Nancy B. Iannucci

Antiproliferative effect of 1-deamino-8-D-arginine vasopressin analogs on human breast cancer cells

BACKGROUND Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe antidiuretic and hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). It is known that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. RESULTS/DISCUSSION Looking for novel analogs with improved anti-tumor activity, positions 4 and 5, at the conformational peptide loop, were substituted. The analog [V(4)Q(5)]dDAVP ([4-valine 5-glutamine] desmopressin) exhibited a significantly higher antiproliferative effect than dDAVP in cultures of MCF-7, a V2R-expressing human breast carcinoma cell line. The chiral isomer of this analog and tetrapeptide fragments corresponding to the loop region were also assessed. CONCLUSION Preclinical evaluation of the anti-tumor activity of [V(4)Q(5)]dDAVP in animal models is warranted.

Improved antimicrobial activity of h-lysozyme (107–115) by rational Ala substitution

The most challenging target in the design of new antimicrobial agents is the development of antibiotic resistance. Antimicrobial peptides are good candidates as lead compounds for the development of novel anti‐infective drugs. Here we propose the sequential substitution of each Ala residue present in a lead peptide with known antimicrobial activity by specific amino acids, rationally chosen, that could enhance the activity of the resultant peptide. Taking the fragment 107–115 of the human lysozyme as lead, two‐round screening by sequentially replacing both Ala residues (108 and 111) by distinct amino acids resulted in a novel peptide with 4‐ and 20‐fold increased antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, respectively. These results reinforce the strategy proposed, which, in combination with simple and easy screening tools, will contribute to the rapid development of new therapeutic peptides required by the market. Copyright

Biological activity of antibacterial peptides matches synergism between electrostatic and non electrostatic forces

Substitution of Ala 108 and Ala 111 in the 107-115 human lysozyme (hLz) fragment results in a 20-fold increased anti-staphylococcal activity while its hemolytic activity becomes significant (30%) at very high concentrations. This analog displays an additional positive charge near the N-terminus (108) and an extra Trp residue at the center of the molecule (111), indicating that this particular amino acid sequence improves its interaction with the bacterial plasma membrane. In order to understand the role of this arrangement in the membrane interaction, studies with model lipid membranes were carried out. The interactions of peptides, 107-115 hLz and the novel analog ([K(108)W(111)]107-115 hLz) with liposomes and lipid monolayers were evaluated by monitoring the changes in the fluorescence of the Trp residues and the variation of the monolayers surface pressure, respectively. Results obtained with both techniques revealed a significant affinity increase of [K(108)W(111)]107-115 hLz for lipids, especially when the membranes containing negatively charged lipids, such as phosphatidylglycerol. However, there is also a significant interaction with zwitterionic lipids, suggesting that other forces in addition to electrostatic interactions are involved in the binding. The analysis of adsorption isotherms and the insertion kinetics suggest that relaxation processes of the membrane structure are involved in the insertion process of novel peptide [K(108)W(111)]107-115 hLz but not in 107-115 hLz, probably by imposing a reorganization of water at the interphases. In this regard, the enhanced activity of peptide [K(108)W(111)]107-115 hLz may be explained by a synergistic effect between the increased electrostatic forces as well as the increased hydrophobic interactions.

Structure‑activity relationship of 1‑desamino‑8‑D‑arginine vasopressin as an antiproliferative agent on human vasopressin V2 receptor‑expressing cancer cells

The synthetic nonapeptide 1‑desamino‑8‑D‑arginine vasopressin (dDAVP) can reduce tumor cell growth through agonist action on the vasopressin V2 receptor. A structure‑antiproliferative activity relationship analysis of dDAVP was performed using the alanine scanning technique on the aggressive MDA‑MB‑231 human breast carcinoma cell line. The results from this analysis demonstrated that the amino acids located at the loop of dDAVP are important for the antiproliferative activity of dDAVP, highlighting the key role of the N‑terminal region of the peptide in the interaction with the tumor cell surface receptor. The findings from this study present novel strategies for designing improved compounds with enhanced stability for cancer therapy.

The novel desmopressin analogue [V4Q5]dDAVP inhibits angiogenesis, tumour growth and metastases in vasopressin type 2 receptor-expressing breast cancer models.

