Characteristic Binding Landscape of Estrogen Receptor-α36 Protein Enhances Promising Cancer Drug Design.

Breast cancer (BC) remains the most common cancer among women worldwide, and estrogen receptor-α expression is a critical diagnostic factor for BC. Estrogen receptor (ER-α36) is a dominant-negative effector of ER-α66-mediated estrogen-responsive gene pathways. ER-α36 is a novel target that mediates the non-genomic estrogen signaling pathway. However, the crystallized structure of ER-α36 remains unavailable for molecular studies. ER-positive and triple-negative BC tumors aggressively resist the FDA-approved drugs; therefore, highly potent structure-based inhibitors with preeminent benefits over toxicity will preferably replace the current BC treatment. Broussoflanol B (BFB), a B. papyrifera bark compound, exhibits potent growth inhibitory activity in ER-negative BC cells by inducing cell cycle arrest. For the first time, we unravel the comparative dynamic events of the enzymes' structures and the binding mechanisms of BFB when bound to the ER-α36 and ER-α66 ligand-binding domain using an all-atom molecular dynamics simulations approach and MM/PBSA-binding-free energy calculations. The dynamic findings have revealed that ER-α36 and ER-α66 LBD undergo timescale "coiling", opening and closing conformations favoring the high-affinity BFB-bound ER-α36 (ΔG = -52.57 kcal/mol) compared to the BFB-bound ER-α66 (ΔG = -42.41 kcal/mol). Moreover, the unbound (1.260 Å) and bound ER-α36 (1.182 Å) exhibit the highest flexibilities and atomistic motions relative to the ER-α66 systems. The RMSF (Å) of the unbound ER-α36 and ER-α66 exhibit lesser stabilities than the BFB-bound systems, resulting in higher structural flexibilities and atomistic motions than the bound variants. These findings present a model that describes the mechanisms by which the BFB compound induces downregulation-accompanied cell cycle arrest at the Gap0 and Gap1 phases.

HIV Protease Hinge Region Insertions at Codon 38 Affect Enzyme Kinetics, Conformational Stability and Dynamics.

HIV-1 protease is essential for the production of mature, infectious virions and is a major target in antiretroviral therapy. We successfully purified a HIV-1 subtype C variant, L38↑N↑L- 4, containing an insertion of asparagine and leucine at position 38 without the four background mutations - K20R, E35D, R57K, V82I using a modified purification protocol. Isothermal titration calorimetry indicated that 50% of the variant protease sample was in the active conformation compared to 62% of the wild type protease. The secondary structure composition of the variant protease was unaffected by the double insertion. The specific activity and kcat values of the variant protease were approximately 50% lower than the wild type protease values. The variant protease also exhibited a 1.6-fold increase in kcat/KM when compared to the wild type protease. Differential scanning calorimetry showed a 5 °C increase in Tm of the variant protease, indicating the variant was more stable than the wild type. Molecular dynamics simulations indicated the variant was more stable and compact than the wild type protease. A 3-4% increase in the flexibility of the hinge regions of the variant protease was observed. In addition, increased flexibility of the flaps, cantilever and fulcrum regions of the variant protease B chain was observed. The variant protease sampled only the closed flap conformation indicating a potential mechanism for drug resistance. The present study highlights the direct impact of a double amino acid insertion in hinge region on enzyme kinetics, conformational stability and dynamics of an HIV-1 subtype C variant protease.

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.

Long noncoding RNAs (lncRNAs) in HIV-mediated carcinogenesis: Role in cell homeostasis, cell survival processes and drug resistance.

