The human positive regulatory domain I binding factor 1 (PRDI-BF1/PRDM1) promotes differentiation of mature B cells into antibody secreting plasma cells. In contrast ectopic expression of PRDM1 in lymphoma cells can lead to inhibition of proliferation or apoptosis. However, little is currently known about the regulation of PRDM1. The first study presented demonstrates that in lymphoma cells stimulation through the B cell receptor rapidly induces endogenous PRDM1 at the level of transcription. This study provides evidence that the PRDM1 promoter is preloaded and poised for activation in the B cell lines. The transcription factor PU.1 is shown to be required for B cell receptor induced expression of PRDM1 in lymphoma cells and in PU.1 positive myeloma cells. Furthermore, activation is associated with loss of the co-repressor TLE4 from the PU.1 complex. The second study establishes the requirement for PRDM1 in Mantle cell lymphoma (MCL) response to Bortezomib. MCL, an aggressive form of B cell lymphoma, has poor disease- free survival rate. The proteasome inhibitor, Bortezomib, is approved for treatment of relapsed and refractory MCL. However, the precise mechanism of action of Bortezomib is not well understood. Bortezomib rapidly induces transcription of PRDM1 along with apoptosis in MCL cell lines and primary MCL tumor samples. Knockdown of PRDM1 inhibits Bortezomib-induced apoptosis, while ectopic expression of PRDM1 alone leads to apoptosis in MCL. MKI67 and PCNA, which are required for proliferation and survival, were identified as novel direct targets of PRDM1 in MCL. Chromatin immunoprecipitation and knockdown studies reveal specific repression of MKI67 and PCNA is mediated by PRDM1 in response to Bortezomib. Furthermore promoter studies demonstrate that PRDM1 functions through a specific site in the proximal promoter region of PCNA and through a distal upstream repression domain on the MKI67 promoter. Together these findings establish PRDM1 as a key mediator of Bortezomib activity in MCL through suppression of proliferation and survival genes. The third study presented demonstrates use of Tandem affinity purification technique followed by mass spectrometry to identify PRDM1 and Reptin52 protein interactions. The observations in this study provide preliminary evidence of novel mechanism of regulation of PRDM1 protein function.

This thesis is focused on enzymatic studies of the secreted aspartic proteases (Saps) from Candida albicans as a tool for discovery of anti-candida drugs. C. albicans causes infections in a number of different locations, which differ widely in the protein substrates available and pH. Since C. albicans needs Saps during virulent growth, these enzymes are good targets for drug development. In order to investigate the catalytic characteristics of Saps and their inhibitor affinities, substrate-based kinetic assays were developed. Due to the low sensitivity of these assays, especially at the sub-optimal pH required to mimic the different locations of infections, these assays were not satisfactory. Therefore, a biosensor assay was developed whereby, it was possible to study interaction between Saps and inhibitors without the need to optimise catalytic efficacy. Furthermore, the biosensor assay allowed determination of affinity, as well as the individual association and dissociation rates for inhibitor interactions. Knowledge about substrate specificity, Sap subsite adaptivity, and the pH dependencies of catalytic efficacy has been accumulated. Also, screening of transition-state analogue inhibitors designed for HIV-1 protease has revealed inhibitors with affinity for Saps. Furthermore, the kinetics and pH dependencies of their interaction with Saps have been investigated. One of these inhibitors, BEA-440, displayed a complex interaction with Saps, indicating a conformational change upon binding and a very slow dissociation rate. A time dependent interaction was further supported by inhibition measurements. The structural information obtained affords possibilities for design of new more potent inhibitors that might ultimately become drugs against candidiasis. The strategy to combine substrate specificity studies with inhibitor screening has led to complementary results that generate a framework for further development of potent inhibitors.

