Squaric esters as peptide stapling reagents.

We report that squaric esters can serve as bifunctional reagents for selective peptide stapling reactions. Formation of the squaric amide staple occurs under mild conditions with amine-containing side chains. We show that short resin-bound peptides are readily stapled on solid phase and that stapling can occur at various relative positions along the peptide and with various amine tether lengths (e.g. Lysine, ornithine, etc). The squaric amide staples are stable to strong acid conditions used to cleave the stapled peptide from the resin and the stapled peptides show an increase in helicity as analyzed through circular dichroism.

Effect of sequencing of androgen deprivation and radiotherapy on prostate cancer growth.

PURPOSE: Androgen deprivation (AD) is frequently combined with radiotherapy (RT); however, the optimal sequence in vivo is currently unknown. Previous published work from our laboratory demonstrated that AD with RT was consistent with at least an additive, and possibly supra-additive, effect with the combined approach. We, therefore, performed additional experiments to elucidate the optimal sequence. METHODS AND MATERIALS: R3327-G Dunning rat prostate tumor cells were grown s.c. in the flanks of Copenhagen rats. Treatment was initiated when the tumor reached approximately 1 cm(3). Temporary AD was performed by a transscrotal orchiectomy followed 14 days later with androgen restoration using s.c. testosterone implants. RT was delivered using (60)Co to 7 Gy. Seven groups, including the controls, were analyzed: Group 1, sham control (Day 0: AD + testosterone); 2, AD control (Day 0: AD, Day 14: testosterone); 3, RT alone on Day 7 (Day 0: AD + testosterone, Day 7 RT); 4, RT alone on Day 3 (Day 0: AD + testosterone, Day 3: RT); 5, RT during AD (Day 0: AD, Day 7: RT, Day 14: testosterone); 6, RT before AD (Day 0: RT, Day 3: AD, Day 17: testosterone); and Group 7, RT after AD (Day 0: AD, Day 14: testosterone, Day 17: RT). The doubling times for tumor growth were calculated for the seven groups from the end of treatment plus 1 day. Differences in doubling time were assessed using analysis of variance, with pair-wise comparisons accomplished using post-hoc Bonferroni tests. RESULTS: An analysis of the differences in the tumor volume doubling time as measured from the end of treatment suggests that Groups 1 and 7 were statistically different from the other groups (p = 0.02). As expected, the sham control group had the shortest doubling time at 5.4 days and Group 7 (14 days of AD administered before RT) had the longest doubling time at 32.6 days. The findings were similar even after excluding an outlying doubling time of 85 days from Group 7 (p < 0.0001). To assess the effect of sequencing further, only Groups 5 through 7 (excluding the outlier) were compared in an analysis of variance with post-hoc Bonferroni tests. Group 7 (RT after AD) demonstrated a significantly longer doubling time than Groups 5 and 6 (p = 0.0024). CONCLUSION: The results suggest that neoadjuvant AD may result in prolonged suppression of tumor growth, even after testosterone replacement.

Anticoagulant activity of synthetic hirudin peptides.

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.

Thrombin-mediated release of factor VIII antigen from human umbilical vein endothelial cells in culture.

We have examined the effects of purified human alpha-thrombin on factor VIII antigen (FVIII-Ag) release by human umbilical vein endothelial cells in culture. Alpha-thrombin induced a time and dose-dependent release of FVIII-Ag into supernatant medium. Alpha-thrombin-mediated FVIII-Ag release was not dependent on protein synthesis and was observed in both serum-free and serum-containing media. FVIII-Ag release, however, was prevented when the serine esterase activity of thrombin was inhibited. Pretreatment of human endothelial cells with alpha-thrombin, but not diisofluorophosphate-thrombin, prevented subsequent FVIII-Ag release by alpha-thrombin. Thrombin-mediated FVII-Ag release was not associated with significant 51Cr release from prelabeled endothelial monolayers. We conclude that alpha-thrombin induces release of preformed FVIII-Ag from human umbilical vein endothelial cells by a receptor-independent, nonlytic mechanism requiring serine esterase activity.

Evaluating service needs in drug-abusing clients.

Patients applying for treatment to the State of Illinois' Chicago Central Intake Facility were interviewed to assess their service needs and treatment expectations; a subgroup was reinterviewed after 4 months. Patients were classified as long-term opioid-dependent, short-term opioid-dependent, or as polydrug nonopioid dependent abusers. We found high levels of housing, employment, and legal needs among all subgroups at both first and second interviews. There were significant differences between drug-use groups in reasons for seeking treatment; opioid-dependent groups gave drug-related reasons more frequently while polydrug abusers cited legal reasons. Length of time patients remained in treatment was not related to service needs.