There is accruing data implicating long non-coding RNAs (lncRNAs) in the development and progression of non-communicable diseases such as cancer. These lncRNAs have been implicated in many diverse HIV-host interactions, some of which are beneficial to HIV propagation. The virus-host interactions induce the expression of HIV-regulated long non-coding RNAs, which are implicated in the carcinogenesis process, therefore, it is critical to understand the molecular mechanisms that underpin these HIV-regulated lncRNAs, especially in cancer formation. Herein, we summarize the role of HIV-regulated lncRNAs targeting cancer development-related processes including apoptosis, cell cycle, cell survival signalling, angiogenesis and drug resistance. It is unclear how lncRNAs regulate cancer development, this review also discuss recent discoveries regarding the functions of lncRNAs in cancer biology. Innovative research in this field will be beneficial for the future development of therapeutic strategies targeting long non-coding RNAs that are regulated by HIV, especially in HIV associated cancers.

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.

Molecular Mechanisms of HIV Protease Inhibitors Against HPV-Associated Cervical Cancer: Restoration of TP53 Tumour Suppressor Activities.

Cervical cancer is a Human Papilloma virus-related disease, which is on the rise in a number of countries, globally. Two essential oncogenes, E6 and E7, drive cell transformation and cancer development. These two oncoproteins target two of the most important tumour suppressors, p53 and pRB, for degradation through the ubiquitin ligase pathway, thus, blocking apoptosis activation and deregulation of cell cycle. This pathway can be exploited for anticancer therapeutic interventions, and Human Immunodeficiency Virus Protease Inhibitors (HIV-PIs) have attracted a lot of attention for this anticancer drug development. HIV-PIs have proven effective in treating HPV-positive cervical cancers and shown to restore impaired or deregulated p53 in HPV-associated cervical cancers by inhibiting the 26S proteasome. This review will evaluate the role players, such as HPV oncoproteins involved cervical cancer development and how they are targeted in HIV protease inhibitors-induced p53 restoration in cervical cancer. This review also covers the therapeutic potential of HIV protease inhibitors and molecular mechanisms behind the HIV protease inhibitors-induced p53-dependent anticancer activities against cervical cancer.

Recombinant expression, purification, and characterization of full-length human BST-2 from Escherichia coli.

HIV-1 virus release from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express® pLysY E. coli culture. Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner, HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli.

Design, synthesis and biological evaluation of novel 2-(5-aryl-1H-imidazol-1-yl) derivatives as potential inhibitors of the HIV-1 Vpu and host BST-2 protein interaction.

Novel ethyl 2-(5-aryl-1H-imidazol-1-yl)-acetates 17 and propionates 18, together with their acetic acid 19 and acetohydrazide 20 derivatives, were designed and synthesized using TosMIC chemistry. Biological evaluation of these newly synthesized scaffolds in the HIV-1 Vpu- Host BST-2 ELISA assay identified seven hits (17a, 17b, 17c, 17g, 18a, 20f and 20g) with greater than 50% inhibitory activity. These hits were validated in the HIV-1 Vpu- Host BST-2 AlphaScreen™ and six of the seven compounds were found to have comparable percentage inhibitory activities to those of the ELISA assay. Compounds 17b and 20g, with consistent percentage inhibitory activities across the two assays, had IC50 values of 11.6 ± 1.1 µM and 17.6 ± 0.9 µM in a dose response AlphaScreen™ assay. In a cell-based HIV-1 antiviral assay, compound 17b exhibited an EC50 = 6.3 ± 0.7 µM at non-toxic concentrations (CC50 = 184.5 ± 0.8 µM), whereas compound 20g displayed antiviral activity roughly equivalent to its toxicity (CC50 = 159.5 ± 0.9 µM). This data suggests that compound 17b, active in both cell-based and biochemical assays, provides a good starting point for the design of possible lead compounds for prevention of HIV-1 Vpu and host BST-2 protein binding in new anti-HIV therapeutics.

Design, synthesis and biological evaluation of imidazole and oxazole fragments as HIV-1 integrase-LEDGF/p75 disruptors and inhibitors of microbial pathogens.

