Characterization of a monoclonal antibody produced in an attempt to mimic the active site of HIV aspartyl protease using haptens based on inhibitor models.

The high binding affinity and specificity of antibodies for a great variety of ligands has been widely exploited in structure-activity relationship studies of biomolecules and more recently in the development of new catalysts for several chemical reactions. It is assumed that antibodies generated against haptenic protease inhibitors would recognize both these haptens and the substrate of the model proteolytic reaction. We have produced antibodies against HIV PRp12 aspartyl protease substrate analogues, chemically modified at the scissile bond, Phe-Pro. Identical chemical modifications have been reported for related HIV protease inhibitors. We finally selected an anti-hapten monoclonal antibody that specifically recognized the substrate and those haptens with both the phenylalanyl side chain and the prolyl pyrrolidine ring. This selectivity of recognition suggests that such an antibody might mimic the catalytic site of the model protease.

Antineoplastic activity of didemnin congeners: nordidemnin and modified chain analogues.

Nordidemnin (2), a natural analogue of the marine cyclodepsipeptide didemnin B (1b), showed cytotoxic activity against L1210 leukemia and antineoplastic activity against P388 leukemia as well as B16 melanoma; nordidemnin (2) was as active as didemnin B (1b). The influence of synthetic modifications in the linear peptidic chain on in vitro and in vivo activity was also studied. Replacement of the terminal lactyl residue by mandelyl and 3-(p-hydroxyphenyl)propionyl residues in compounds 3 and 4, respectively, did not affect the cytotoxic activity against L1210 leukemia (ID50 of 1.1 nM and 1.2 nM, respectively) or the in vivo activity against P388 leukemia. Unlike these aromatic substituants, the lipophilic palmityl residue induced a dramatic loss in cytotoxic activity. The inverted chirality of the MeLeu joining residue in compound 6 caused a marked reduction in the in vitro activity.

Synthesis and biological activity of chimeric structures derived from the cytotoxic natural compounds dolastatin 10 and dolastatin 15.

The natural cytotoxic compounds dolastatins 10 and 15 exhibit great similarities in structure and in their biological activity profiles. Two compounds (1 and 2) formed by interchanging the dolaisoleuine residue of dolastatin 10 and the MeVal-Pro dipeptide of dolastatin 15 were synthesized in order to evaluate the possible equivalence of these units. These compounds can be considered as chimeras of dolastatins 10 and 15 formed by the N-terminal part of the former and the C-terminal part of the latter and vice versa. Both analogues exhibited a marked decrease in their cytotoxic activity but showed similar differential cytotoxicity with regard to the cell lines assayed compared with the parent compounds. HT-29 cell line was the least sensitive one. However, this activity was in the nanomolar level and close to that of vincristine. The differences in their effect on tubulin polymerization were less pronounced. We confirmed the already known crucial role of the Dil residue in this assay. The nonequivalence of the Dil unit and the MeVal-Pro dipeptide probably reflects modification in the relative positions of the N-dimethylamino and the phenyl moieties.

Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell-cell adhesion.

The spleen tyrosine kinase Syk has predominantly been studied in hematopoietic cells in which it is involved in immunoreceptor-mediated signaling. Recently, Syk expression was evidenced in numerous nonhematopoietic cells and shown to be involved in tumor formation and progression. The Syk downstream signaling effectors in nonhematopoietic cells remain, however, to be uncovered, and were investigated using MS-based quantitative phosphoproteomics. Two strategies, based on the inhibition of the Syk catalytic activity and on the loss of Syk expression were employed to identify phosphotyrosine-dependent complexes. Quantitative measurements were obtained on 350 proteins purified with phosphotyrosine affinity columns using the SILAC method. Forty-one proteins are dependent on both Syk expression and catalytic activity and were selected as signaling effectors. They are involved in a variety of biological processes such as signal transduction, cell-cell adhesion and cell polarization. We investigated the functional involvement of Syk in cell-cell adhesion and demonstrated the phosphorylation of E-cadherin and alpha-catenin. In addition, Syk is localized at cell-cell contacts, and Syk-mediated phosphorylation of E-cadherin seems to be important for the proper localization of p120-catenin at adherens junctions. Identification of the biochemical pathways regulated by Syk in human cancer cells will help to uncover its role in tumor formation and progression.

