The primary goals of our research are to understand the virology and enzymology of human T-cell leukemia virus type I (HTLV-I) that will lead to the development of treatments for patients infected with HTLV-I. HTLV-I is an oncogenic virus of the Retroviridae family and is the causative agent of adult T-cell leukemia/lymphoma (ATL), tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). HTLV-I has been classified as a dangerous emerging pathogen by the Centers for Disease Control and Prevention with at least 20 million people infected with the virus. This is a significant problem because there are currently no effective treatments to control HTLV-I infection and prevent or treat HTLV-I induced ATL and TSP/HAM. The protease is necessary for retroviral maturation and replication and is, therefore, an attractive target for inhibitor design. Investigation of peptide mimetic compounds incorporating the tetrahedral intermediate of aspartyl protease catalyzed cleavage are crucial for the development of lead inhibitors. Compounds containing statine, 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), or hydroxyethylamine (HEA) are presented in this work. The best compound was a statine-based inhibitor, which had a Ki = 29 +/- 4 nM and 88% inhibition against an HTLV-I protease native substrate in a FRET assay.

Cysteine proteases are a class of proteolytic enzymes, which are involved in a series of metabolic and catabolic processes, such as protein turnover, digestion, blood coagulation, apoptosis, fertilization and cell differentiation, and the immune response system. The development of novel potent and selective inhibitors for cysteine proteases has therefore gained increasing attention among medicinal chemists. In this thesis we have reported the design, synthesis, and evaluation of several peptidyl inhibitors for clan CA and clan CD cysteine proteases. We have continued the investigation of dipeptidyl vinyl sulfones as potent and selective inhibitors for dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, which is involved in the processing of intracellular proteases, such as granzymes. We have found that DPPI tolerates negatively charged amino acid residues in the P2 position with inhibition rates of 7,600 M-1s-1. Dipeptidyl vinyl sulfones with positively charged amino acid residues at the P1 position, however, do not inhibit DPPI at all. A second project focused on the epoxidation of the double bond of the vinyl sulfone moiety of the dipeptidyl vinyl sulfones. Instead of epoxidizing the double bond, we found that an isomerization had occurred. The newly formed compounds were determined to be allyl sulfones. We tested this new class of inhibitors with clan CA proteases and obtained inhibition rates of 560 M-1s-1 for Cbz-Leu-Phe-AS-Ph with calpain I. Two new classes of compounds for the clan CD protease S. mansoni legumain were designed, synthesized, and evaluated. Aza-peptidyl epoxides were found to be potent and selective inhibitors of S. mansoni legumain with IC50’s as low as 45 nM. Aza-peptide Michael acceptors were derived from the aza-peptide epoxide design and synthesized in an analogous fashion. The aza-peptide Michael acceptors inhibited S. mansoni legumain with even lower IC50’s, as low as 10 nM. However, the aza-peptide Michael acceptors react with thioalkylating agents contained in the buffer, such as DTT. The rates of degradation were determined spectroscopically, and half-lives of 3 to 20 minutes were measured. This observation gave us insights into the enzymatic mechanism and allowed us to determine the point of attack for the legumain active site cysteine thiol.

In this study, hydrolytic enzymes from sunn pest (Eurygaster integriceps) and cotton bollworm (Helicoverpa armigera) midguts were identified and characterized in terms of their optimum pH, Km and Vmax values. Hydrolytic activities were also tested for inhibition by several protease and alpha-amylase inhibitors which can be used for the development of insect resistant plants through transgenic technologies. For sunn pest midgut, a low proteolytic activity, belonging mostly to trypsin-like and leucine aminopeptidase-like proteases, and a very high alpha-amylase activity was found in sunn pest midgut, reflecting its high carbohydrate diet. Proteolytic activities could not be inhibited by natural protease inhibitors (SBTI and aprotinin) but inhibited significantly by a general serine protease inhibitor PMSF and metalloprotease inhibitors CdCl2 and CuCl2. alpha-Amylase activity of sunn pest midgut is resistant to inhibition by bean alpha-amylase inhibitor, but inhibited by chickpea, wheat and maize alpha-amylase inhibitors by 26 %, 37 % and 40 %, respectively. For cotton bollworm midgut, a very high proteolytic activity, belonging to serine and metalloprotease type, was detected. alpha-Amylase activity was lower compared to sunn pest midgut, but there were higher and diverse type of proteases, might be reflecting its wide range of host preference. Proteolytic activity was significantly inhibited by both natural protease inhibitors (SBTI and aprotinin). It was also inhibited by several synthetic protease inhibitors (PMSF, E-64, TPCK, CdCl2, CuCl2, Chymostatin). alpha-Amylase activity was inhibited by 60 % by wheat alpha-amylase inhibitor, while maize, chickpea and bean alpha-amylase inhibitors had no effect on cotton bollworm midgut alpha-amylase activity.