Le traitement du VHC connaît une évolution rapide avec le développement d'antiviraux à action directe plus efficaces et mieux tolérés qui vont modifier les stratégies thérapeutiques, les facteurs prédictifs de réponse et les modalités de suivi des patients. Notre travail s'intéresse aux paramètres de suivi du traitement que sont les dosages pharmacologiques de ribavirine et le suivi des cinétiques virales lors d'une trithérapie. L'étude d'une cohorte prospective incluant 186 patients sous trithérapie par IP montre que 60% d'entre-eux présentent une SVR12 et que les facteurs prédictifs sont le génotype de l'IL28B et la réponse au précédent traitement. Une diminution de la filtration glomérulaire réversible est également observée. La mesure du taux résiduel de ribavirine permet de réduire les risques hématologiques chez des patients insuffisants rénaux, la réalisation de l'ASC témoigne d'une moins bonne exposition à la ribavirine chez des patients co-infectés par le VIH/VHC et la biodisponibilité de la ribavirine et la sévérité des anémies augmentent chez des patients traités par télaprévir. Au sein de la cohorte CUPIC, la négativation ou la diminution >50-70% de la charge virale initiale à S2 de trithérapie sont fortement prédictives de la SVR12. Cette mesure à S2 permet aussi de dépister les échappements viraux précoces. La place de la ribavirine est importante dans les associations thérapeutiques actuelles et futures. Sa surveillance pharmacologique peut avoir un intérêt au cours de futures multi-thérapies exposant à d'éventuelles interactions médicamenteuses / The rapid development of new direct antiviral agents (DAA) against HCV gives hope of more potent and well tolerated treatments. These new compounds will deeply modify therapeutic schedules, virological response prognostic factors and patients’ monitoring. The aim of our work was to define the relevance of ribavirin plasma concentration and viral kinetics monitoring during triple therapy. The study of a prospective cohort including 186 patients under triple therapy showed an SVR12 rate of 60%. Associated predictive factors were IL-28B genotype and previous treatment response. A reversible decrease of glomerular filtration rate was also observed. Ribavirin plasma concentration monitoring reduced hematological risks among patients with renal insufficiency. Early ribavirin plasma exposure showed an underexposure among HIV/HCV patients and ribavirin biodisponibility with severe anemia increased among telaprevir-treated patients. Within the CUPIC cohort, the initial viral load undetectability or decrease up to 50% or 70% at week 2 of triple therapy were predictive of SVR12. Moreover, this week 2 viral load assessment allowed the detection of early viral breakthrough. Ribavirin still plays a major role in current and future therapeutic strategies. Ribavirin monitoring could also be important during future multi-drug therapy that could be associated with drug interactions

<p>Ten years ago, the first protease inhibitor targeting the human immunodeficiency virus (HIV) was approved for clinical use. Highly active antiretroviral therapy (HAART), which combined protease and reverse transcriptase inhibitors, quickly became the standard therapy for treating patients infected with HIV and Acquired Immune Deficiency Syndrome (AIDS). Nevertheless, last year the AIDS pandemic reached its highest level ever. Many infected patients, mainly in the developing countries, are still without treatment. Among those patients who receive treatment, an increase in drug resistance and new-infection with drug-resistant strains are seen. To come to terms with these problems, new drugs that are efficient against resistant strains and can be produced at low cost are needed.</p><p>In this study, we have focused our research efforts on cyclic sulfamides active as HIV-1 protease inhibitors. Distinctive to this compound class, as compared to the inhibitors so far approved for clinical use, was the incorporation of a water mimic that displaces the structural water (W301) observed in the X-ray crystal co-complexes. The first part of the study was aimed at understanding the rationale behind the nonsymmetric binding mode that the inhibitor adopted when bound to the enzyme. Symmetric and nonsymmetric inhibitors were synthesized and the structure-activity relationships and preferable binding modes were rationalized with the help of Comparative Molecular Field Analysis (CoMFA).</p><p>In the second part of the study, an attempt was made to reduce the size of these inhibitors. As a result, the traditional P1/P1' substituents were removed, while the P2/P2' substituents were elongated in an attempt to reach between the binding sites. The design hypothesis was shown to be successful and inhibitors possessing nanomolar activity were identified.</p>

<p>This thesis is focused on enzymatic studies of the secreted aspartic proteases (Saps) from <i>Candida albicans</i> as a tool for discovery of anti-<i>candida</i> drugs. <i>C. albicans</i> causes infections in a number of different locations, which differ widely in the protein substrates available and pH. Since <i>C. albicans</i> needs Saps during virulent growth, these enzymes are good targets for drug development.</p><p>In order to investigate the catalytic characteristics of Saps and their inhibitor affinities, substrate-based kinetic assays were developed. Due to the low sensitivity of these assays, especially at the sub-optimal pH required to mimic the different locations of infections, these assays were not satisfactory. Therefore, a biosensor assay was developed whereby, it was possible to study interaction between Saps and inhibitors without the need to optimise catalytic efficacy. Furthermore, the biosensor assay allowed determination of affinity, as well as the individual association and dissociation rates for inhibitor interactions.</p><p>Knowledge about substrate specificity, Sap subsite adaptivity, and the pH dependencies of catalytic efficacy has been accumulated. Also, screening of transition-state analogue inhibitors designed for HIV-1 protease has revealed inhibitors with affinity for Saps. Furthermore, the kinetics and pH dependencies of their interaction with Saps have been investigated. One of these inhibitors, BEA-440, displayed a complex interaction with Saps, indicating a conformational change upon binding and a very slow dissociation rate. A time dependent interaction was further supported by inhibition measurements. The structural information obtained affords possibilities for design of new more potent inhibitors that might ultimately become drugs against candidiasis. The strategy to combine substrate specificity studies with inhibitor screening has led to complementary results that generate a framework for further development of potent inhibitors.</p>