Desmopressin (dDAVP) is a safe haemostatic agent with previously reported antitumour activity. It acts as a selective agonist for the V2 vasopressin membrane receptor (V2r) present on tumour cells and microvasculature. The purpose of this study was to evaluate the novel peptide derivative [V4Q5]dDAVP in V2r-expressing preclinical mouse models of breast cancer. We assessed antitumour effects of [V4Q5]dDAVP using human MCF-7 and MDA-MB-231 breast carcinoma cells, as well as the highly metastatic mouse F3II cell line. Effect on in vitro cancer cell growth was evaluated by cell proliferation and clonogenic assays. Cell cycle distribution was analysed by flow cytometry. In order to study the effect of intravenously administered [V4Q5]dDAVP on tumour growth and angiogenesis, breast cancer xenografts were generated in athymic mice. F3II cells were injected into syngeneic mice to evaluate the effect of [V4Q5]dDAVP on spontaneous and experimental metastatic spread. In vitro cytostatic effects of [V4Q5]dDAVP against breast cancer cells were greater than those of dDAVP, and associated with V2r-activated signal transduction and partial cell cycle arrest. In MDA-MB-231 xenografts, [V4Q5]dDAVP (0.3 μg/kg, thrice a week) reduced tumour growth and angiogenesis. Treatment of F3II mammary tumour-bearing immunocompetent mice resulted in complete inhibition of metastatic progression. [V4Q5]dDAVP also displayed greater antimetastatic efficacy than dDAVP on experimental lung colonisation by F3II cells. The novel analogue was well tolerated in preliminary acute toxicology studies, at doses ≥300-fold above that required for anti-angiogenic/antimetastatic effects. Our data establish the preclinical activity of [V4Q5]dDAVP in aggressive breast cancer, providing the rationale for further clinical trials.

Purification of Neutral Protease by Dye Affinity Chromatography

Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme™, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25°C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8°C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25°C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.

Structural glance into a novel anti-staphylococcal peptide.

Novel antimicrobial peptides are valuable molecules for developing anti‐infective drugs to counteract the contemporary spread of microbial drug‐resistance. Here we focus on a novel peptide (RKWVWWRNR‐NH2) derived from the fragment 107–115 of the human lysozyme that displays a 20‐fold increase in anti‐staphylococcal activity. The conformational analysis of this peptide and its interaction with model lipidic phases—as assayed by circular dichroism and fluorescence spectroscopy—show a noteworthy spectral change, which might be related to its anti‐staphylococcal activity. The secondary structure of peptide [K108W111] 107–115 hLz was dramatically affected through a single substitution at position 111 (Ala by Trp). Therefore, this conformational change might improve the interaction of the novel peptide with the bacterial plasma membrane. These results highlight the role of peptide secondary structure and the distribution of polar/nonpolar residues for the effective interaction of this peptide with the bacterial plasma membrane, a key step for triggering its lethal effect. This knowledge may contribute to the rational design of a new generation of antimicrobial peptides with increased efficacy developed from natural sources by simple screening tools.

Isolation of Trypsin from Bovine Pancreas Using Immobilized Benzamidine and Peptide CTPR Ligands in Expanded Beds

Abstract Peptide CTPR and p‐amino benzamidine (PAB) immobilized on Streamline™ were utilized as the chromatographic matrices for trypsin purification from bovine pancreas. By using a clarified pancreas extract, maximum capacity for CTPR‐Streamline was 47.4 mg/mL and for PAB‐Streamline 78.9 mg/mL while Kd values were 0.39 and 0.38 respectively. Dynamic capacity was 23.0 and 46.0 mg/mL for CTPR‐ and PAB‐Streamline respectively. When the purification process was applied to unclarified pancreas extract in the expanded‐bed adsorption mode, 80% trypsin recovery with a purification factor of 18.7 was achieved. Cationic and anionic trypsin obtained from the affinity column were separated by ion‐exchange chromatography.

Abstract P2-09-15: Effect of [4-valine 5-glutamine] desmopressin, a novel vasopressin analog, on human breast cancer cell growth in two-dimensional (2D) and three-dimensional (3D) cultures

