Sara Rubinraut

Inhibition of human neuroblastoma growth by a specific VIP antagonist

The 28-amino-acid neuropeptide, vasoactive intestinal peptide (VIP), is a potent mitogen during embryonic development and plays a vital role in brain growth. VIP is also mitogenic for tumor cells, including the human neuroblastoma (NMB). Northern blot analysis has revealed VIP mRNA transcripts in NMB. We now report VIP-like immunoreactivity within these neuroblastoma cells that increased during logarithmic growth and decreased after attaining confluency. About 106 seeded cells secreted 5–40 pg of VIP-like immunoreactivity into the medium. These results suggest an autocrine role for VIP in the regulation of neuroblastoma growth. A VIP hybrid antagonist (neurotensin6–11 VIP7–28) that has been shown to inhibit lung cancer proliferation was now tested for inhibition of neuroblastoma growth. Receptor binding studies indicated that the hybrid antagonist displaced [125I]-VIP binding in the neuroblastoma cells (EC50=5×10−6M). Furthermore, as measured by thymidine incorporation and by cell counts, the potent VIP hybrid antagonist inhibited neuroblastoma multiplication in a dose-dependent manner. In conclusion, VIP may be an important regulator of growth of nerve cell progenitors and of tumors derived from neuronal origin and intervening with VIP function may lead to improved treatment of cancer.

Engineering prolonged-acting prodrugs employing an albumin-binding probe that undergoes slow hydrolysis at physiological conditions

Here we describe the design and application of OSu-FMS-MAL-S-(CH(2))(15)-COOH, an agent that associates with albumin while linked to a peptide or a protein with sufficient affinity (Ka=2 to 2.6 x 10(5)M(-1)) to protract the action of short- lived peptides and proteins in vivo. Under physiological conditions this probe undergoes spontaneous hydrolysis with the concomitant reactivation of inactive conjugates. Intravenously administered (125)I-labeled-Insulin-FMS-MAL-S-(CH(2))(15)-COOH to rats shows half-life of 17+/-2h, exceeding 5.2 times that obtained with intravenously administered (125)I-labeled Insulin. In mice this derivative facilitates glucose-lowering effect over a period of 24h, yielding AUC five times greater than that obtained by a similar dose of insulin-detemir. Similarly, subcutaneous administration of Exendin-4-FMS-MAL-S-(CH(2))(15)-COOH into mice facilitated prolonged and stable reduction in glucose level, yielding a t(1/2) value of 28+/-2h, exceeding the effect of exendin-4 4.7 folds. The inactive derivative gentamicin-FMS-MAL-S-(CH(2))(15)-COOH regained its full antibacterial potency upon incubation at physiological conditions yielding a t(1/2) value of 7.1+/-0.2h. In conclusion, the albumin-binding probe we introduced enables to prolong the action of any amino containing molecule in vivo, without the drawback of inactivation that often occurs upon such derivatization.

VIP-derived sequences modified by N-terminal stearyl moiety induce cell death: the human keratinocyte as a model

Vasoactive intestinal peptide (VIP) is a recognized growth factor affecting many cell types. We have previously developed a series of lipophilic VIP analogues containing an N‐terminal covalently attached stearyl moiety. The current studies identified stearyl‐Nle17‐VIP and stearyl‐Nle17‐neurotensin6–11VIP7–28, acting at μM concentrations, as cytotoxic to human keratinocytes. The core C‐terminal active VIP‐derived peptide, stearyl‐Lys‐Lys‐Tyr‐Leu‐NH2 (St‐KKYL‐NH2), was identified as being responsible for the observed cytotoxicity. Cytotoxicity coincided with marked reduction in intracellular cyclic GMP and was abolished by co‐treatment with the endonuclease inhibitor, aurine‐tricarboxylic acid, suggesting apoptotic mechanisms. Stearyl‐VIP derivatives thus offer lead compounds for future drug development against hyperproliferative skin conditions.

Protection Against Developmental Deficiencies by a Lipophilic VIP Analogue

Stearyl-Nle-VIP (SNV) is a novel agonist of vasoactive intestinal peptide (VIP) exhibiting a 100-fold greater potency than the parent molecule and specificity for a receptor associated with neuronal survival. Here, the developmental and protective effects of SNV were investigated in vivo using two models of developmental retardation, hypoxia and cholinergic blockade. In both cases chronic administration of SNV during development provided protective effects. Water maze experiments on the weaned animals have demonstrated a prophylactic action for SNV and enhancement of spatial memory in animals exposed to a cholinotoxin. SNV may act by providing neuroprotection, thereby improving cognitive functions. This work is dedicated to Prof. R.J. Wurtman whose inspiration and leadership in the field of neuroscience and cognition is beyond comparison.

