PubMed Health⌕ Search

Biomedical subjects

M Brunetti

Publications and source records attributed to M Brunetti.

At least 19 recordsLinked to original sources
PubMed2006

Immunohistochemical characterization of Kisselev nodules (ectopic lymphoid follicles) in wild boar (Sus scrofa L.).

This paper describes the histopathological features and cellular distribution of T lymphocytes (CD3), B cells (CD79), follicular dendritic cells (FDC) and macrophages (alpha-1-antitrypsin, lysozyme) in lymphoid aggregates (Kisselev nodules) found in the lung, kidney and liver of wild boar (Sus scrofa L.). The distribution of immunoreactive cells, tested for antibodies, was similar to that found in the cortex of lymph nodes: lymphoid follicles with germinal centers mainly consisting of CD79+ B cells with sparse interfollicular tissue (CD3+ T lymphocytes). This finding and the association of these structures with helminthic infections suggests that local humoral immunity is central to the organism's response to parasitic challenge. The presence of follicular dendritic cells confirms the high degree of organization of these lymphoid-like structures. The role of other pathogenic factors and the induction of chronic inflammatory reaction in these ectopic lymphoid sites is also discussed.

Animals↗
PubMed2005

Human brain activation during passive listening to sounds from different locations: an fMRI and MEG study.

Recent animal and human studies indicate the existence of a neural pathway for sound localization, which is similar to the "where" pathway of the visual system and distinct from the sound identification pathway. This study sought to highlight this pathway using a passive listening protocol. We employed fMRI to study cortical areas, activated during the processing of sounds coming from different locations, and MEG to disclose the temporal dynamics of these areas. In addition, the hypothesis of different activation levels in the right and in the left hemispheres, due to hemispheric specialization of the human brain, was investigated. The fMRI results indicate that the processing of sound, coming from different locations, activates a complex neuronal circuit, similar to the sound localization system described in monkeys known as the auditory "where" pathway. This system includes Heschl's gyrus, the superior temporal gyrus, the supramarginal gyrus, and the inferior and middle frontal lobe. The MEG analysis allowed assessment of the timing of this circuit: the activation of Heschl's gyrus was observed 139 ms after the auditory stimulus, the peak latency of the source located in the superior temporal gyrus was at 156 ms, and the inferior parietal lobule and the supramarginal gyrus peaked at 162 ms. Both hemispheres were found to be involved in the processing of sounds coming from different locations, but a stronger activation was observed in the right hemisphere.

Acoustic Stimulation↗
PubMed2004

Audit of a shared-care program for persons with diabetes: baseline and 3 annual follow-ups.

In Italy, data on shared-care programs for diabetes are lacking. We described the characteristics of type 2 diabetic population assisted in general practice and evaluated 3 years of follow-up outcomes and performance indicators in a shared-care program in Modena, Italy (1998-2001); only well-controlled diabetic patients were considered. Forty-nine percent of territorial GPs adhered to the project (257 out of 521) and 77% of them sent 6409 paired baseline and follow-up datasheets. Altogether, 97.8% patients had type 2 diabetes, mean age 68.6+/-11.7 years, disease duration 9.6+/-7.5 years, BMI 28.6+/-4.8 kg/m2, HbA(1c) 7.6%+/-1.6%, 16.1% of them were disabled. Among the non-disabled patients, 23.6% had optimal glycemic control (HbA(1c) < or =6.5%); at baseline the prevalence of micro- and macrovascular diabetic complications was: 8.2% microalbuminuria and 2.4% macroalbuminuria plus nephropathy, 11.0% nonproliferative and 3.0% preproliferative retinopathy, 7.0% neuropathy, 1.8% diabetic foot; 8.5% angina, 6.9% TIA or stroke, 6.3% infarction, 5.2% intermittent claudication, 4.1% heart failure. Among the disabled patients 27.9% had optimal glycemic control, but they had more diabetic complications. The performance indicators significantly improved over the 3-year study period: glycemic control indicators increased from 66%-75% to 83%-90% and micro- and macrovascular indicators from 59%-65% to 75%-81%. The outcome indicators also improved: mean HbA(1c) value changed from 7.6%+/-1.6% to 7.3%+/-1.3% and the percentage of people with HbA(1c)< or =6.5% significantly improved over time. Similar trends were observed in both disabled and non-disabled diabetic patients.

