A new structural class of serine protease inhibitors revealed by the structure of the hirustasin–kallikrein complex

AbstractBackground: Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin.Results: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 Å resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described.Conclusions: The crystal structure of the kallikrein–hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel β sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine

Efficacy, safety, and dose of Pafuramidine, a new oral drug for treatment of first stage sleeping sickness, in a phase 2a clinical study and phase 2b randomized clinical studies

Sleeping sickness (human African trypanosomiasis [HAT]) is caused by protozoan parasites and characterized by a chronic progressive course, which may last up to several years before death. We conducted two Phase 2 studies to determine the efficacy and safety of oral pafuramidine in African patients with first stage HAT.; The Phase 2a study was an open-label, non-controlled, proof-of-concept study where 32 patients were treated with 100 mg of pafuramidine orally twice a day (BID) for 5 days at two trypanosomiasis reference centers (Angola and the Democratic Republic of the Congo [DRC]) between August 2001 and November 2004. The Phase 2b study compared pafuramidine in 41 patients versus standard pentamidine therapy in 40 patients. The Phase 2b study was open-label, parallel-group, controlled, randomized, and conducted at two sites in the DRC between April 2003 and February 2007. The Phase 2b study was then amended to add an open-label sequence (Phase 2b-2), where 30 patients received pafuramidine for 10 days. The primary efficacy endpoint was parasitologic cure at 24 hours (Phase 2a) or 3 months (Phase 2b) after treatment completion. The primary safety outcome was the rate of occurrence of World Health Organization Toxicity Scale Grade 3 or higher adverse events. All subjects provided written informed consent.; Pafuramidine for the treatment of first stage HAT was comparable in efficacy to pentamidine after 10 days of dosing. The cure rates 3 months post-treatment were 79% in the 5-day pafuramidine, 100% in the 7-day pentamidine, and 93% in the 10-day pafuramidine groups. In Phase 2b, the percentage of patients with at least 1 treatment-emergent adverse event was notably higher after pentamidine treatment (93%) than pafuramidine treatment for 5 days (25%) and 10 days (57%). These results support continuation of the development program for pafuramidine into Phase 3

Cardiac Alterations in Human African Trypanosomiasis (T.b. gambiense) with Respect to the Disease Stage and Antiparasitic Treatment

In Human African Trypanosomiasis (HAT), neurological symptoms dominate and cardiac involvement has been suggested. Because of increasing resistance to the available drugs for HAT, new compounds are desperately needed. Evaluation of cardiotoxicity is one parameter of drug safety, but without knowledge of the baseline heart involvement in HAT, cardiologic findings and drug-induced alterations will be difficult to interpret. The electrocardiogram (ECG) is a tool to evaluate cardiac involvement and the risk of arrythmias. We analysed the ECG of 465 HAT patients and compared them with the ECG of 61 healthy volunteers. In HAT patients the QTc interval was prolonged. This comprises a risk of fatal arrhythmias if new drugs with antiarrhythmic potential will be used. Further, repolarization changes and low voltage were more frequent than in healthy controls. This could be explained by an inflammation of the heart. Treatment of HAT was associated with appearance of repolarization changes but not with a QTc prolongation. These changes appear to be associated with the disease, but not with a specific drug. The main conclusion of this study is that heart involvement is frequent in HAT and mostly well tolerated. However, it can become relevant, if new compounds with antiarrhythmic potential will be used

Efficacy and safety of pafuramidine versus pentamidine maleate for treatment of first stage sleeping sickness in a randomized, comparator-controlled, international phase 3 clinical trial

Sleeping sickness (human African trypanosomiasis [HAT]) is a neglected tropical disease with limited treatment options that currently require parenteral administration. In previous studies, orally administered pafuramidine was well tolerated in healthy patients (for up to 21 days) and stage 1 HAT patients (for up to 10 days), and demonstrated efficacy comparable to pentamidine.; This was a Phase 3, multi-center, randomized, open-label, parallel-group, active control study where 273 male and female patients with first stage Trypanosoma brucei gambiense HAT were treated at six sites: one trypanosomiasis reference center in Angola, one hospital in South Sudan, and four hospitals in the Democratic Republic of the Congo between August 2005 and September 2009 to support the registration of pafuramidine for treatment of first stage HAT in collaboration with the United States Food and Drug Administration. Patients were treated with either 100 mg of pafuramidine orally twice a day for 10 days or 4 mg/kg pentamidine intramuscularly once daily for 7 days to assess the efficacy and safety of pafuramidine versus pentamidine. Pregnant and lactating women as well as adolescents were included. The primary efficacy endpoint was the combined rate of clinical and parasitological cure at 12 months. The primary safety outcome was the frequency and severity of adverse events. The study was registered on the International Clinical Trials Registry Platform at www.clinicaltrials.gov with the number ISRCTN85534673.; The overall cure rate at 12 months was 89% in the pafuramidine group and 95% in the pentamidine group; pafuramidine was non-inferior to pentamidine as the upper bound of the 95% confidence interval did not exceed 15%. The safety profile of pafuramidine was superior to pentamidine; however, 3 patients in the pafuramidine group had glomerulonephritis or nephropathy approximately 8 weeks post-treatment. Two of these events were judged as possibly related to pafuramidine. Despite good tolerability observed in preceding studies, the development program for pafuramidine was discontinued due to delayed post-treatment toxicity

The probability of refractory cases.

<p>The probability of refractory cases.</p

Treatment-emergent adverse events experienced by pafuramidin treated patients in the Phase 2a study.

<p>Treatment-emergent adverse events experienced by pafuramidin treated patients in the Phase 2a study.</p

Treatment-emergent adverse events experienced by ≥3 patients in any treatment group in the Phase 2b studies.

<p>Treatment-emergent adverse events experienced by ≥3 patients in any treatment group in the Phase 2b studies.</p

Parasitological cure rates (primary and secondary efficacy endpoints).

<p>Parasitological cure rates (primary and secondary efficacy endpoints).</p