'Suicide' of crickets harbouring hairworms: a proteomics investigation.

Despite increasing evidence of host phenotypic manipulation by parasites, the underlying mechanisms causing infected hosts to act in ways that benefit the parasite remain enigmatic in most cases. Here, we used proteomics tools to identify the biochemical alterations that occur in the head of the cricket Nemobius sylvestris when it is driven to water by the hairworm Paragordius tricuspidatus. We characterized host and parasite proteomes during the expression of the water-seeking behaviour. We found that the parasite produces molecules from the Wnt family that may act directly on the development of the central nervous system (CNS). In the head of manipulated cricket, we found differential expression of proteins specifically linked to neurogenesis, circadian rhythm and neurotransmitter activities. We also detected proteins for which the function(s) are still unknown. This proteomics study on the biochemical pathways altered by hairworms has also allowed us to tackle questions of physiological and molecular convergence in the mechanism(s) causing the alteration of orthoptera behaviour. The two hairworm species produce effective molecules acting directly on the CNS of their orthoptera hosts.

Comparison of different immunoenzymatic methods for the determination of the fine specificity and affinity constants of polyclonal antibodies against pseudopeptide haptens.

We evaluated two phosphinopeptides and one phosphonopeptide, which are transition state analogs of a proteolytic reaction, for their ability to generate murine polyclonal antibodies. The specificity of these antisera was determined by indirect and competitive ELISA. Cross-reactivity analysis by these ELISA showed that the antisera recognized selectively haptens containing a phosphate group. The pseudopeptides recognized by the antisera in the indirect ELISA were not the same, however, as those recognized in the competitive ELISA. The differences between these results are probably due to the presentation forms of the hapten, i.e., passively adsorbed in the former ELISA format and soluble in the latter. The affinity of the antibodies was then determined by using two methods based on competitive ELISA, one described by Friguet et al. and the other by Seligman. The dissociation constant (Kd) values calculated by the two methods, for an antiserum and its homologous hapten, are similar. However, only the middle portion of the inhibition scale in Seligman's method gave access to reliable values. Nevertheless, the Seligman representation allowed us to underscore the large range of affinity constants of the polyclonal antibodies.

5-Hydroxytryptamine1A receptor synthetic peptides. Mechanisms of adenylyl cyclase inhibition.

The 5-hydroxytryptamine1A receptor (5-HT1AR) is a G-protein-coupled receptor negatively coupled to adenylyl cyclase (AC). We have studied the functional domains of 5-HT1AR using synthetic peptides to block or mimic receptor function. The entire second intracellular loop (5-HT1AR-i2) and the carboxyl end of the third intracellular loop (5-HT1AR-i3-C) strongly inhibited forskolin-stimulated AC activity. These effects were not additive with those of 5-HT. Like 5-HT, the peptides 5-HT1AR-i3-C and -i2 weakly inhibited AIF4- and Mn2+ stimulated AC activity. 5-HT1AR binding assays indicated that peptides could interact with the same G-protein pool as the 5-HT1AR. 5-HT1AR-i3-C- and -i2-stimulated [35S]guanosine 5'-O-(thiotriphosphate) binding on Go/Gi proteins. Only 5-HT1AR-i3-C partially adopted an alpha-helical conformation in solution. These data show that different domains in the 5-HT1AR second and third intracellular loops can couple to and activate Gi proteins in order to mediate AC inhibition. Peptide-induced AC inhibition was not sensitive to pertussis toxin as opposed to the 5-HT1AR-mediated effect. Our data show that the 5-HT1AR and the 5-HT1AR peptides activate Gi proteins in a slightly different manner.

Effect of nordidemnin on the cell cycle of sea urchin embryos. Role in synthesis and phosphorylation of proteins and in polyphosphoinositide turnover in mitosis progression.