Cysteine proteases are important drug targets due to their involvement in many biological processes such as protein turnover, digestion, blood coagulation, apoptosis, cell differentiation, cell signaling, and the immune response. In this thesis, we have reported the design, synthesis and evaluation of clan CA and clan CD cysteine protease inhibitors. Aza-peptidyl Michael acceptor and epoxide inhibitors for asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE) and the hard tick, Ixodes ricinus (IrAE) were designed and synthesized. SARs were similar, but with some notable exceptions. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased inhibitory potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues in the P2 position. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors and, for some of these compounds, second order inhibition rate constants are the fastest yet discovered. We have also synthesized aza-peptidyl Michael acceptor and epoxide inhibitors for the parasitic cysteine proteases; cruzain, rhodesain. We have found that monosubstituted amides were favored over disubstituted amides indicating the involvement of the amide hydrogen in a H-bond network. We have shown that aza-peptide epoxides were as potent as Michael acceptors and we have obtained compounds with IC50 values as low as 20 nM. We have worked on the synthesis of heterocyclic peptidyl α-ketoamides, peptidyl ketones and aza-peptidyl ketones as calpain inhibitors. We have synthesized peptidyl α-ketoamides with nucleotide bases in the primed region to create compounds that can cross the blood-brain barrier. We have improved the potency by introducing a hydrophobic group on the adenine ring. We have obtained compounds with Ki values in the nanomolar range. We have designed peptidyl aminoketones as a new class of inhibitors for calpain. Peptidyl aminoketones were less potent than peptidyl α-ketoamides but still reasonable inhibitors of calpain that have the potential to cross the BBB.

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Burkitt lymphoma (BL) was the first tumor found to be strongly associated with Epstein-Barr virus (EBV). Almost 100% of the lymphoma cells are cycling, necessitating dose- and time-intense multi-agent chemotherapy regimens to achieve a cure of the disease. Whilst standard risk BL can be cured with this approach, high risk BL with leukaemic and CNS disease has significantly inferior survival. The intensive chemotherapy regimen causes considerable toxicity to the patients and relapse of BL is largely incurable. Thus, novel therapeutic approaches for high risk and relapsed BL are needed. Histone deacetylase inhibitors (HDACis) represent a novel class of drugs with potent anti-cancer effect in a wide range of malignancies. In the first part of this study, we tested HDACis of different classes for their ability to inhibit cell proliferation and activate the lytic cycle of EBV in a panel of EBV-positive BL cells of different latent viral gene expression patterns (type I, Wp-restricted and type III latency with highly restrictive, partial and full spectrum of EBV latent gene expression, respectively). Different HDACis could inhibit proliferation of EBV-positive BL cells in a time- and dose-dependent manner but only weakly activate the viral lytic cycle indicating that the drugs’ cytotoxic effect is independent of the EBV lytic cycle. Of note, BL cells of Wp-restricted or type III latency were more resistant to killing by HDACis than those of latency I, suggesting a possible link between relative resistance to the drug and expression of the latent viral genes. Bortezomib, a proteasome inhibitor, may have synergistic action with HDACis on lymphoid malignancies. We hypothesized that Bortezomib could potentiate the killing of EBV-positive BL cells by HDACis. In the second part, we tested the effect of combination of a FDA-approved HDACi, suberoylanilide hydroxamic acid (SAHA) and Bortezomib in the same panel of BL cells and also EBV-transformed lymphoblastoid cell lines (LCLs) which represent an in-vitro model of EBV-associated post-transplant lymphoproliferative disorder (PTLD). Interestingly, combination of SAHA and Bortezomib significantly enhanced the killing of BL cells of Wp-restricted or type III latency. Furthermore, the resistance to either SAHA or Bortezomib alone in contrast to synergistic killing by the combination of the two drugs could be observed in LCLs which also have the type III latency pattern. Compared with either drug alone, combination of SAHA and Bortezomib induced enhanced apoptosis in Wp-restricetd BL cells and LCLs as shown by the increase in the percentage of annexin V-positive cell, sub-G1 population and the proteolytic cleavage of apoptotic markers including PARP, caspase-3 and -9. The drug combination hyper-acetylated histone and induced cell cycle arrest. Combination of SAHA and Bortezomib was further shown to suppress the growth of BL xenograft in nude mice. In conclusion, our data indicated that expression of partial or full spectrum of viral latent genes in EBV-positive BL cells of Wp-restricted or type III latency confers resistance of the tumor cells to cytotoxic effect of HDACis. Bortezomib could potentiate SAHA-induced apoptosis of both BL cells and LCLs and might overcome mechanism of drug resistance. