N-Methylated cyclic RGD peptides as highly active and selective alpha(V)beta(3) integrin antagonists.

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.

[Left ventricular function assessment using a floating catheter in dynamic and isometric (handgrip) stress in normal and heart infarct patients]. / Linksventrikuläre Funktionsprüfung mittels Einschwemmkatheter während dynamischer und isometrischer (Handgriff-)Belastung bei Normalen und bei Herzinfarktpatienten.

UNLABELLED: Left ventricular function was examined in a control group of 8 healthy men (average age 26 +/- 6.2 years) and was compared to 40 post-MI patients (average age 50 +/- 8.2 years, 39 men, one woman). It was measured using the floating-catheter technique under isometric exercise (50% of maximal voluntary contraction using both hands) and also under dynamic exercise (progressive supine bicycle-ergometry). In the control group, there was a significant increase in heart rate, arterial blood-pressure and the average PCP from rest to isometric exercises. PCP was noted to stay within its normal range (less than 20 mm Hg) under both modes of exercise. There was no significant difference comparing the effects of dynamic and isometric exercise (isometric: PCP 13.3 +/- 3.3 mm Hg; dynamic: PCP 11.8 +/- 3.6 mm Hg). In Group I (20 post-MI-patients showing ischemia during exercise-ECG) PCP stayed normal under isometric exercise (PCP 18.8 +/- 10.2 mm Hg). Under dynamic exercise, their PCP increased up to a pathological level (PCP 24.9 +/- 10.1 mm Hg, p less than 0.01). In Group II (20 post-MI-patients with lacking proof of ischemia during exercise-ECG) there was no significant difference between the increase in PCP under isometric vs dynamic exercise (isometric: PCP 18.7 +/- 13.4 mm Hg; dynamic: PCP 18.5 +/- 10.0 mm Hg). CONCLUSION: The results lead to the conclusion that isometric exercise alone (50% of maximal voluntary contraction using both hands, handgrip) can replace dynamic exercise in the diagnosis of LV malfunction in post-MI patients who do not show ischemia during exercise-ECG.

NMR spectroscopy reveals the solution dimerization interface of p53 core domains bound to their consensus DNA.

The p53 protein is a transcription factor that acts as the major tumor suppressor in mammals. The core DNA-binding domain is mutated in about 50% of all human tumors. The crystal structure of the core domain in complex with DNA illustrated how a single core domain specifically interacts with its DNA consensus site and how it is inactivated by mutation. However, no structural information for the tetrameric full-length p53-DNA complex is available. Here, we present novel experimental insight into the dimerization of two p53 core domains upon cooperative binding to consensus DNA in solution obtained by NMR. The NMR data show that the p53 core domain itself does not appear to undergo major conformational changes upon addition of DNA and elucidate the dimerization interface between two DNA-bound core domains, which includes the short H1 helix. A NMR-based model for the dimeric p53 core-DNA complex incorporates these data and allows the conclusion that the dimerization interface also forms the actual interface in the tetrameric p53-DNA complex. The significance of this interface is further corroborated by the finding that hot spot mutations map to the H1 helix, and by the binding of the putative p53 inhibitor 53BP2 to this region via one of its ankyrin repeats. Based on symmetry considerations it is proposed that tetrameric p53 might link non-contiguous DNA consensus sites in a sandwich-like manner generating DNA loops as observed for transcriptionally active p53 complexes.

Solution structure, dimerization, and dynamics of a lipophilic alpha/3(10)-helical, C alpha-methylated peptide. Implications for folding of membrane proteins.

The solution structure and the dimerization behavior of the lipophilic, highly C(alpha)-methylated model peptide, mBrBz-Iva(1)-Val(2)-Iva(3)-(alphaMe)Val(4)-(alphaMe)Phe(5)-(alphaMe)Val(6)-Iva(7)-NHMe, was studied by NMR spectroscopy and molecular dynamics simulations. The conformational analysis resulted in a right-handed 3(10)/alpha-helical equilibrium fast on the NMR time scale with a slight preference for the alpha-helical conformation. The NOESY spectrum showed intermolecular NOEs due to an aggregation of the heptapeptide. In addition, temperature-dependent diffusion measurements were performed to calculate the hydrodynamic radius. All these findings are consistent with an antiparallel side-by-side dimerization. The structure of the dimeric peptide was calculated with a simulated annealing strategy. The lipophilic dimer is held together by favorable van der Waals interactions in the sense of a bulge fitting into a groove. The flexibility of the helical conformations concerning an alpha/3(10)-helical equilibrium is shown in a 3 ns molecular dynamics simulation of the resulting dimeric structure. Both overall helical structures of each monomer and the antiparallel mode of dimerization are stable. However, transitions were seen of several residues from a 3(10)-helical into an alpha-helical conformation and vice versa. Hence, this peptide represents a good model in which two often-discussed aspects of hierarchical transmembrane protein folding are present: i <-- i + 3 and i <-- i + 4 local H-bonding interactions cause a specific molecular shape which is then recognized as attractive by other surrounding structures.