<p>HIV-1 protease inhibitors are important in the most frequently used regimen for the treatment of HIV/AIDS, the highly active antiretroviral therapy (HAART). For patients with access to this treatment, an HIV infection is no longer lethal, but rather a manageable, chronic infection. However, the HIV-1 protease inhibitors are generally associated with serious shortcomings such as adverse events, development of drug resistance and poor pharmacokinetic properties. Most of the approved inhibitors suffer from high protein binding, rapid metabolism and/or low membrane permeability. </p><p>In this project, novel HIV-1 protease inhibitors comprising a rarely used tertiary alcohol in the transition-state mimic were designed, synthesized and evaluated. The rationale behind the design was to achieve ‘masking’ of the tertiary alcohol by for example, intramolecular hydrogen bonding, which was believed could enhance transcellular transport. </p><p>A reliable synthetic protocol was developed and a series of highly potent inhibitors was obtained exhibiting excellent membrane permeation properties in a Caco-2 cell assay. However, the cellular antiviral potencies of these compounds were low. In an attempt to improve the anti-HIV activity, microwave-accelerated, palladium-catalyzed cross-coupling reactions and aminocarbonylation of aryl bromide precursors were employed to produce P1'-extended test compounds. Inhibitors demonstrating up to six times higher antiviral effect were obtained, the best derivatives having para 3- or 4-pyridyl elongations in P1'.</p><p>Fast metabolic degradation was observed in liver microsome homogenate, which is believed, at least partly, to be attributable to benzylic oxidation of the indanol P2 group of the inhibitors. To enable facile variation of the P2 side chain a new synthetic route was developed using an enantiomerically pure, benzyl-substituted epoxy carboxylic acid as the key intermediate. Cyclic and amino-acid-residue-derived P2 groups were evaluated, and inhibitors equipotent to the series containing an indanol moiety were produced.</p>

Ten years ago, the first protease inhibitor targeting the human immunodeficiency virus (HIV) was approved for clinical use. Highly active antiretroviral therapy (HAART), which combined protease and reverse transcriptase inhibitors, quickly became the standard therapy for treating patients infected with HIV and Acquired Immune Deficiency Syndrome (AIDS). Nevertheless, last year the AIDS pandemic reached its highest level ever. Many infected patients, mainly in the developing countries, are still without treatment. Among those patients who receive treatment, an increase in drug resistance and new-infection with drug-resistant strains are seen. To come to terms with these problems, new drugs that are efficient against resistant strains and can be produced at low cost are needed. In this study, we have focused our research efforts on cyclic sulfamides active as HIV-1 protease inhibitors. Distinctive to this compound class, as compared to the inhibitors so far approved for clinical use, was the incorporation of a water mimic that displaces the structural water (W301) observed in the X-ray crystal co-complexes. The first part of the study was aimed at understanding the rationale behind the nonsymmetric binding mode that the inhibitor adopted when bound to the enzyme. Symmetric and nonsymmetric inhibitors were synthesized and the structure-activity relationships and preferable binding modes were rationalized with the help of Comparative Molecular Field Analysis (CoMFA). In the second part of the study, an attempt was made to reduce the size of these inhibitors. As a result, the traditional P1/P1' substituents were removed, while the P2/P2' substituents were elongated in an attempt to reach between the binding sites. The design hypothesis was shown to be successful and inhibitors possessing nanomolar activity were identified.

