Mrudula Donepudi

Insights into the Regulatory Mechanism for Caspase-8 Activation

In the death receptor induced apoptotic pathway, caspase-8 autocatalytically cleaves itself at specific cleavage sites. To better understand the regulatory mechanisms behind caspase-8 activation, we compared active wild-type caspase-8 (wtC8) and an uncleavable form of procaspase-8 (uncleavable C8). We demonstrate that wtC8 predominantly exists as a monomer and dimerizes in a concentration and inhibitor binding-dependent fashion. The K(D) for dimeric wtC8 is approximately 50 micro M and decreases when inhibitor bound. Uncleavable C8 is mainly monomeric, but a small amount that dimerizes is as active as wtC8. Inhibitor binding does not favor dimerization but induces active site rearrangements in uncleavable C8. Our findings suggest that dimerization is the crucial factor for caspase-8 activation.

Structure and zymogen activation of caspases

Apoptosis is primarily executed by active caspases, which are derived from the inactive zymogens. Structural and biochemical studies of caspases-1, -3, -7, -8 and -9 have greatly enhanced our understanding of the structure, function, and specificity of the active form of these enzymes. Only recently, the structures of procaspase-7 and biochemical studies of procaspase-9 and -8 have provided insight into the process of procaspase activation. The mechanism of zymogen activation requires limited proteolysis as for many other proteases. In addition, self-activation through oligomerization has been demonstrated for the initiator caspases-8, -9 and -10. These studies provide a structural mechanism for caspase activation, substrate/inhibitor binding, and contribute to the understanding of the biological role of caspases in the processes of apoptosis.

Human caspases in vitro: expression, purification and kinetic characterization.

A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer. Furthermore, we observed that not all caspases show a linear relationship between enzymatic activity and protein concentration, which explains the discrepancy between published values of specific activities from different laboratories. Our broad kinetic analysis allows the conclusion that the dependency of caspase activities on protein concentration is an effect of concentration-dependent dimerization, which can also be influenced by kosmotropic salts. The protocol recommendations as an outcome of this work will yield higher reproducibility regarding expression and purification of human caspases and contribute to standardization of enzyme kinetic data.

Work productivity among treatment-naïve patients with genotype 1 chronic hepatitis C infection receiving telaprevir combination treatment

Work productivity is impacted in hepatitis C virus (HCV)‐infected patients and has been linked to treatment. In two Phase 3 trials, ADVANCE and ILLUMINATE, treatment‐naïve genotype 1 chronic HCV‐infected patients received 12‐week telaprevir (T) with 24 (T12PR24)‐ or 48 (T12PR48)‐week peginterferon alfa‐2a/ribavirin. The objective of this analysis was to examine the impact of chronic HCV infection and its treatment with combination therapy on work productivity. The 5‐item, self‐reported work productivity questionnaire (WPQ) was administered in Phase 3 trials to assess unemployment status, days unable to work due to HCV/treatment, reduced hours worked and impact on productivity in prior 4 weeks. Descriptive statistics and multivariate regression analyses were employed in analyses of pooled trial data. About 1147 patients were included; 22% (n = 255) were unemployed at baseline, with 8% being unemployed due to health reasons. At week 12, there were no differences by treatment regimen in the number of days unable to work. At week 48, improvements were observed earlier among patients receiving the shorter duration of T combination treatment. Mean (95% CI) change from baseline in days unable to work was −0.48 (−0.85, −0.11) days for T12PR24, 1.43 (0.63, 2.24) days for T12PR48 and 1.24 (0.18, 2.30) days for PR48 with placebo. Predictors of days unable to work were identified and include demographic characteristics, pretreatment and on‐treatment levels of fatigue, as well regional variation. In post hoc analyses of the ADVANCE and ILLUMINATE trials, work productivity decreased during the initial 12 weeks regardless of treatment group.

904 HEALTH-RELATED QUALITY-OF-LIFE AMONG GENOTYPE 1 TREATMENT-EXPERIENCED CHRONIC HEPATITIS C PATIENTS: POST-HOC ANALYSES FROM THE REALIZE STUDY

did not return baseline until Week 60. Over the entire study, fatigue was lower for simeprevir-treated patients than controls (p < 0.001). EQ5D-VAS plots showed an expected inverse trend to FSS. FSS demonstrated high internal consistency (Cronbach’s a 0.95), reasonable test-retest reliability (intra-class correlation 0.74) and concurrent validity (correlation between FSS total score and EQ5D-VAS at Week 24; −0.63).

Sa1048 Health-Related Quality-of-Life Among Genotype 1 Treatment-NaïVE Chronic Hepatitis C Patients Receiving Telaprevir Combination Treatment: Post-Hoc Analyses of Data From the Advance Trial

combined with less than 1 log10 decline of HBV DNA at week 12 is the best early predictor of nonresponder revealed very high negative predictive value (NPV) and sensitivity (100% and 100%, respectively) with higher positive predictive value (PPV) and specificity (40% and 44%, respectively). Conclusions: This study suggests that treatment with peginterferon alpha-2a had high rate of HBsAg loss even in genotype C. Combination of HBsAg level and Hepatitis B viral load reduction at week 12 is very useful to predict SVR. With very high NPV, these values may be useful for early stopping rule.