Multiple Actions of a Hybrid PACAP Antagonist: Neuronal Cell Killing and Inhibition of Sperm Motilitya

Abstract: Pituitary stimulating adenylate cyclase (PACAP) is a major regulatory peptide with two active molecular forms: PACAP‐27 and PACAP‐38. Both molecular forms promote neuronal survival and protect against neurotoxicity. Based on our previous hybrid peptide strategy in designing vasoactive intestinal peptide (VIP) antagonists, novel PACAP analogues were synthesized (neurotensin6‐11 PACAP7‐27 and neurotensin6‐11 PACAP7‐38). In addition to the hybrid modification, the methionine in position 17 was replaced by norleucine (Nle). Treatment of rat cerebral cortical cultures for five days with the putative PACAP antagonists (1 nM) resulted in a 35‐45% reduction in neuronal cell counts as compared to controls. Neuronal cell death was already obtained at picomolar concentrations for the neurotensin6‐11PACAP7‐27 antagonist with 70% death at 10−8 M. Co‐administration of the PACAP hybrid analogue with picomolar amounts of PACAP‐27 or Nle17‐PACAP‐27 attenuated the reduction in neuronal cell counts. While the protective effects of both analogues exhibited a peak at 1 pM concentrations, the Nle‐containing agonist displayed a broader range of active concentrations (10−12 M‐10−9 M)

Polytuftsin: A potential precursor for slow release of the phagocytosis stimulating peptide tuftsin

1. Polytuftsin (Thr-Lys-Pro-Arg)n, was synthesized through polycondensation of an amino-free and carboxyl-activated derivative of tuftsin, H2N-Thr-Lys(Z)-Pro-Arg(Tos)-OSu, following suitable deprotection and fractionation steps. 2. Digestion of polytuftsin by trypsin, as well as by normal human serum, at 37 degrees C, yielded free tuftsin. 3. Polytuftsin affected the decreased formation of lung-metastasis, in B16 melanoma treated mice and prolonged the survival of animals more efficiently than tuftsin. 4. Tuftsin was found to be totally degraded by serum enzymes within approximately 60 min at 37 degrees C.

Novel extended and branched N-terminal analogs of VIP

The effects of vasoactive intestinal peptide (VIP) are primarily mediated through VPAC1 and VPAC2, receptors that are preferentially coupled to adenylate cyclase activation. As a large majority of the potent VIP antagonists have modifications in the N-terminal domain of the peptide, the effect of multiplication of this domain on VIP was examined with the aim of possibly amplifying peptide-receptor (VPAC1) activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the N-terminal domain that was obtained by either linear tandem extension or by parallel branching. Circular dichorism (CD) analysis revealed that these extended/branched peptides maintained an alpha helical structure in organic environment, similar to VIP. A specific branched VIP analog was found to be slightly more potent towards VPAC1-related cAMP production as compared to VIP. This analog could have potential therapeutic value in several disorders, similar to VIP. Two branched N-terminal VIP sequences demonstrated superior receptor binding and activation as compared to two N-terminals in tandem. The results suggest that correct alignment of the VIP N-terminal region is important for receptor binding and activation. However, increased receptor binding was not directly associated with increased cAMP production suggesting steric dynamic interactions.

Newly designed modifier prolongs the action of short-lived peptides and proteins by allowing their binding to serum albumin.

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.

Novel analogs of VIP with multiple C-terminal domains.

The effect of multiplication of the N-terminal domain of vasoactive intestinal peptide (VIP) on the binding activity of the peptide was recently evaluated. A VIP analog with multiple N-terminal domains was found to be slightly more potent as compared to [Nle(17)]VIP towards VIP receptor type 1 (VPAC1)-related cAMP production. Here, the effect of multiplication of the C-terminal domain of VIP was evaluated with the aim of possibly amplifying peptide-receptor (VPAC1) binding and activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the C-terminal domain that was obtained by either a simple linear tandem extension or by a unique branching methodology. Results show that despite significant alterations in the C-terminal domain of VIP that is considered essential to induce potent receptor binding, few peptides demonstrated only slight reduction in receptor binding and activation in comparison to [Nle(17)]VIP. Furthermore, a specific branched VIP analog with multiple C-terminal domains was equipotent to [Nle(17)]VIP in the cAMP production assay. Therefore, it is concluded that the association between the VIP ligand to the VIP receptor could be tolerable to size increases in the C-terminal region of the VIP ligand and multiplication of the C-terminal does not increase activity.

Ozonolysis by microwave discharge of oxygen of naphthalene adsorbed on florisil

Microwave discharge of O2 produces O(3P) atoms which form O3 on florisil surface. Reaction with adsorbed naphthalene results in o-formylcinnamaldehydes as the main product.