Synthesis, antiviral activity, and conformational studies of a P3 aza-peptide analog of a potent macrocyclic tripeptide HCV protease inhibitor.

BILN 2061 is a macrocyclic tripeptide inhibitor of hepatitis C virus NS3-4A protease that has shown efficacy in the clinic for treating patients infected with HCV. We have synthesized a P3 aza-peptide analog of a potent macrocyclic tripeptide inhibitor closely related to BILN 2061. This aza-derivative was found to be >2 orders of magnitude less active than the parent macrocycle in both isolated enzyme (HCV NS3-4A) and HCV subgenomic replicon assays. NMR studies of P3 aza-peptides revealed these compounds adopt a beta-turn conformation stabilized by an intramolecular H-bonding interaction. Molecular models of these structures indicate a D-like configuration of the P3 aza-residue. Thus, the configurationally undefined nature at P3 in the aza-peptide allows the compound to adopt an H-bond stabilized conformation that is substantially different from that necessary for tight binding to the active site of HCV NS3 protease.

Decreased E-cadherin but not beta-catenin expression is associated with vascular invasion and decreased survival in head and neck squamous carcinomas.

OBJECTIVE: We wished to correlate the expression of E-cadherin and beta-catenin in squamous carcinomas of the head and neck to outcome and other clinicopathologic variables. STUDY DESIGN AND SETTING: This retrospective study was carried out in a tertiary care setting. The tumors of 45 patients who had their head and neck squamous carcinoma primarily treated by resection were evaluated immunohistochemically with antibodies to E-cadherin and beta-catenin. Thirty-two tumors arose in the oral cavity, 9 tumors originated in the larynx, and 4 tumors began in the hypopharynx. Patient outcome and the clinicopathologic variables of tumor site, tumor stage, cervical lymph node status, tumor differentiation, perineural invasion, and vascular invasion were correlated to immunohistochemical expression of E-cadherin and beta-catenin. RESULTS: Low expression of E-cadherin in the tumors was significantly associated with decreased overall survival (P = 0.004), disease-free survival (P = 0.007), and vascular invasion (P = 0.02) but not with other clinicopathologic variables. beta-catenin expression was not significantly associated with any of the studied clinicopathologic variables. CONCLUSION: Decreased E-cadherin but not beta-catenin expression is associated with decreased survival in patients with head and neck squamous carcinomas. SIGNIFICANCE: Detection of loss of E-cadherin expression may help predict which patients with head and neck squamous cell carcinoma will experience a worse outcome compared to patients whose tumors have not lost this tumor suppressor. EBM RATING: C-4.

Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease.

Maturation of the hepatitis C virus (HCV) polyprotein occurs by a series of proteolytic processes catalyzed by host cell proteases and the virally encoded proteases NS2 and NS3. Although several peptidomimetic inhibitors of NS3 protease have been published, only a few small molecule inhibitors have been reported. In an effort to improve screening efficiency by minimizing the spectral interference of various test compounds, a substrate that contains the longer wavelength fluorescence resonance energy transfer (FRET) pair, TAMRA/QSY-7, was devised. For the optimized substrate T-Abu-Q, with sequence Ac-Asp-Glu-Lys(TAMRA)-Glu-Glu-Abu-Psi(COO)Ala-Ser-Lys(QSY-7)-amide, the kinetic parameters with HCV NS3 protease are K(m)=30 microM, k(cat)=0.6s(-1), and k(cat)/K(m)=20,100s(-1)M(-1). We show that this substrate is suitable for inhibitor analysis and mechanistic studies so long as the substrate concentration is low enough (0.5 microM) to avoid complications from high inner filter effects. The substrate is especially useful with ultra-high-density screening formats, such as microarrayed compound screening technology, because there is less spectral interference from the compounds being tested than with more traditional (EDANS/DABCYL) FRET protease substrates. The merits of the new substrate, as well as potential applications of this FRET pair to other protease substrates, are discussed.

Ultrarapid staining for cutaneous melanoma: study and protocol.

