Complicações renais e urológicas incluindo nefrolitíase, cristalúria, cólica renal e lombalgia, são eventos adversos bem conhecidos do indinavir (IDV), um inibidor de protease (IP) largamente utilizado no tratamento de pacientes infectados com o vírus da imunodeficiência humana (HIV). Prévios estudos em ratos demonstraram que o IDV, um potente IP capaz de provocar uma sustentada supressão da carga viral do HIV, induz vasoconstricção renal, diminui a filtração glomerular (RFG) e reduz a excreção urinária de nitrito (NO2-), sugerindo que a vasoconstricção causada pelo IDV deve ser mediada pelo óxido nítrico (NO). Os objetivos deste estudo foram investigar a ocorrência de insuficiência renal (clearance de creatinina < 80ml/min) em pacientes com infecção pelo HIV tratados com terapia anti-retroviral altamente potente incluindo o inibidor de protease IDV, e mensurar a excreção urinária de nitrato (NO3-) nestes pacientes, comparando-os com outro grupo de pacientes tratados com efavirenz (EFV), um inibidor de transcriptase reversa não-análogo de nucleosídeo (NNRTI). No período compreendido entre março de 2000 e outubro de 2003, estudamos 36 pacientes infectados pelo HIV que estavam em terapia com IDV na dose de 800 mg de 8/8 horas por pelo menos 12 meses. Os pacientes foram avaliados para uma variedade de parâmetros clínicos e laboratoriais: idade, peso, tempo de infecção, tempo de uso de IDV, uso de sulfametoxazol-trimetoprim (SMX-TMP) ou sulfadiazina, exames bioquímicos (colesterol total, triglicérides, magnésio, sódio, potássio e creatinina), exame do sedimento urinário, clearance de creatinina, osmolaridade urinária, volume urinário de 24 h, fração de excreção de sódio (FENa), fração de excreção de potássio (FEK) e fração de excreção de água (FEH2O). NO3 urinário foi mensurado em 18 pacientes recebendo terapia anti-retroviral com IDV e 8 pacientes recebendo terapia com EFV. Leucocitúria ocorreu em 78.8% dos pacientes tratados com IDV. Clearance de creatinina diminuído foi observado em 21 pacientes e foi associado com menor peso e uso de derivados de sulfa. Nestes pacientes com diminuição da função renal, também detectamos menor osmolaridade urinária e uma FEH2O mais alta. A excreção urinária de NO3- foi significativamente menor nos pacientes tratados com IDV (908 ± 181) quando comparados aos pacientes do grupo EFV (2247 ± 648, p<0.01). Nossos resultados mostram que insuficiência renal ocorreu em 58% dos pacientes tratados com IDV e foi associada com menor peso corpóreo e uso de derivados de sulfa. A menor excreção urinária de NO3- e as alterações na osmolaridade e FEH2O sugerem que o IDV diminui a produção de óxido nítrico e causa dano tubular, respectivamente. Sugerimos então que os pacientes em uso de IDV sejam monitorados routineiramente para função renal através do clearance de creatinina. / Renal and urological complications including nephrolithiasis, crystalluria, renal colic and flank pain are significant side effects of the HIV protease inhibitor indinavir (IDV), and IDV has been widely used in the treatment of human immunodeficiency virus (HIV) infection. Previous studies in rats demonstrated that IDV, a potent protease inhibitor that causes profound and sustained supression of HIV replication, also induces renal vasoconstriction, decreases glomerular filtration rate (GFR) and reduces urinary excretion of nitrite (NO2-), suggesting that IDV-vasoconstriction may be mediated by nitric oxide (NO). The objectives of this study were to investigate the occurrence of renal failure (creatinine clearance <80ml/min) in human HIV patients treated with highy active antiretroviral therapy (HAART), including IDV, and to measure urinary excretion of nitrate (NO3-) in those patients, comparing it with that of another group of patients treated with the non-nucleoside reverse-transcriptase inhibitor efavirenz (EFV). From March 2000 through October 2003, we evaluated 36 patients infected with HIV who was receiving IDV 800 mg q8h for at least 12 months. The patients were assessed for a variety of clinical and laboratory parameters including age, body weight, duration of infection, time of IDV