Aged↗
PubMed2004

Once-weekly dosing of recombinant human erythropoietin alpha in patients with myelodysplastic syndromes unresponsive to conventional dosing.

BACKGROUND: Once-weekly dosing of recombinant human erythropoietin (rhEPO) in patients with myelodysplastic syndromes (MDS) has not been investigated thoroughly. We performed a clinical trial to evaluate the effects of this new dosing regimen in patients with MDS who were unresponsive to the conventional three-times-weekly schedule. PATIENTS AND METHODS: Forty-eight patients with low- or intermediate-risk MDS were enrolled in a 12-week study. rhEPO alpha (rhEPOalpha) was administered once-weekly by subcutaneous injection with a starting dose of 40,000 U fixed dose. The drug dosage was increased to 60,000 U fixed dose if after 6 weeks there was no or suboptimal erythroid response. RESULTS: Clinically significant responses were seen in 13 (27%) patients, with 11 improving their response after dose escalation of rhEPOalpha. Only one patient (case 23) maintains a response after a follow-up period of 14 months. All other patients had responses lasting between 10 and 43 weeks, with a median time to relapse of 20 weeks. Treatment was well tolerated, with no relevant adverse events. Response to therapy was associated with significantly higher concentrations of circulating erythroid blast-forming units and a decrease of the bone marrow fraction of apoptic CD34+ cells. CONCLUSIONS: Once-weekly rhEPOalpha therapy results in an improvement of erythropoiesis in a subset of MDS patients who are unresponsive to conventional dosing, and may act by inhibiting apoptosis of erythroid precursors. These results warrant further investigation of this dosing regimen either alone or in combination with other agents.

Aged↗
PubMed2004

Characteristics of Type 2 diabetic patients cared for by general practitioners either with medical nutrition therapy alone or with hypoglycaemic drugs.

The essential role of medical nutrition therapy (MNT) for people with diabetes is widely recognised, and its exclusive use is recommended in mild diabetes according to a stepwise therapeutic approach. We describe the characteristics of MNT-treated Type 2 diabetic patients (vs drugs) cared for by general practitioners (GPs) in order to check that appropriate differences did exist between the two groups, by auditing the data from our local shared-care program for diabetes. We had 16,000 diabetic patients (out of 630,000 inhabitants); 6,800 of them (42.5%) cared for by GPs. Thirty-one percent (2,079 out of 6,800 patients cared for by GPs) were treated with MNT and 69% with drugs. The MNT-treated patients (vs drugs) were younger (66.1 +/- 10.7 vs 67.7 +/- 11.0 yr, p<0.01), had shorter disease duration (8.2 +/- 6.6 vs 11.2 +/- 7.6 yr, p<0.01), lower HbA1c (7.0 +/- 1.1 vs 7.8 +/- 1.6%, p<0.01) and body mass index (BMI) (28.6 +/- 4.6 vs 29.0 +/- 4.9 kg/m2, p<0.01). They had less prevalence of high blood triglycerides (25.4% vs 29.0%, p<0.01). MNT-treated patients had less micro-albuminuria (5.3% vs 8.8%, p<0.01); less retinopathy both non-proliferant (6.5% vs 11.1%, p<0.01), and pre-proliferant (6.8% vs 12.7%, p<0.01), and proliferant (7.0% vs 12.9%, p<0.01); less peripheral neuropathy (3.9% vs 8.3%, p<0.01); and diabetic foot (1.0% vs 2.0%, p<0.01). They had less chronic heart failure (2.7% vs 4.6%, p<0.01), and claudicatio intermittens (3.3% vs 5.3%, p<0.01). In conclusion, the Type 2 diabetic patients cared for by GPs using MNT appropriately had a less severe form of diabetes.

Age Factors↗
PubMed2003

Changes in long-term glycemic control and performance indicators in a cohort of type 2 diabetic patients cared for by general practitioners: findings from the "Modena Diabetes Project".