HIV-1 protease inhibitors are important in the most frequently used regimen for the treatment of HIV/AIDS, the highly active antiretroviral therapy (HAART). For patients with access to this treatment, an HIV infection is no longer lethal, but rather a manageable, chronic infection. However, the HIV-1 protease inhibitors are generally associated with serious shortcomings such as adverse events, development of drug resistance and poor pharmacokinetic properties. Most of the approved inhibitors suffer from high protein binding, rapid metabolism and/or low membrane permeability. In this project, novel HIV-1 protease inhibitors comprising a rarely used tertiary alcohol in the transition-state mimic were designed, synthesized and evaluated. The rationale behind the design was to achieve ‘masking’ of the tertiary alcohol by for example, intramolecular hydrogen bonding, which was believed could enhance transcellular transport. A reliable synthetic protocol was developed and a series of highly potent inhibitors was obtained exhibiting excellent membrane permeation properties in a Caco-2 cell assay. However, the cellular antiviral potencies of these compounds were low. In an attempt to improve the anti-HIV activity, microwave-accelerated, palladium-catalyzed cross-coupling reactions and aminocarbonylation of aryl bromide precursors were employed to produce P1'-extended test compounds. Inhibitors demonstrating up to six times higher antiviral effect were obtained, the best derivatives having para 3- or 4-pyridyl elongations in P1'. Fast metabolic degradation was observed in liver microsome homogenate, which is believed, at least partly, to be attributable to benzylic oxidation of the indanol P2 group of the inhibitors. To enable facile variation of the P2 side chain a new synthetic route was developed using an enantiomerically pure, benzyl-substituted epoxy carboxylic acid as the key intermediate. Cyclic and amino-acid-residue-derived P2 groups were evaluated, and inhibitors equipotent to the series containing an indanol moiety were produced.

Hepatitis C is a global health problem that affects approximately 120–180 million people. This viral infection causes serious liver diseases and the therapy available suffers from low efficiency and severe side effects. Consequently, there is a huge unmet medical need for new therapeutic agents to combat the hepatitis C virus (HCV). Inhibition of the viral NS3 protease has recently emerged as a promising approach to defeat this infection, and the first HCV NS3 protease inhibitors have now entered clinical trials. In this project, several novel HCV NS3 protease inhibitors have been designed, synthesized and biochemically evaluated. Inhibitors with various P1 C-terminal functional groups intended as potential bioisosteres to the carboxylic acid found in product-based inhibitors have been revealed. Special focus has been placed on establishing structure–activity relationships of inhibitors containing the promising P1 C-terminal acyl sulfonamide group. The properties of the acyl sulfonamide functionality that are important for producing potent inhibitors have been identified. In addition, the advantages of the acyl sulfonamide group compared to the carboxylic acid have been demonstrated in both enzymatic and cell-based assays. Besides the acyl sulfonamide functionality, the acyl cyanamide and the acyl sulfinamide groups have been identified as new carboxylic acid bioisosteres in HCV NS3 protease inhibitors. The synthetic work included the development of a fast and convenient methodology for the preparation of aryl acyl sulfonamides. The use of microwave heating and Mo(CO)6 as a solid carbon monoxide source provided aryl acyl sulfonamides from aryl halides in excellent yields. This method was subsequently used in the decoration of novel HCV NS3 protease inhibitors comprising a non-natural P1 moiety. This new class of compounds can be used as lead structures in a future optimization process aimed at producing more drug-like HCV NS3 protease inhibitors.

The hepatitis C virus (HCV), with a global prevalence of roughly 2%, is among the most serious diseases today. Among the more promising HCV targets is the NS3 protease, for which several drug candidates have entered clinical trials. In this work, computational methods have been developed and applied to the design of inhibitors of the HCV NS3 protease. Comparative molecular field analysis (CoMFA) modeling and molecular docking are the two main computational tools used in this work. CoMFA is currently the most widely used 3D-QSAR method. Methodology for improving its predictive performance by evaluating 6120 combinations of non-default parameters has been developed. This methodology was tested on 9 data sets for various targets and found to consistently provide models of enhanced predictive accuracy. Validation was performed using q2, r2pred and response variable randomization. Molecular docking was used to develop SARs in two series of inhibitors of the HCV NS3 protease. In the first series, preliminary investigations indicated that replacement of P2 proline with phenylglycine would improve potency. Docking suggested that phenylglycine-based inhibitors may participate in two additional interactions but that the larger, more flexible phenylglycine group may result in worse ligand fit, explaining the loss in potency. In the second series, β-amino acids were explored as α-amino acid substitutes. Although β-amino acid substitution may reduce the negative attributes of peptide-like compounds, this study showed that β-amino acid substitution resulted in reduced potency. The P3 position was least sensitive to substitution and the study highlighted the importance of interactions in the oxyanion hole. Finally, docking was used to provide the conformations and alignment necessary for a CoMFA model. This CoMFA model, derived using default settings, had q2 = 0.31 and r2pred = 0.56. Application of the optimization methodology provided a more predictive model with q2 = 0.48 and r2pred = 0.68.