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

There is an increasing number of patients failing second line highly active antiretroviral therapy (AZT, DDI and LPV/r) in South Africa, where HIV-1 subtype C predominates. Mutations at gag cleavage sites (CS) have been found to correlate with resistance mutations in protease (PR). Therefore, it is important to collect data on subtype C protease and gag sequences from patients as these mutations may affect the efficacy of protease inhibitor (PI) containing drug regimens. In this study, 30 subtype-C infected second-line failures were genotyped using the ViroSeqTM resistance genotyping kit and the gag region from these isolates were then characterised. These sequences were then compared to 30 HIV-1 subtype C infected first-line failures (PI-naïve) and subtype B, C and group M naïve sequences that were downloaded from the Los Alamos Sequence Database. Amino acid diversity at the CS was measured using Mega version 4.0. To investigate the effect of CS mutations on replication capacity, a mutation was introduced by site-directed mutagenesis (Stratagene’s QuikChange Site-Directed Mutagenesis kit). Of the 30 second-line failures that we genotyped, only 16 had resistance mutations in PR and 23 in gag. The most frequent major PI mutations were: I54V/L, M46I, V82A, and I84V and in gag CS were V390L/I and A431V. Interestingly the A431V mutation significantly correlated with protease mutations M46I/L, I54V and V82A. The virus carrying the A431V mutation in vitro was found to have a lower replication capacity compared to the wild type. These findings emphasize the need for further investigation of gag mutations and their contribution to the evolution of HIV resistance to PIs. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2010.

Introduction Angiogenesis, the growth of new blood vessels from the pre-existing vasculature, is a pre-requisite for tumour growth and metastasis. Tumour-angiogenesis is regulated by various pro- and anti-angiogenic factors released by both endothelial and tumour cells, as well as by the micro-environment. Numerous studies have implicated various systems in the acquisition of the angiogenic phenotype. The present study sought to investigate the role of the kallikrein-kinin system (KKS) in tumour-angiogenesis. The kallikreins consist of two serine proteases, plasma and tissue kallikrein (TK), involved in the release of kinin peptides by enzymatic cleavage of kininogens. Stimulation of the cognate bradykinin receptors (BKR), B1R and B2R, mediates the mitogenic and vasoactive properties of kinins. In addition, TK activates matrix metallo-proteinases (MMPs) involved in extracellular matrix (ECM) degradation. The expression profiles of TK and kinins have been found to be dys-regulated in numerous human cancers, and several studies have demonstrated the involvement of the KKS in growth and metastasis of prostate tumours. Further, previous in vitro models in our laboratory have established an association between the KKS and prostate tumour-angiogenesis. In those studies it was postulated that the up-regulated TK (produced by endothelial and tumour cells) stimulated endothelial cell proliferation. Thus, the aim of the present study was to define the effects of the KKS and seek a direct correlation with angiogenesis using in vitro models with tumour conditioned medium (CM), kinin receptor agonists and antagonists. Methods Ethical approval for this project was granted by the Biomedical Research Ethics Committee, University of KwaZulu-Natal (reference number BE152/08). Micro-vascular endothelial cells represent a suitable in vitro angiogenic model and dermal micro-vascular endothelial cells (dMVECs) were obtained commercially for this purpose. The tumour model used in this study was an immortalised prostate cancer (DU145) cell line. The CM model involves the treatment of one cell line with the metabolites of another. In the angiogenic