BACKGROUND AND AIM: General practitioners (GPs) are being increasingly asked to play a key role in the shared care of people with diabetes mellitus, but data concerning the effects of this in Italy are still scarce. We therefore evaluated the 4-year follow-up changes in outcomes and performance indicators in type 2 diabetic patients cared for by GPs in the framework of the "Modena Diabetes Project". METHODS AND RESULTS: Seventy-four percent of the local GPs participating in the project (387 out of 521) sent 5260 paired baseline and follow-up datasheets. The baseline characteristics of the type 2 diabetic patients (49.6% male) were a mean age of 67.3+/-11.2 years, a mean disease duration of 10.9+/-7.4 years, a mean BMI of 28.7+/-4.8 kg/m2, and a mean HbA1c level 7.56+/-1.52%. After four years follow-up, the individual before/after match-paired outcomes revealed an improvement in glycemic control: HbA1c levels significantly decreased to 7.39+/-1.31%, and the percentage of patients with HbA1c level of <6.5% significantly increased from 15.7% to 22.1%. There was also a significant decrease in body weight (from 78.3+/-14.8 to 77.6+/-14.6 kg) and BMI (from 28.8+/-4.8 to 28.5+/-4.9 kg/m2). The time trends of glycemic control significantly improved during the 4-year follow up, but those of the body weight and BMI values did not. Furthermore, the percentages of performance indicators matching the expected rate of recurrence per each year of follow-up significantly improved during the study period. CONCLUSIONS: Long-term glycemic control and the performance indicators relating to the type 2 diabetic patients participating in our shared care programme progressively improved.

Aged↗
PubMed2002

Home based management in multiple sclerosis: results of a randomised controlled trial.

BACKGROUND: Home based medical care is a popular alternative to standard hospital care but there is uncertainty about its cost-effectiveness. OBJECTIVES: To compare the effectiveness and the costs of multidisciplinary home based care in multiple sclerosis with hospital care in a prospective randomised controlled trial with a one year follow up. METHODS: 201 patients with clinically definite multiple sclerosis were studied. They were randomised in a ratio 2:1 to an intervention group (133) or a control group (68). They were assessed at baseline and one year after randomisation with validated measures of physical and psychological impairment and quality of life (SF-36 health survey). The costs to the National Health Service over the one year follow up were calculated by a cost minimisation analysis. RESULTS: There were no differences in functional status between the home based care group and the hospital group. There was a significant difference between the two groups favouring home based management in four SF-36 health dimensions-general health, bodily pain, role-emotional, and social functioning (all p < or = 0.001). The cost of home based care was slightly less (822 euros/patient/year) than hospital care, mainly as a result of a reduction in hospital admissions. CONCLUSIONS: Comprehensive planning of home based intervention implemented by an interdisciplinary team and designed specifically for people with multiple sclerosis may provide a cost-effective approach to management and improve the quality of life.

Adult↗
PubMed2002

[Indications and short term results of subfascial endoscopic perforator surgery (SEPS)].

BACKGROUND: The aim of this study is to evaluate diagnostic methods, indications and surgical technique in SEPS procedure and to analyze short term results. METHODS: Eighteen patients affected by chronic venous insufficiency (CVI) have been analyzed. According to NAVS (North American Vascular Society) classification three patients were included in class 6 (C6), 3 (C5), 6 (C4), 2 (C3) and 4 (C2). From 2 to 5 selective subfascial endoscopic ligation of perforator veins, especially I and II Cockett perforator veins' were performed. In 7 cases, total stripping of the great (6) or less (1) saphenous vein was associated with SEPS procedure. After the operation, an elastic bandage of the lower limbs was performed and a medical treatment with LMVH was started. RESULTS: In 6 patients of the C2 and C3 groups, neither recurrence or pathological reflux were observed at clinical examination and at color duplex. In 12 patients of C4, C5 and C6 groups a reduction of the perimalleolar oedema was observed. In the last 3 patients, with leg ulceration, a resolution of the lesion in 2 cases, and a reduction in diameter in the last one, were observed. CONCLUSIONS: SEPS is particularly advised in those patients belonging to C5 and C6 groups, especially in presence of leg ulceration. This operations is suggested also in patients with CVI and incontinence of perforator veins detected by at color duplex. This diagnostic investigation seems to be adequate in the diagnosis of CVI and in the mapping of perforator veins of the leg.