We describe here the synthesis of libraries of novel 1-subtituted-5-aryl-1H-imidazole, 5-aryl-4-tosyl-4,5-dihydro-1,3-oxazole and 5-aryl-1,3-oxazole fragments via microwave (MW)-assisted cycloaddition of para-toluenesulfonylmethyl isocyanide (TosMIC) to imines and aldehydes. The compounds obtained were biologically evaluated in an AlphaScreen HIV-1 IN-LEDGF/p75 inhibition assay with six imidazole-based compounds (16c, 16f, 17c, 17f, 20a and 20d) displaying more than 50% inhibition at 10 µM, with IC50 values ranging from 7.0 to 30.4 µM. Additionally the hypothesis model developed predicts all active scaffolds except 20d to occupy similar areas as the N-heterocyclic (A) moiety and two aromatic rings (B and C) of previously identified inhibitor 5. These results indicate that the identified compounds represent a viable starting point for their use as templates in the design of next generation inhibitors targeting the HIV-1 IN and LEDGF/p75 protein-protein interaction. In addition, the in vitro antimicrobial properties of these fragments were tested by minimum inhibitory concentration (MIC) assays showing that compound 16f exhibited a MIC value of 15.6 µg/ml against S. aureus, while 17f displayed a similar MIC value against B. cereus, suggesting that these compounds could be further developed to specifically target those microbial pathogens.

Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli.

Functional roles of HIV-1 Vpu and CD74: Details and implications of the Vpu-CD74 interaction.

HIV-1 Vpu has a variety of functions, including CD4 degradation and the downregulation of MHCII. Downregulation of the MHCII occurs through Vpu binding to the cytoplasmic domain of CD74, the chaperone for antigen presentation. The CD74 cytoplasmic domain also plays a vital role in cell signaling through the activation of an NF-κB signal cascade for the maturation, proliferation and survival of B cells as well as by binding the macrophage inhibitory factor. In view of these functions, it follows that the Vpu-CD74 interaction has multiple downstream consequences for the immune system as it not only impairs foreign antigen presentation but may also have an effect on signal transduction cascades. It is thought that Vpu specifically targets intracellular CD74 while other HIV-1 proteins cannot. Therefore, this protein-protein interaction would be a potential drug target in order to reduce viral persistence. We review the functional importance and specific binding site of Vpu and CD74.

Synthesis and evaluation of 3-hydroxy-3-phenylpropanoate ester­AZT conjugates as potential dual-action HIV-1 Integrase and Reverse Transcriptase inhibitors.

Novel 3-hydroxy-3-phenylpropanoate ester-azidothymidine (AZT) conjugates have been prepared using Baylis-Hillman methodology, and their potential as dual-action HIV-1 Integrase and Reverse Transcriptase inhibitors has been explored using enzyme inhibition and computer modelling techniques; their activity and HeLa cell toxicity have been compared with those of their cinnamate ester analogues.

The evaluation of statins as potential inhibitors of the LEDGF/p75-HIV-1 integrase interaction.

Lovastatin was identified through virtual screening as a potential inhibitor of the LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the purified recombinant protein-protein interaction (IC50 = 1.97 ± 0.45 µm) more effectively than seven other tested statins. None of the eight statins, however, yielded antiviral activity in vitro, while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand transfer activity (31.65% at 100 µm). A correlation between lipophilicity and increased cellular toxicity of the statins was observed.

Novel furocoumarins as potential HIV-1 integrase inhibitors.

A series of seven novel, rationally designed N-substituted 3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propanamides have been prepared as potential HIV-1 integrase (IN) inhibitors via a five-step pathway commencing with resorcinol and diethyl 2-acetylglutarate, and the HIV-1 IN inhibition potential of these compounds has been examined relative to raltegravir, a known HIV-1 IN inhibitor.

Modification of HIV-1 reverse transcriptase and integrase activity by gold(III) complexes in direct biochemical assays.