Background: Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). This receptor is expressed in endothelium and also in some tumor cells. V2R stimulation is associated with increased levels of cyclic AMP and cyclic AMP-dependent protein kinase activation. We previously reported that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. This nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity. Such positions belong to the conformational peptide loop which has a key role in ligand-receptor interaction. After an extensive in vitro and in vivo characterization, novel peptide compound [4-valine 5-glutamine] dDAVP ([V4Q5]dDAVP) exerted a remarkable and improved antitumor effect in comparison to parental peptide dDAVP. [V4Q5]dDAVP inhibited tumor growth in both syngeneic F3II and xenogeneic MDA-MB-231 breast cancer models, modulating tumor aggressiveness and vasculature. In this study, to further understand direct cytostatic action of this novel vasopressin analog on tumor cells, we assessed the effect of [V4Q5]dDAVP on clonogenic and three dimensional growth of V2R-expressing human breast carcinoma cell lines. Methods and results: We used MCF7 and triple negative MDA-MB-231 human breast cancer cell lines to conduct the experiments. Vasopressin V2 membrane receptor expression in tumor cell lines was assessed by RT-PCR and immunofluorescence. We first examined peptide ability to modify in vitro low density cell growth by the clonogenic growth assay. Incubation with [V4Q5]dDAVP (100-1500 nM) during one week significantly reduced the number of tumor cell colonies showing a concentration dependant manner and an IC50 value of 1135 nM. To assess the effect of novel analog on three dimensional growth of cancer cells we used two different assays. The first one evaluates the ability of basement membrane extract (BME)-embedded single cells to proliferate and form spheroids. The second assay evaluates growth of single fully established spheroids (Approx. 300 mm diameter) transplanted individually on a non-adherent surface. [V4Q5]dDAVP, at a concentration of 1000 nM, was able to significantly reduce the number of BME-embedded spheroids formed from single cells after 14 days of treatment. In a similar manner, incubation of established and isolated spheroids with [V4Q5]dDAVP using the same concentration for 9 days, caused a decrease in multicellular spheroids final diameter. Cellular metabolic activity analysis by the MTS assay showed no significant differences between treated and control groups. Conclusions: We report for the first time the inhibitory effect of [V4Q5]dDAVP on colony and spheroid formation and growth of human breast carcinoma cells. Our results using three dimensional models suggest that V2R stimulation in cancer cells may affect early stages of microtumor establishment. Further analysis of involved signaling pathways in treated 3D cultures is needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-15.

Abstract 2321: The novel vasopressin analog [4-valine 5-glutamine] desmopressin inhibits tumor growth and angiogenesis in V2 receptor-expressing breast cancer xenografts

Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe antidiuretic and hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). This receptor is expressed in kidney collecting tubes, endothelium and also in some tumor cells. We previously reported that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. This cyclic nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity (Iannucci et al., Fut. Med. Chem. 2011). Such positions belong to the conformational peptide loop which has a key role in ligand-receptor interaction. In this study, we evaluated the effects of the novel analog [4-valine 5-glutamine] dDAVP ([V4Q5]dDAVP) on cell proliferation and migration in vitro, and tumor growth and angiogenesis in vivo using the MDA-MB-231 triple-negative V2R-expressing human breast cancer cell line. We first examined peptide ability to modify in vitro cell growth of MDA-MB-231 cells by the MTT assay. Incubation with [V4Q5]dDAVP analog (100-1500 nM) during 72 h significantly reduced cell proliferation showing a better performance at high concentrations than the parental compound dDAVP. When studying the effect on cell cycle, flow cytometric analysis showed that a 24-h treatment with [V4Q5]dDAVP resulted in an arrest of MDA-MB-231 cells in G0/G1 phase. Next, we used wound migration and transwell chemotaxis assays to study the ability of [V4Q5]dDAVP to inhibit tumor cell motility. After a 16-h treatment, MDA-MB-231 migration and chemotaxis was impaired by 30-60%. To asses the in vivo effect of the compound on tumor growth, nu/nu mice where implanted s.c. with MDA-MB-231 cells. Three weekly i.v. doses of [V4Q5]dDAVP (0.3 µg/kg/dose) reduced by 40% the subcutaneous tumor volume compared with vehicle-treated controls after a 4- week treatment. Finally, to evaluate the in vivo angiostatic effect, a modified Matrigel plug assay was used. Intravenous administration of [V4Q5]dDAVP (0.3 µg/kg/dose thrice a week) significantly reduced by 18% MDA-MB-231 cell-induced angiogenesis as measured by hemoglobin content in plugs recovered from mice 14 days after tumor cell inoculation. These in vivo effects are probably due to a dual action of [V4Q5]dDAVP involving angiogenesis inhibition mechanisms and direct cytostatic action. In conclusion, this study indicates that the novel peptide compound [V4Q5]dDAVP exerts a remarkable and improved antitumor and antiangiogenic effect in comparison to parental peptide dDAVP. Increased hidrophobicity at the conformational peptide loop by replacing Gln for Val may play a key role on ligand-receptor interaction, thus enhancing agonist effect on V2R present in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2321. doi:1538-7445.AM2012-2321