BACKGROUND: Immunohistochemistry has revolutionized the use of frozen sections for the removal of melanoma. OBJECTIVE: Two studies were performed to address the best and fastest way to stain melanocytes in frozen tissue sections. MATERIALS AND METHODS: Fifty-six standing cones were taken from sun-damaged white skin and stained with combinations of anti-S-100, anti-HMB-45, anti-Melan-A, and azure B with appropriate controls. RESULTS: Anti-Melan-A was the best immunohistochemical stain. Azure B stains melanin granules metachromatically green and allows differentiation of melanocytes and melanophages. Ultrarapid staining worked as well as rapid staining. CONCLUSION: Dako's EnVision ultrarapid staining protocol and azure B counterstain represent a dramatic improvement and should be considered the easily obtainable histologic standard when excising cutaneous melanoma.

Perineural and vascular invasion in oral cavity squamous carcinoma: increased incidence on re-review of slides and by using immunohistochemical enhancement.

CONTEXT: Perineural invasion and vascular invasion may be adverse prognostic factors in patients with oral cavity squamous cell carcinoma. However, the incidence of perineural and vascular invasion varies in the literature, and the use of immunohistochemistry to enhance their detection has not been evaluated in oral cavity squamous cell carcinomas. OBJECTIVE: To determine if the previously assessed incidence of perineural and vascular invasion in cases of oral cavity squamous cell carcinoma would be increased by re-review of the original routinely hematoxylin-eosin-stained sections as well as review of slides stained immunohistochemically with S100 and CD31 to enhance visualization of nerves and vessels. DESIGN: Forty cases of oral cavity squamous cell carcinoma in which the status of perineural and vascular invasion had been part of the original pathology report were reviewed. All original routinely stained slides were reviewed as well as S100- and CD31-stained sections of each case's tissue blocks that contained tumor. RESULTS: Perineural invasion was identified in 30% (12/ 40) of tumors in the original reports, 62% (25/40) of the authors' re-review of the same slides, and 82% (33/40) when cases were stained with S100. Vascular invasion was identified in 30% (12/40) of tumors in the original reports, 35% (14/40) of the authors' re-review of the same slides, and 42% (17/40) when cases were stained with CD31. False-positive and false-negative results were common in the original reports. The number of foci of both types of invasion was related to its discovery in the original reports. Vascular invasion, but not perineural invasion, was significantly associated with death at 5-year follow-up. CONCLUSIONS: Although careful re-review of routinely stained slides will detect a significant number of cases of perineural and vascular invasion, immunohistochemical enhancement further improves the accuracy of the determination.

Management of membranoproliferative glomerulonephritis type II with plasmapheresis.

Membranoproliferative glomerulonephritis type II (MPGN II) is a rare kidney disease identified microscopically by electron-dense deposits surrounded by complement component C3 in glomerular basement membranes. MPGN II usually leads to renal failure, and patients with MPGN II experience a high rate of recurrence following renal transplantation. No treatment modalities have been proven successful if recurrence does occur. The sera of most patients with MPGN II contain complement C3 nephritic factor (C3NF), an IgG autoantibody directed against C3 convertase (C3bBb) that results in constitutive breakdown of C3. C3NF may be important in the pathogenesis of the disease. Since C3NF is IgG, we predicted that C3NF could be removed from the serum through plasmapheresis. We describe the use of long-term plasmapheresis to maintain good renal function in a 15-year-old girl with rapidly progressive recurrent MPGN II. After 73 plasmapheresis procedures over 63 weeks, her serum creatinine remained stable, and her creatinine clearance trended upward. Serial biopsies of the transplanted kidney demonstrated persistent MPGN II but no development of tubular atrophy. During the course of therapy, serum C3NF activity decreased; furthermore, C3NF activity was detected in the removed plasma. We have shown that plasmapheresis is a safe and effective method for delaying the onset of chronic renal failure in recurrent MPGN II. The efficacy may be due to the removal of serum C3NF.