Nordidemnin (NorD) is a cyclic depsipeptide isolated from a Caribbean tunicate. This drug is thought to affect cell proliferation by acting on protein, RNA, and DNA syntheses. We studied the ability of NorD to arrest sea urchin embryos at the prophase stage. We tested whether NorD could alter the synthesis and phosphorylation of proteins as well as polyphosphoinositide (PPI) metabolism, as the activation of these processes is necessary for progression through the cell cycle. The dose-response effect on protein synthesis and cell cleavage suggests that NorD acts in the same way as emetine, a well-known protein synthesis inhibitor. We observed that treatment of eggs with emetine or NorD, even at concentrations that inhibited DNA and protein synthesis as well as phosphorylation of proteins, led to modifications in the incorporation of 32P into phosphatidylinositol phosphate and phosphatidylinositol bisphosphate without any alteration in the chemical amounts of these lipids. However, fluctuations in the PPI messenger system that occur during the cell cycle were maintained in the presence of either drug. We suggest that proteins and PPI are linked in an intricate network to control mitosis and cellular proliferation.

Synthesis and antiproliferative activity of a cyclic analog of dolastatin 10.

A cyclic analog of the natural antiproliferative compound dolastatin 10 was synthesized by introducing an ester link between the N- and C-terminal residues which were modified accordingly. The final macrolactonization was performed by using isopropenyl chloroformate and DMAP as reagents. This analog exhibits submicromolar antiproliferative activity against the L1210 and HT29 cell lines and inhibits in vitro tubulin polymerization (IC50, 39 microM).

Dual effects of nordidemnin on WRK1 cells: inhibition of phosphoinositide metabolism and cell proliferation.

Nordidemnin (NorD), a cyclodepsipeptide isolated from marine invertebrates, exhibits antiproliferative and antitumoral properties identical to didemnin B on many cell lines. On WRK1 cells, a rat mammary tumor cell line, NorD considerably reduced the vasopressin-stimulated accumulation of inositol phosphates. This effect was more pronounced on dividing cells and of weak amplitude on quiescent ones. It was observed with nanomolar concentrations of NorD and became significative after 3 hr of incubation at 37 degrees C. The maximal effect was observed after a 14-hr incubation period. In contrast, the inactive analog epinordidemnin, as well as the structurally related immunosuppressive cyclosporin A, had no significant effect on phosphoinositide metabolism. More detailed analysis demonstrated that NorD reduced the amounts of all intracellular inositol phosphate isomers, including inositol pentakisphosphate and inositol hexakisphosphate. Vasopressin-stimulated inositol (1,4,5)-trisphosphate accumulation was reduced by 80% and, as a consequence, the intracellular calcium mobilization was strongly affected. Similarly, NorD reduced both the level of inositol (1,4,5)-trisphosphate and the intracellular free calcium concentration of unstimulated cells. NorD blocked phosphoinositide metabolism by reducing the myoinositol transporter and, by a consequence, the pool of inositol lipids. NorD also strongly inhibited WRK1 cell proliferation with the same EC50 as that observed for the effect on phosphoinositide metabolism. Epinordidemnin, which was unable to inhibit inositol phosphate accumulation, had no effect on cell growth. Cyclosporin A, which slightly inhibited WRK1 cell growth, did not significantly affect the calcium-phosphatidylinositol cascade. Taken together, these results suggest that NorD might interfere with WRK1 cell growth by inhibiting phosphoinositide turnover.

Development and standardization of an immuno-quantified solid phase assay for HIV-1 aspartyl protease activity and its application to the evaluation of inhibitors.

The catELISA technique was modified and standardized for measuring HIV-1 aspartyl protease activity and evaluating the potency of synthetic peptide inhibitors. This immuno-quantified solid phase assay combines the use of an immobilized C-terminal biotinylated peptide as substrate, a crude enzyme preparation, and a highly specific antiserum elicited against the C-terminal product of the enzyme reaction. A standard curve of this C-terminal product was constructed to determine the enzyme activity. This assay, which requires less enzyme and substrate, is more sensitive than the conventional HPLC method. The amounts of C-terminal peptide produced in solution as determined from ELISA and HPLC standard curves were comparable. Analogues of peptidomimetics designed in our laboratory were assayed for their potency to inhibit the enzyme. One of them, H4, which is a hydroxyethylamine isostere of the Phe-Pro peptide bond, was a powerful inhibitor.