model, dMVECs were exposed to increasing concentrations of DU145 CM. Stimulation was further augmented with BKR agonists. Specific BKR antagonists were used to test the specificity of stimulation. In addition, vascular endothelial growth factor (VEGF) was tested as a positive proliferation control. The potential of these agents to induce proliferation and migration was determined using the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and a modified Boyden chamber assay, respectively. Previous studies investigating the pro-angiogenic effects of CM differed, in many respects, in terms of their models and methodologies. In an attempt to fully explore the pro-mitogenic effects of CM on endothelial cells, various modifications, as well as alternate endothelial and tumour cell types, were employed in the present study. The mitogenic and migratory effect of BKR agonists and antagonists on DU145 cells was also assessed. Further, the tumour model was expanded to investigate the autocrine potential of the KKS, by investigating the effect of DU145 CM on DU145 migration. Results In the angiogenic model, although the addition of DU145 CM elicited a statistically significant increase in micro-vascular endothelial cell proliferation, this increase was very small (<10%) and not dose-dependent. Pre-incubation of dMVECs with a B1R or B2R antagonist did not influence this small effect of CM on proliferation. In addition, neither B1R nor B2R agonists, at any concentration, produced any significant proliferative effect on endothelial cells. In contrast to these findings VEGF, a well-known mitogen, was able to stimulate proliferation of dMVECs. Migration assays revealed that DU145 CM failed to stimulate endothelial cell motility. Further, neither BKR agonist displayed any chemo-attractant potential in those assays. The most important finding was in the tumour model, where stimulation with a B1R agonist significantly enhanced proliferation and especially migration of DU145 cells. In addition, pre-treatment with a B1R antagonist abolished both these effects. B2R agonists could not produce the same positive effect as the B1R agonist on growth and migration of prostate tumour cells. DU145 CM did not prove to be a migratory stimulus for DU145 cells at any concentration. Discussion Previous studies in our laboratory have shown prostate-tumour CM to promote proliferation of endothelial cells and have postulated that TK up-regulation may be the reason for this. However, the present study could not reproduce this effect of CM. Further, BKR antagonists had no notable or consistent effect on the minimal promotion of proliferation that had been produced by DU145 CM. In addition, selective BKR agonists failed to induce proliferation or migration of endothelial cells, key events in the angiogenic cascade. Although in contrast to some studies, the present study was unable to implicate the KKS in angiogenesis, tumour neo-vascularisation is a consequence of several angiogenic factors functioning together as opposed to a single, isolated factor. For example, we were able to demonstrate a positive mitogenic effect of VEGF on endothelial cells and it may be this as well as other factors in the CM that are responsible for the small proliferation we observed. Up-regulation of kallikreins and kinins in tumours may enhance fundamental events in tumourigenesis in an autocrine manner, and bradykinin (BK) has previously been shown to promote tumour growth in mouse models. Our study supported the involvement of the KKS in tumourigenesis. Although CM from DU145 cells did not self-stimulate the migration of these cells, a B1R agonist enhanced both proliferation and migration, an effect that was also abrogated by the relevant antagonist, indicating a role for kinins. In contrast to the findings of another study, stimulation of the B2R failed to significantly promote tumour growth or motility. However, this is not an unexpected finding because it is thought that the ubiquitous B2R mediates physiological effects in the prostate while the inducible B1R plays a role in prostate cancer pathology. In summary, this study lends support to the ongoing exploration of BKR antagonists as possible candidates in the development of alternate approaches to cancer therapy. This may be particularly beneficial to hormone-independent tumours, such as those of the prostate, for which there exists few effective treatment options. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