Gold(I) and gold(III) complexes have been previously investigated for potential biomedical applications including as anti-HIV agents. The oxidising nature of some gold(III) complexes yields well-documented cellular toxicity in cell-based assays but the effect in direct biochemical assays has not been fully investigated. In this study, gold(III) complexes were evaluated in HIV-1 reverse transcriptase and HIV-1 integrase biochemical assays. The gold(III) tetrachlorides KAuCl(4) and HAuCl(4) yielded sub-micromolar IC(50)'s of 0.947 and 0.983µM in the direct HIV-1 RT assay, respectively, while IC(50)'s ranging from 0.461 to 8.796µM were obtained for seven selected gold(III) complexes. The gold(III) tetrachlorides were also effective inhibitors of integrase enzymatic activity with >80% inhibition obtained at a single dose evaluation of 10µM. RT inhibition was decreased in the presence of a reducing agent (10mM DTT) and against the M184V HIV-1 RT mutant, while none of the gold(III) complexes were effective inhibitors in cell-based antiviral assays (SI values <5.95). Taken together, the findings of this study demonstrate that gold(III) complexes modify HIV-1 enzyme activity in direct biochemical assays, most likely through protein oxidation.

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.

Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1.

Molecular docking and ANS-displacement experiments indicated that 8-anilinonaphthalene sulfonate (ANS) binds the hydrophobic site (H-site) in the active site of dimeric class Mu rGST M1-1. The naphthalene moiety provides most of the van der Waals contacts at the ANS-binding interface while the anilino group is able to sample different rotamers. The energetics of ANS binding were studied by isothermal titration calorimetry (ITC) over the temperature range of 5-30 degrees C. Binding is both enthalpically and entropically driven and displays a stoichiometry of one ANS molecule per subunit (or H-site). ANS binding is linked to the uptake of 0.5 protons at pH 6.5. Enthalpy of binding depends linearly upon temperature yielding a DeltaC(p) of -80+/-4 cal K(-1) mol(-1) indicating the burial of solvent-exposed nonpolar surface area upon ANS-protein complex formation. While ion-pair interactions between the sulfonate moiety of ANS and protein cationic groups may be significant for other ANS-binding proteins, the binding of ANS to rGST M1-1 is primarily hydrophobic in origin. The binding properties are compared with those of other GSTs and ANS-binding proteins.

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 alpha-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.

Residue 219 impacts on the dynamics of the C-terminal region in glutathione transferase A1-1: implications for stability and catalytic and ligandin functions.

The C-terminal region in class alpha glutathione transferases (GSTs) modulates the catalytic and nonsubstrate ligand binding functions of these enzymes. Except for mouse GST A1-1 (mGST A1-1), the structures of class alpha GSTs have a bulky aliphatic side chain topologically equivalent to Ile219 in human GST A1-1 (hGST A1-1). In mGST A1-1, the corresponding residue is an alanine. To investigate the role of Ile219 in determining the conformational dynamics of the C-terminal region in hGST A1-1, the residue was replaced by alanine. The substitution had no effect on the global structure of hGST A1-1 but did reduce the conformational stability of the C-terminal region of the protein. This region could be stabilized by ligands bound at the active site. The catalytic behavior of hGST A1-1 was significantly compromised by the I219A mutation as demonstrated by reduced enzyme activity, increased K(m) for the substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), and reduced catalytic efficiencies. Inhibition studies also indicated that the binding affinities for product and substrate analogues were dramatically decreased. The affinity of the mutant for GSH was, however, only slightly increased, indicating that the G-site was unaltered by the mutation. The binding affinity and stoichiometry for the anionic dye 8-anilino-1-naphthalene sulfonate (ANS) was also not significantly affected by the I219A mutation. However, the lower DeltaC(p) for ANS binding to the mutant (-0.34 kJ/mol per K compared with -0.84 kJ/mol per K for the wild-type protein) suggests that ANS binding to the mutant results in the burial of less hydrophobic surface area. Fluorescence data also indicates that ANS bound to the mutant is more prone to quenching by water. Overall, the data from this study, together with the structural details of the C-terminal region in mGST A1-1, show that Ile219 is an important structural determinant of the stability and dynamics of the C-terminal region of hGST A1-1.