Adult↗
PubMed2001

Role of charged residues in the catalytic mechanism of hepatitis C virus NS3 protease: electrostatic precollision guidance and transition-state stabilization.

Maturational cleavage of the hepatitis C virus polyprotein involves the viral chymotrypsin-like serine protease NS3. The substrate binding site of this enzyme is unusually flat and featureless. We here show that NS3 has a highly asymmetric charge distribution that is characterized by strong positive potentials in the vicinity of its active site and in the S5/S6 region. Using electrostatic potential calculations, we identified determinants of this positive potential, and the role of six different residues was explored by site-directed mutagenesis. Mutation of residues in the vicinity of the active site led to changes in k(cat) values of a peptide substrate indicating that basic amino acids play a role in the stabilization of the transition state. Charge neutralization in the S5/S6 region increased the K(m) values of peptide substrates in a manner that depended on the presence of negatively charged residues in the P5 and P6 positions. K(i) values of hexapeptide acids spanning P6-P1 (product inhibitors) were affected by charge neutralization in both the active site region and the S5/S6 region. Pre-steady-state kinetic data showed that the electrostatic surface potential is used by this enzyme to enhance collision rates between peptidic ligands and the active site. Calculations of the interaction energies of protease-substrate or protease-inhibitor complexes showed that electrostatic interaction energies oppose the formation of a tightly bound complex due to an unfavorable change in the desolvation energy. We propose that desolvation costs are minimized by avoiding the formation of individual ion pair interactions through the use of clusters of positively charged residues in the generation of local electrostatic potentials.

Amino Acid Sequence↗
PubMed2001

Characterization of the hepatitis C virus NS2/3 processing reaction by using a purified precursor protein.

The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Amino Acid Sequence↗
PubMed2001

Health-related quality of life (HR-QOL) and regulatory issues. An assessment of the European Agency for the Evaluation of Medicinal Products (EMEA) recommendations on the use of HR-QOL measures in drug approval.

Interest in measuring qualitative aspects of life that are most closely related to health and healthcare has increased in recent years. Methods of describing patients' subjective health status now incorporate standardised measures, and several psychometric measures are available. Despite the thousands of empirical and conceptual papers in the medical and pharmacological literature on health-related quality of life (HR-QOL), the value of such measures in the regulatory process is still being debated. We conducted an assessment to understand and document the position of the European Agency for the Evaluation of Medicinal Products (EMEA) on the use of HR-QOL measures in studies conducted for regulatory purposes. Official documents produced and circulated by the EMEA containing recommendations on trial design, conduct and analysis for sponsors and scientific experts were independently reviewed by authors to document the position of the Agency on the specific topic of HR-QOL. All documents found in the Agency website on 30 September 1999 were identified and then assessed to: (i) identify diseases or drugs for which formal HR-QOL assessment is recommended; (ii) identify measures and methods recommended; and (iii) evaluate the reliability of recommendations across documents. Of the 189 documents retrieved, none focused directly on health-related quality of life. A few explicit recommendations were identified for 13 specific drugs or conditions. These recommendations were mostly general and vague, and used nonstandard terminology. In addition, terminology and recommendations were not consistent across documents and, in at least one case, were in contrast with the US Food and Drug Administration (FDA) guidelines. EMEA guidelines incorporating quality-of-life outcomes are welcomed but it is obvious that more detailed guidance is required. Closer collaboration between the EMEA and the FDA is also recommended. Experts from different disciplines should be involved in the preparation of such documents to assure the necessary technical expertise and the representativeness of the various counterparts.

Drug Approval↗
PubMed2001

Cost-effectiveness analysis of n-3 polyunsaturated fatty acids (PUFA) after myocardial infarction: results from Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto (GISSI)-Prevenzione Trial.