The objective of this dissertation is to study the mechanism by which HIV protease inhibitors enter into the central nervous system (CNS) and breast milk of rats, and what effects MDR modulators have on the distribution and metabolism of HIV protease inhibitors. The transporter P-glycoprotein (P-gp) has been shown to limit the distribution of HIV protease inhibitors into the CNS of rodents. This thesis examined the effects of GF120918, an MDR modulator, on the CNS distribution of amprenavir, an HIV protease inhibitor, in rats. GF120918 significantly increased the unbound CNS concentrations of amprenavir without altering the unbound blood concentrations of amprenavir. The results of these studies show that GF120918 can inhibit P-gp at the blood brain barrier (BBB) to increase the unbound CNS concentration of amprenavir and potentially other HIV protease inhibitors. Many first generation MDR modulators inhibited both P-gp transport and CYP3A metabolism. Therefore, a principal goal of this thesis was to determine if GF120918 could selectively inhibit P-gp transport without inhibiting CYP3A metabolism. Using in vitro (human) and in vivo (rat) studies, GF120918 selectively inhibited P-gp at the BBB without inhibiting CYP3A metabolism. The transporter MRP1 has been shown to both transport HIV protease inhibitors and expressed in the CNS. Studies contained in the thesis have shown that mrp1 is not localized to the BBB of rats, therefore, mrp1 is unlikely to play a significant role in the distribution of HIV protease inhibitors into the CNS of rats. The distribution of nelfinavir, an HIV protease inhibitor, into rat breast milk was studied in the thesis as a first approach in understanding the extent to which HIV protease inhibitors can accumulate into milk. The concentration of nelfinavir in rat milk was approximately half that of plasma. P-gp protein expression was detected in lactating rat mammary tissue. However, GF120918 showed no effect on the distribution of nelfinavir into rat milk suggesting that P-gp does not play a significant role in the distribution of HIV protease inhibitors into milk.

According to the 2012 UNAIDS global report, sub-Saharan Africa hosts 69% of the world’s total population living with HIV, South Africa being the most affected with a reported 24% incidence rate. To date, extensive research is being conducted globally, particularly involving anti-HIV treatment that targets the retroviral enzymes: reverse transcriptase, integrase and protease. The discovery of inhibitors to HIV protease which disrupts virion protein assembly has made this enzyme a prime target of anti-retroviral therapies, thus there exists a concerted research initiative to identify compounds with HIV protease inactivation potential. This study employs HIV protease that is isolated and purified from a genetically modified HIV protease overexpressing Escherichia coli strain to monitor the inhibitory capacity of new lead compounds. Optimized growth conditions for HIV protease production displayed that the use of chemically defined media resulted in higher yields of the enzyme. Recent research studies have shown that peptide-based cage and glycosylated compounds displayed HIV protease inhibitor activity in cell free enzymatic reactions that are comparable to commercially available HIV protease inhibitors. However, in contrast it has also been reported that these inhibitors are inactive in whole T-cell assays, when employing HIV infected CD4 cells. It is a well-known fact that potential new chemical entities that do not possess oral bioavailability, in terms of their absorption properties, are not successful candidates within the drug discovery industry. Following this, the current study was designed to determine if inefficient membrane permeability of these promising anti-HIV protease lead compounds could result in their inactivity in whole T-cell assays. Two different methods were considered, a cell-based method using the Madin Darby Canine Kidney strain I (MDCKI) cell line and a non-cell based method, the parallel artificial membrane permeability assay (PAMPA). MDCKI cells have been extensively used to form monolayers that mimic human intestinal membranes whilst the PAMPA utilizes an artificial lipid membrane composition on a filter support. Data from permeability assays using the novel chemically synthesized inhibitors have been compared to commercially available drugs, antipyrine, metoprolol and caffeine, which displayed efficient membrane permeability characteristics, thereby validating the assay. The results indicated that novel cage-derived and glycosylated peptide inhibitors do not possess sufficient passive diffusion properties which may explain their inactivity in whole T-cell assays. / M.Sc. University of KwaZulu-Natal, Durban 2014.