treatment, trimethoprim/sulfamethoxazole (TMP/SMX) or sulfadiazine use, biochemistry (total cholesterol, triglycerides, magnesium, sodium, potassium and creatinine), urinalysis, creatinine clearance, urine osmolality, 24-hour urine volume, fractional excretion of sodium (FENa), potassium (FEK) and water (FEH2O). Urinary NO3 was measured in 18 IDV-treated patients and compared with that of 8 EFV-treated patients. Leukocyturia occurred in 78.8% of the IDV-treated patients. Reduced creatinine clearance was observed in 21 patients and was associated with lower body weight and sulfa-derivated use. In these renal failure patients, we also detected a lower osmolality and a higher FEH2O. Excretion of NO3- was significantly lower in IDV-treated patients (908 ± 181) than in EFV-treated patients (2247 ± 648, p<0.01). Our data show that renal failure occurred in 58% of IDV-treated patients and was associated with lower body weight and sulfa administration. The lower NO3- excretion suggests that this drug decreases nitric oxide production, and the alterations in osmolality and FEH2O indicate that it also causes tubular damage. Based on our findings, we suggest that the renal function of patients under IDV treatment should be closely monitored with creatinine clearance.

Complicações renais e urológicas incluindo nefrolitíase, cristalúria, cólica renal e lombalgia, são eventos adversos bem conhecidos do indinavir (IDV), um inibidor de protease (IP) largamente utilizado no tratamento de pacientes infectados com o vírus da imunodeficiência humana (HIV). Prévios estudos em ratos demonstraram que o IDV, um potente IP capaz de provocar uma sustentada supressão da carga viral do HIV, induz vasoconstricção renal, diminui a filtração glomerular (RFG) e reduz a excreção urinária de nitrito (NO2-), sugerindo que a vasoconstricção causada pelo IDV deve ser mediada pelo óxido nítrico (NO). Os objetivos deste estudo foram investigar a ocorrência de insuficiência renal (clearance de creatinina < 80ml/min) em pacientes com infecção pelo HIV tratados com terapia anti-retroviral altamente potente incluindo o inibidor de protease IDV, e mensurar a excreção urinária de nitrato (NO3-) nestes pacientes, comparando-os com outro grupo de pacientes tratados com efavirenz (EFV), um inibidor de transcriptase reversa não-análogo de nucleosídeo (NNRTI). No período compreendido entre março de 2000 e outubro de 2003, estudamos 36 pacientes infectados pelo HIV que estavam em terapia com IDV na dose de 800 mg de 8/8 horas por pelo menos 12 meses. Os pacientes foram avaliados para uma variedade de parâmetros clínicos e laboratoriais: idade, peso, tempo de infecção, tempo de uso de IDV, uso de sulfametoxazol-trimetoprim (SMX-TMP) ou sulfadiazina, exames bioquímicos (colesterol total, triglicérides, magnésio, sódio, potássio e creatinina), exame do sedimento urinário, clearance de creatinina, osmolaridade urinária, volume urinário de 24 h, fração de excreção de sódio (FENa), fração de excreção de potássio (FEK) e fração de excreção de água (FEH2O). NO3 urinário foi mensurado em 18 pacientes recebendo terapia anti-retroviral com IDV e 8 pacientes recebendo terapia com EFV. Leucocitúria ocorreu em 78.8% dos pacientes tratados com IDV. Clearance de creatinina diminuído foi observado em 21 pacientes e foi associado com menor peso e uso de derivados de sulfa. Nestes pacientes com diminuição da função renal, também detectamos menor osmolaridade urinária e uma FEH2O mais alta. A excreção urinária de NO3- foi significativamente menor nos pacientes tratados com IDV (908 ± 181) quando comparados aos pacientes do grupo EFV (2247 ± 648, p<0.01). Nossos resultados mostram que insuficiência renal ocorreu em 58% dos pacientes tratados com IDV e foi associada com menor peso corpóreo e uso de derivados de sulfa. A menor excreção urinária de NO3- e as alterações na osmolaridade e FEH2O sugerem que o IDV diminui a produção de óxido nítrico e causa dano tubular, respectivamente. Sugerimos então que os pacientes em uso de IDV sejam monitorados routineiramente para função renal através do clearance de creatinina. / Renal and urological complications including