OBJECTIVE: To estimate the cost effectiveness of treatment with n-3 polyunsaturated fatty acids (PUFA) for secondary prevention after myocardial infarction (MI). DESIGN AND SETTING: The cost-effectiveness analysis of n-3 PUFA treatment after MI was based on morbidity and mortality data and the use of resources obtained prospectively during the 3.5 year follow-up period of the Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto (GISSI)-Prevenzione study. The cost-effectiveness analysis took into account the incremental number of life-years gained and the incremental costs for hospital admissions, diagnostic tests and drugs, applying a 5% discount rate. The value for money of n-3 PUFA treatment was assessed using the cost-effectiveness ratio and the number needed to treat (NNT) approach. PERSPECTIVE: Third-party payer. MAIN OUTCOME MEASURES AND RESULTS: The incremental cost-effectiveness ratio for n-3 PUFA in the basecase scenario was 24,603 euro (EUR, 1999 values) per life-year gained (95% confidence interval: 22,646 to 26,930). Sensitivity analysis included the analysis of extremes, producing estimates varying from EUR15,721 to EUR52,524 per life-year gained. 172 patients would need to be treated per year with n-3 PUFA, at an annual cost of EUR68,000, in order to save 1 patient. This is comparable with the NNT value, and associated annual cost for simvastatin, but less costly than that for pravastatin. CONCLUSIONS: The cost effectiveness of long term treatment with n-3 PUFA is comparable with other drugs recently introduced in the routine care of secondary prevention after MI. Since the clinical benefit provided by n-3 PUFA is additive, this therapy should be added to the established routine practice, with additive costs.

Cost-Benefit Analysis↗
PubMed2001

Cost effectiveness of ramipril in patients with non-diabetic nephropathy and hypertension: economic evaluation of Ramipril Efficacy in Nephropathy (REIN) Study for Germany from the perspective of statutory health insurance.

BACKGROUND: In the Ramipril Efficacy In Nephropathy (REIN) trial, ramipril significantly lowered the rate of reaching the combined end-point of doubling of baseline serum creatinine levels or end-stage renal failure (ESRF). OBJECTIVE: To determine the additional cost per patient-year of chronic (long term) dialysis avoided (PYCDA) when the ACE inhibitor, ramipril, was added to conventional treatment of patients with non-diabetic nephropathy and hypertension. STUDY PERSPECTIVE: Statutory Health Insurance (SHI) provider in Germany. DESIGN AND SETTING: Data from the REIN Study were used in a cost-effectiveness analysis (CEA). A modelling approach was used, which was based on secondary analysis of published data, and costs were those incurred by the SHI provider (i.e. SHI expenses). In the base-case analysis, average case-related SHI expenses were applied and PYCDA were quantified using the cumulative incidence of ESRF as observed in the REIN trial. MAIN OUTCOME MEASURES AND RESULTS: The incremental cost-effectiveness ratios (ICERs) of ramipril varied between about -76,700 deutschmarks (DM) and -DM81,900 per PYCDA (DM 1 approximately equals 0.55 US dollars; 1999 values), according to the treatment periods of 1 year and 3 years, respectively. In the sensitivity,analysis, the robustness of the model and its results were shown when the extent of influence of different model variables on the base-case results was investigated. First, probabilities of ESRF and PYCDA were estimated according to the Weibull method. Second, the influence of the model variables on the target variable was quantified using a deterministic model. Third, the dependency of the target variable (ICER) on random variables was described in a simulation. The cost for chronic dialysis had by far the greatest impact on the target variable, which was 28 times greater than the impact of clinical effectiveness of ramipril, i.e. the number of PYCDA. There were net savings per PYCDA with ramipril treatment after 1, 2 and 3 years: 95% of the 10,000 simulation steps resulted in savings of between DM69 500 and D94,600 per PYCDA after 3 years. CONCLUSIONS: Results from this evaluation show that ramipril offers enormous savings from the perspective of the SHI provider (third-party payer) in Germany when added to the conventional treatment of patients with non-diabetic nephropathy and hypertension.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2001

[Indication for short stripping of the great saphenous vein. Results and indications].

