Design of peptidomimetics that inhibit the association of phosphatidylinositol 3-kinase with platelet-derived growth factor-beta receptor and possess cellular activity.

Phosphorylated tyrosine residues of growth factor receptors that associate with intracellular proteins containing src-homology 2 (SH2) domains are integral components in several signal transduction pathways related to proliferative diseases such as cancer, atherosclerosis, and restenosis. In particular, a phosphorylated pentapeptide [pTyr751-Val-Pro-Met754-Leu (pTyr = phosphotyrosine)] derived from the primary sequence of platelet-derived growth factor-beta (PDGF-beta) receptor blocks the association of the C-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) to PDGF-beta receptor with an IC50 of 0.445 +/- 0.047 microM. Further evaluation of the structure-activity relationships for pTyr751-Val-Pro-Met-Leu resulted in the design of smaller peptidomimetics with enhanced affinity including Ac-pTyr-Val-Ala-N(C6H13)2 (IC50 = 0.076 +/- 0.010 microM). In addition, the phosphotyrosine residue was replaced with a difluorophosphonate derivative [4-phosphono(difluoromethyl)phenylalanine (CF2Pmp)] which has been shown to be stable to cellular phosphatases. The extracellular administration of either CF2Pmp-Val-Pro-Met-Leu or Ac-CF2Pmp-Val-Pro-Met-NH2 in a whole cell assay resulted in specific inhibition of the PDGF-stimulated association from the C-terminal SH2 domain of the p85 subunit of PI 3-kinase to the PDGF-beta receptor in a dose-dependent manner. These compounds were also effective in inhibiting GLUT4 translocation, c-fos expression, and cell membrane ruffling in single-cell microinjection assay.

Bacterial expression and characterization of human recombinant apolipoprotein(a) kringle IV type 9.

Elevated plasma lipoprotein(a) [Lp(a)] is an independent risk factor for several vascular diseases. Lp(a) particles are generated through the formation of a disulfide bond between Cys4057 of kringle IV type 9, (KIVt9), of the multikringle apolipoprotein(a) [apo(a)] and a cysteine in apoB-100 low-density lipoprotein (LDL). To better understand this interaction, we have expressed and purified KIVt9 from Escherichia coli as a His-Tag fusionprotein. Dithiothreitol (DTT)-treated purified KIVt9 migrated as a single approximately 17. 3-kDa band on SDS-PAGE gels. Without DTT, an additional band twice the molecular weight of KIVt9 was observed. The double-size band presumably resulted from dimerization of individual kringles, through their unpaired cysteine residues, since a mutation Cys4057 --> Ser ([Ser4057]KIVt9) abolished dimer formation. Using a gel-shift assay, we showed that KIVt9 could couple to 14-amino-acid apoB-100 synthetic peptides (apoB3732-3745 and apoB4319-4332) containing Cys3734 or Cys4326. Both of these apoB-100 cysteines have been reported to associate with apo(a) to generate Lp(a). In the presence of either apoB-100 peptide, KIVt9 was shifted to a higher molecular weight that was consistent with the covalent addition of a 1.2-kDa apoB-100 peptide. Identical apoB-100 peptides in which the cysteine residues were replaced by alanine ([Ala3734]apoB3732-3745 and [Ala4326]apoB4319-4332) had no effect in the gel-shift assay. Furthermore, [Ser4057]KIVt9 did not covalently interact with apoB3732-3745 or apoB4319-4332. These results indicated that KIVt9 couples to the Cys-apoB-100 peptides through a disulfide linkage. This system may be suitable for further investigating the apo(a)/apoB-100 coupling reaction and the structure of KIVt9 through X-ray crystallographic studies.

Structure-activity relationships of cysteine-lacking pentapeptide derivatives that inhibit ras farnesyltransferase.

Mutational activation of ras has been found in many types of human cancers, including a greater than 50% incidence in colon and about 90% in pancreatic carcinomas. The activity of both native and oncogenic ras proteins requires a series of post-translational processing steps. The first event in this process is the farnesylation of a cysteine residue located in the fourth position from the carboxyl terminus of the ras protein, catalyzed by the enzyme farnesyltransferase (FTase). Inhibitors of FTase are potential candidates for development as antitumor agents. Through a high-volume screening program, the pentapeptide derivative PD083176 (1), Cbz-His-Tyr(OBn)-Ser(OBn)-Trp-DAla-NH2, was identified as an inhibitor of rat brain FTase, with an IC50 of 20 nM. Structure-activity relationships were carried out to determine the importance of the side chain and chirality of each residue. This investigation led to a series of potent FTase inhibitors which lack a cysteine residue as found in the ras peptide substrate. The parent compound (1) inhibited the insulin-induced maturation of Xenopus oocytes (concentration: 5 pmol/oocyte), a process which is dependent on the activation of the ras pathway.

Structure-activity studies of phosphorylated peptide inhibitors of the association of phosphatidylinositol 3-kinase with PDGF-beta receptor.

Phosphorylated pentapeptides derived from Tyr751 of the PDGF-beta receptor (pTyr751-Val-Pro-Met-Leu, pTyr = phosphotyrosine) were prepared to examine their ability to inhibit the association of the C-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) with the PDGF-beta receptor. Peptidic analogs were prepared to examine the importance of the amine and carboxy terminus and specific amino acids via alanine/D-amino acid scans and site specific modifications. Several of these peptides had submicromolar activity. In particular, it was shown that neutralization of the amine and carboxy terminus led to analogs with enhanced activity. In addition, it was determined that only minimal modifications were allowed for pTyr and Met, while the other positions were quite tolerant of modification.

Potent pseudopeptide bombesin-like agonists and antagonists. Correlation of ordered conformation of bombesin analogs to receptor activity.

Bombesin-like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13-14 and 8-9, respectively, with psi[CH2S] and psi[CH2N(CH3)]. [Phe13 psi[CH2S]Leu14]bombesin and [Phe8 psi[CH2S]-Leu9]litorin bound to the murine pancreatic bombesin/gastrin releasing peptide receptor with similar dissociation constants (Kd = 3.9 and 3.4 nM, respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, Kd = 1.6 nM and isomer II, Kd = 0.89 nM. Further oxidation to the sulfone decreased potency ([Phe8 psi[CH2SO2]Leu9]litorin, Kd = 9.9 nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a psi[CH2N(CH3)] at the 8-9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (Kd = 0.1 nM) and [Leu9]litorin (Kd = 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH-dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pKa = 6.3 and 8.5). The lower pKa is found in the agonist analogs but not in the psi [CH2S]-containing antagonist.