The alpha(V)beta(3) integrin receptor plays an important role in human tumor metastasis and tumor-induced angiogenesis. The in vivo inhibition of this receptor by antibodies or by cyclic peptides containing the RGD sequence may in the future be used to selectively suppress these diseases. Here we investigate the influence of N-methylation of the active and selective alpha(V)beta(3) antagonist cyclo(RGDfV) (L1) on biological activity. Cyclo(RGDf-N(Me)V-) (P5) was found to be even more active than L1 and is one of the most active and selective compounds in inhibiting vitronectin binding to the alpha(V)beta(3) integrin. Its high-resolution, three-dimensional structure in water was determined by NMR techniques, distance geometry calculations, and molecular dynamics calculations, providing more insight into the structure-activity relationship.

Double‐tailed lipo‐tetragastrin derivatives of increasing fatty acid chain length were used to identify the minimum size of the fatty acid moieties (≥C10) that restricts the access to the CCK‐B/gastrin (CCK: cholecystokinin) receptor via a membrane‐bound pathway. Then dimyristoyl‐mercaptoglycerol/maleoyl‐gastrin adducts of increasing peptide chain length were synthesized to define the minimal peptide size required for receptor binding affinities comparable to those of underivatized gastrin peptides despite anchorage of the lipid tails in the membrane bilayer. The experimental results indicated that most of the little‐gastrin sequence, i.e., 2–17, is needed for optimal interaction of the molecule with the binding cleft of the receptor. From these data experimentally based restraints could be derived for docking of lipo‐gastrin onto a CCK‐B/gastrin receptor model applying molecular dynamics simulations and energy minimizations. In the receptor‐bound state some of the secondary structure elements of gastrin as determined by nmr analysis of gastrin‐peptides in low dielectric constant media are retained. The N‐terminal gastrin portion interacts in a more or less extended conformation with the receptor surface, and upon a sharp kink at the Ala‐Tyr dipeptide portion the C‐terminal pentapeptide amide part inserts deeply into the helix bundle. Besides Arg‐57 on top of helix 1 of the receptor, for which no potential interaction with the ligand could be detected, the other amino acid residues identified by mutagenesis studies as involved in gastrin recognition were found to interact with the C‐terminal portion of gastrin. Even taking into account the strong limitations of such a model system, it represents an interesting tool for rationalizing the experimental results of the extensive structure‐function studies performed previously on gastrin and to delineate more precisely the putative ligand binding site on the extracellular face of the receptor. © 1997 John Wiley & Sons, Inc. Biopoly 41: 799–817, 1997

Cyclo(PheN2-Tyr-D-Trp-Lys-Val-PheC3)-Thr-NH2 (PTR 3046), a backbone-cyclic somatostatin analogue, was synthesized by solid-phase methodology. The binding characteristics of PTR 3046 to the different somatostatin receptors, expressed in CHO cells, indicate high selectivity to the SSTR5 receptor. PTR 3046 is highly stable against enzymatic degradation as determined in vitro by incubation with rat renal homogenate and human serum. The biological activity of PTR 3046 in vivo was determined in rats. PTR 3046 inhibits bombesin- and caerulein-induced amylase and lipase release from the pancreas without inhibiting growth hormone or glucagon release. The major conformation of PTR 3046 in CD3OH, as determined by NMR, is defined by a type II' beta-turn at D-Trp-Lys and a cis amide bond at Val-PheC3.

Toward establishing the general efficacy of using trisubstituted cyclopropanes as peptide mimics to stabilize extended peptide structures, the cyclopropanes 20a-d were incorporated as replacements into 9-13, which are analogues of the known HIV-1 protease inhibitors 14 and 15. The syntheses of 20a-d commenced with the Rh2[5(S)-MEPY]4-catalyzed cyclization of the allylic diazoesters 16a-d to give the cyclopropyl lactones 17a-d in high enantiomeric excess. Opening of the lactone moiety using the Weinreb protocol and straightforward refunctionalization of the intermediate amides 18a-d gave 20a-d. A similar sequence of reactions was used to prepare the N-methyl-2-pyridyl analogue 28. Coupling of 20a-d and 28 with the known diamino diol 22 delivered 9-13. Pseudopeptides 9-12 were found to be competitive inhibitors of wild-type HIV-1 protease in biological assays having Kis of 0.31-0.35 nM for 9, 0.16-0.21 nM for 10, 0.47 nM for 11, and 0.17 nM for 12; these inhibitors were thus approximately equipotent to the known inhibitor 14(IC50 = 0.22 nM) from which they were derived. On the other hand 13 (Ki = 80 nM) was a weaker inhibitor than its analogue 15 (Ki = 0.11 nM). The solution structures of 9 and 10 were analyzed by NMR spectroscopy and simulated annealing procedures that included restraints derived from homo- and heteronuclear coupling constants and NOEs; because of the molecular symmetry of9 and 10, a special protocol to treat the NOE data was used. The final structure was checked by restrained and free molecular dynamic calculations using an explicit DMSO solvent box. The preferred solution conformations of 9 and 10 are extended structures that closely resemble the three-dimensional structure of 10 bound to HIV-1 protease as determined by X-ray crystallographic analysis of the complex. This work convincingly demonstrates that extended structures of peptides may be stabilized by the presence of substituted cyclopropanes that serve as peptide replacements. Moreover, the linear structure enforced in solution by the two cyclopropane rings in the pseudopeptides 9-12 appears to correspond closely to the biologically active conformation of the more flexible inhibitors 14 and 15. The present work, which is a combination of medicinal, structural, and quantum chemistry, thus clearly establishes that cyclopropanes may be used as structural constraints to reduce the flexibility of linear pseudopeptides and to help enforce the biologically active conformation of such ligands in solution.