BACKGROUND: The aim of surgical therapy of varicose veins is the elimination of reflux from the deep to superficial system at the saphenous crosse and perforant vessel and conservation of the superficial venous system due to possible surgical procedures for arterial revascularization. This latter condition leads to an extension of indications for short stripping procedures, although the venous distal segment may undergo hypoplastic degeneration not compatible for revascularization purposes. Another important reason is the minor incidence of neurologic complication due to saphenous nerve lesion which may occur during long saphenous stripping. METHODS: From January 1994 to June 1999, we considered 233 patients (182 women, 51 men); 180 cases underwent long saphenous stripping procedures, whereas 53 a short stripping of GSV. The incidence of neurologic complications of the saphenous nerve were recorded in 11.6% of the patients treated with the standard procedure, whereas no such complication was observed in all cases treated with the short stripping procedure. RESULTS: ECD follow-up performed for a period of three months from the surgical procedure revealed the patency of the residual saphenous vein, with a minimum diameter of 3 mm, in 28 patients (56.6%). CONCLUSIONS: Our opinion is to extend the indication for short stripping of the saphenous vein to all cases where the distal saphenous trunk is not involved, when the ECD shows a pathological ostial reflux, a truncular reflux limited to the thigh, which may be associated with incontinence of the perforant vein of Dodd.

Female↗
PubMed2000

Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential.

Flavonoids are a class of polyphenolic compounds widely distributed in the plant kingdom, which display a variety of biological activities, including chemoprevention and tumor growth inhibition. Our aim was to investigate the effects of several polyphenols on the growth and metastatic potential of B16-BL6 melanoma cells in vivo. Intraperitoneal administration of quercetin, apigenin, (-)-epigallocathechin-3-gallate (EGCG), resveratrol, and the anti-estrogen tamoxifen, at the time of i.m. injection of B16-BL6 cells into syngeneic mice, resulted in a significant, dose-dependent delay of tumor growth, without toxicity. The relative descending order of potency was EGCG > apigenin = quercetin = tamoxifen > resveratrol > control. Furthermore, polyphenols significantly potentiated the inhibitory effect of a non-toxic dose of cisplatin. When tested for the ability to inhibit lung colonization, quercetin, apigenin, and tamoxifen (but not EGCG or resveratrol) significantly decreased the number of B16-BL6 colonies in the lungs in a dose-dependent manner, with quercetin and apigenin being more effective than tamoxifen. Interestingly, quercetin, apigenin, and tamoxifen (but not EGCG or resveratrol) significantly decreased the invasion of B16-BL6 cells in vitro, with quercetin and apigenin being more effective than tamoxifen. This suggests that anti-invasive activity is one of the mechanisms underlying inhibition of lung colonization by quercetin and apigenin. In conclusion, quercetin and apigenin inhibit melanoma growth and invasive and metastatic potential; therefore, they may constitute a valuable tool in the combination therapy of metastatic melanoma.

Animals↗
PubMed2000

Probing the active site of the hepatitis C virus serine protease by fluorescence resonance energy transfer.

A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.

Amino Acid Sequence↗
PubMed2000

Alpha-ketoacids are potent slow binding inhibitors of the hepatitis C virus NS3 protease.

The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the five cleavage events that occur in the nonstructural region of the HCV polyprotein. We here show that hexapeptide, tetrapeptide, and tripeptide alpha-ketoacids are potent, slow binding inhibitors of this enzyme. Their mechanism of inhibition involves the rapid formation of a noncovalent collision complex in a diffusion-limited, electrostatically driven association reaction followed by a slow isomerization step resulting in a very tight complex. pH dependence experiments point to the protonated catalytic His 57 as an important determinant for formation of the collision complex. K(i) values of the collision complexes vary between 3 nM and 18.5 microM and largely depend on contacts made by the peptide moiety of the inhibitors. Site-directed mutagenesis indicates that Lys 136 selectively participates in stabilization of the tight complex but not of the collision complex. A significant solvent isotope effect on the isomerization rate constant is suggestive of a chemical step being rate limiting for tight complex formation. The potency of these compounds is dominated by their slow dissociation rate constants, leading to complex half-lives of 11-48 h and overall K(i) values between 10 pM and 67 nM. The rate constants describing the formation and the dissociation of the tight complex are relatively independent of the peptide moiety and appear to predominantly reflect the intrinsic chemical reactivity of the ketoacid function.

Alanine↗