nephrolithiasis, crystalluria, renal colic and flank pain are significant side effects of the HIV protease inhibitor indinavir (IDV), and IDV has been widely used in the treatment of human immunodeficiency virus (HIV) infection. Previous studies in rats demonstrated that IDV, a potent protease inhibitor that causes profound and sustained supression of HIV replication, also induces renal vasoconstriction, decreases glomerular filtration rate (GFR) and reduces urinary excretion of nitrite (NO2-), suggesting that IDV-vasoconstriction may be mediated by nitric oxide (NO). The objectives of this study were to investigate the occurrence of renal failure (creatinine clearance <80ml/min) in human HIV patients treated with highy active antiretroviral therapy (HAART), including IDV, and to measure urinary excretion of nitrate (NO3-) in those patients, comparing it with that of another group of patients treated with the non-nucleoside reverse-transcriptase inhibitor efavirenz (EFV). From March 2000 through October 2003, we evaluated 36 patients infected with HIV who was receiving IDV 800 mg q8h for at least 12 months. The patients were assessed for a variety of clinical and laboratory parameters including age, body weight, duration of infection, time of IDV treatment, trimethoprim/sulfamethoxazole (TMP/SMX) or sulfadiazine use, biochemistry (total cholesterol, triglycerides, magnesium, sodium, potassium and creatinine), urinalysis, creatinine clearance, urine osmolality, 24-hour urine volume, fractional excretion of sodium (FENa), potassium (FEK) and water (FEH2O). Urinary NO3 was measured in 18 IDV-treated patients and compared with that of 8 EFV-treated patients. Leukocyturia occurred in 78.8% of the IDV-treated patients. Reduced creatinine clearance was observed in 21 patients and was associated with lower body weight and sulfa-derivated use. In these renal failure patients, we also detected a lower osmolality and a higher FEH2O. Excretion of NO3- was significantly lower in IDV-treated patients (908 ± 181) than in EFV-treated patients (2247 ± 648, p<0.01). Our data show that renal failure occurred in 58% of IDV-treated patients and was associated with lower body weight and sulfa administration. The lower NO3- excretion suggests that this drug decreases nitric oxide production, and the alterations in osmolality and FEH2O indicate that it also causes tubular damage. Based on our findings, we suggest that the renal function of patients under IDV treatment should be closely monitored with creatinine clearance.

Viral proteases play crucial roles in the life cycle and maturation of many viruses by processing the viral polyprotein after translation and in some cases cleaving host proteins associated with the immune response. The essential role of viral proteases makes them attractive therapeutic targets. In this thesis, I provide an introductory summary of viral proteases, their structure, mechanism, and inhibition, while the breadth of this thesis focuses on the Hepatitis C virus (HCV) NS3/4A and Zika virus (ZIKV) NS2B/NS3 viral proteases. HCV NS3/4A protease inhibitors (PIs) have become a mainstay in combination therapies. However, drug resistance remains a major problem against these PIs. In this thesis, I applied insights from the HCV substrate envelope (SE) model to develop strategies for designing PIs that are less susceptible to resistance. Also, I used the HCV NS3/4A protease as a model system to decipher the molecular mechanism and role of fluorination in HCV PIs potency and drug resistance. The drug design strategies described in this thesis have broad applications in drug design. The ZIKV is an emerging global threat, and currently, with no treatment available. In this thesis, I described the discovery, biochemical and antiviral evaluation of novel noncompetitive quinoxaline-based inhibitors of the ZIKV NS2B/NS3 protease. The inhibitors are proposed to interfere with NS2 binding to NS3, thereby preventing the protease from adopting the closed and active conformation. The inhibitors from this work will serve as lead compounds for further inhibitor development toward the goal of developing antivirals.

