC testing in resource-limited regions and/or LICs. Diagnosis of breast cancer at an early stage will improve the survival rates of the patients. This review covers the analysis of the current state of NABLFA technologies used in developing countries to reduce the scourge of breast cancer