Indiana University-Purdue University Indianapolis (IUPUI) / The Lyme disease agent, Borrelia burgdorferi, has a complex system that allows it to thrive in the harsh and distinct environments of its tick vector and mammalian host. Although it has been known for some time that the Borrelia oxidative stress regulator protein (BosR) plays a necessary role in mammalian infectivity and functions as a transcriptional regulator of alternative sigma factor RpoS, very little is known about its mechanism of action, other than the suggestion that BosR activates rpoS transcription by binding to certain upstream regions of the gene. In our studies, we performed protein degradation assays and luciferase reporter assays for further understanding of BosR function. Our preliminary findings suggest that BosR is post-transcriptionally regulated by an unknown protease and may not need to bind to any rpoS upstream regions in order to activate transcription. We also describe the construction of luciferase reporter systems that will shed light on BosR’s mechanism of action. We postulate the provocative possibility that unlike its homologs Fur and PerR in other bacterial systems, BosR may not utilize a DNA-binding mechanism in order to fulfill its role as a transcriptional regulator to modulate virulence gene expression.

Die Therapie der HIV Infektion besteht aus Kombination mehrerer antiretroviraler Substanzen und birgt ein erhöhtes Risiko an Arzneimittelwechselwirkungen. Das bekannte Problem der Virusresistenz kann zudem durch Enzyminduktion begünstigt werden. Das Ziel der vorliegenden Arbeit lag in Untersuchungen zu Einflüssen der Virustatika auf die Expression von Cytochrom P450 Enzymen: 1A1, 1B1, 3A4 sowie der P-Glykoproteins (MDR1) an immortalisierten Zellsystemen. Die Protease Inhibitoren Indinavir, Nelfinavir, Ritonavir und Saquinavir induzierten die Regulation der mRNA Expression über den Aryl-Kohlenwasserstoff-Rezeptor (AhR) und den Pregnan-X-Rezeptor (PXR) dosisabhängig und signifikant. Die Nukleosidischen Reverse Transkriptase Inhibitoren Zalcitabin, Zidovudin und Lamivudin sowie der Nicht-Nukleosidische Inhibitor Nevirapin zeigten induktive Eigenschaften nur für die AhR Zielgene CYP1A1 und CYP1B1. Amprenavir und Efavirenz aktivierten die PXR-Regulation. Die möglichen Auswirkungen der Induktion der untersuchten Gene wurden ausführlich diskutiert. Die molekularen Grundlagen der interindividuell variierenden Aktivität von CYP3A wurden in einer Probandenstudie untersucht. Es wurden die mRNA Expression in den Leukozyten, die Aktivität des Enzyms und einige bekannte Polymorphismen unter Einwirkung von Rifampicin untersucht und diskutiert. / The therapy of HIV infection requires a combination of several antiretroviral substances accompanying risk factors for drug-drug interactions. Moreover, virus resistance can be promoted by enzyme induction caused by antiretroviral drugs. The aim of the study was to investigate the influences of antiretroviral substances on the expression of cytochrome P450 enzymes: 1A1, 1B1, 3A4 and p-glycoprotein (MDR1) using immortalized cell systems. The protease inhibitors indinavir, nelfinavir, ritonavir and saquinavir induced significantly the regulation of mRNA expression through the aryl hydrocarbon receptor (AhR) and the pregnane-x-receptor (PXR) in a concentration-dependent manner. The nucleoside reverse transcriptase inhibitors zalcitabine, zidovudine and lamivudine and the non-nucleoside reverse transcriptase inhibitor nevirapine showed inductive properties only for the AhR target genes CYP1A1 and CYP1B1.Amprenavir and efavirenz activated the PXR target genes. Potentially effects of the described induction are discussed. In a second part of the work, the molecular mechanisms of the individual varying activity of the CYP3A enzyme were investigated applying an in vivo study. CYP3A4 mRNA expression and rifampicin mediated induction in leucocytes were correlated with systemic enzyme activity under induction and known polymorphisms.