Molecular properties that influence the oral bioavailability of drug candidates.

Oral bioavailability measurements in rats for over 1100 drug candidates studied at SmithKline Beecham Pharmaceuticals (now GlaxoSmithKline) have allowed us to analyze the relative importance of molecular properties considered to influence that drug property. Reduced molecular flexibility, as measured by the number of rotatable bonds, and low polar surface area or total hydrogen bond count (sum of donors and acceptors) are found to be important predictors of good oral bioavailability, independent of molecular weight. That on average both the number of rotatable bonds and polar surface area or hydrogen bond count tend to increase with molecular weight may in part explain the success of the molecular weight parameter in predicting oral bioavailability.

Internal mobility of cyclic RGD hexapeptides studied by 13C NMR relaxation and the model-free approach.

The internal mobility of three isomeric cyclic RGD hexapeptides designed to contain two beta-turns in defined positions, cyclo(Arg-Gly-Asp-Gly-D-Pro-Pro) (I), cyclo(Arg-Gly-Asp-D-Pro-Gly-Pro) (II) and cyclo(Arg-Gly-Asp-D-Pro-Pro-Gly) (III), have been studied by 13C NMR longitudinal and transverse relaxation experiments and measurements of steady-state heteronuclear (1H)-13C NOE enhancement with 13C at natural abundance. The data were interpreted according to the model-free formalism of Lipari and Szabo, which is usually applied to data from macromolecules or larger sized peptides with overall rotational correlation times exceeding 1 ns, to yield information about internal motions on the 10-100 ps time scale. The applicability of the model-free analysis with acceptable uncertainties to these small peptides, with overall rotational correlation times slightly below 0.

The soluble form of E-selectin is an asymmetric monomer. Expression, purification, and characterization of the recombinant protein.

The gene coding for a soluble form of human E-selectin (sE-selectin) has been expressed in Chinese hamster ovary (CHO) cells. Cells seeded into a hollow fiber reactor secreted protein at a level of 160 mg/liter. The protein was purified to > 95% pure and low endotoxin (< 2 ng/mg), using physiological pH and buffers.

Conformations of cyclic pentapeptide endothelin receptor antagonists.

The solution conformations in methanol and chloroform of the endothelin A receptor antagonists cyclo(dV-L-dW-dD-P), 1, and cyclo(dV-N alpha-MeL-dW-dD-P), 2, have been studied by NMR spectroscopy at room temperature and below. In these solvents, both peptides were found to have a well defined peptide backbone conformation composed of a type II beta turn at the Leu-D-Trp and a gamma' turn at Pro. This conformation is in agreement with results reported for 1 in other solvents and consistent with the expected location of the N-methyl substituent in that backbone.

Effects of C alpha-methyl substitution on the conformation of linear GnRH antagonist analogs.

We have examined the effect of C alpha-methyl groups on the conformational ensemble of GnRH analog peptides by comparing 1H 2D NMR data from two analogs, Ac-D-Nal1-D-4-Cl-C alpha-Me-Phe2-D-Pal3-Ser4-Tyr5-D-Arg6-Leu7-Arg8-Pro9-D-Al a10- NH2 (1) and Ac-D-Nal1-D-4-Cl-C alpha-Me-Phe2-D-Pal3-Ser4-C alpha-Me-Tyr5-D-Arg6- Leu7-C alpha-Me-Arg8-Pro9-D-Ala10-NH2 (2). The two additional C alpha-methyl groups in residues 5 and 8 of 2 do not influence significantly the pattern of the observable main chain NOE intensities, or of the backbone HN proton chemical shifts, which indicates that they do not produce global changes in the conformational ensemble of the peptide. A local change induced by the substitution was observed in the conformation at D-Arg8-Pro9.

Conformational mobility in cyclic oligopeptides.

Analysis of two isomeric cyclic hexapeptides of composition (Asp, Arg, Gly2, Pro, D-Pro) by a nuclear Overhauser effect constrained distance geometry conformation search yielded a narrowly defined backbone conformation for one and considerable ambiguity about the conformation in part of the other. Preliminary 13C relaxation studies of these peptides suggest that it is possible that this difference may correspond to a physical difference in internal mobility. In connection with this observation, other experimental evidence bearing on the backbone conformational mobility of cyclic oligopeptides with 4-10 residues, frequently considered to have well-defined backbones, is reviewed.

Conformation of cyclic octapeptides. VI. Structure of cyclo-bis-(-L-alanyl-glycyl-L-prolyl-L-phenylalanyl-) tetrahydrate.

C38H48N8O8.4H2O, Mr = 816.9, monoclinic, P21, a = 10.

NMR identification of protein surfaces using paramagnetic probes.

Paramagnetic agents produce line broadening and thus cancellation of anti phase cross-peak components in two-dimensional correlated nuclear magnetic resonance spectra. The specificity of this effect was examined to determine its utility for identifying surface residues of proteins. Ubiquitin and hen egg white lysozyme, for which X-ray crystal structures and proton NMR assignments are available, served as test cases.

Solution structures of human transforming growth factor alpha derived from 1H NMR data.

The 600-MHz 1H NMR spectrum of the des-Val-Val mutant of human transforming growth factor alpha (TGF-alpha) was reassigned at pH = 6.3. The conformation space of des-Val-Val TGF-alpha was explored by distance geometry embedding followed by restrained molecular dynamics refinement using NOE distance constraints and some torsion angle constraints derived from J-couplings.

5,5-Dimethylthiazolidine-4-carboxylic acid (DTC) as a proline analog with restricted conformation.

Spectroscopic evidence is presented for the lack of intramolecular hydrogen bonding in a simple peptide derivative of 5,5-dimethylthiazolidine-4-carboxylic acid (Dtc). The infrared spectrum of Boc-Pro-Ile-OMe 1 in nonpolar solvents displays two N-H stretching bands at 3419 and 3330 cm-1 in CCl4 and one at 3417 and 3328 cm-1 in CHCl3. The low frequency band at 3328-3330 cm-1 may be assigned to conformations with an intramolecular hydrogen bond between the Ile N-H and Boc C = O.

N.m.r studies of internal mobility in a cyclic tetrapeptide.

The conformation of cyclo(D-Phe-D-Pro-Ala-Pro) is reported. Measurements of spin-lattice relaxation in the rotating frame indicate that this peptide is conformationally less mobile on the microsecond time scale than larger cyclic peptides previously studied. Libration of the Pro-Ala and Pro-Phe peptide bond planes is suggested as the source of the small exchange contributions to 1/T1p.

Molecular mechanics investigation of the flexibility of some cyclic peptides.

Conformation space near the crystal conformations of proline-containing cyclic octapeptides and cyclic hexapeptides of C2 sequence symmetry, e.g. cyclo-(Gly-Pro-D-Phe)2 and cyclo-(D-Ala-Gly-Pro-D-Phe)2, was explored using molecular mechanics.

Conformations of cyclic octapeptides. 4. Diastereomers of cyclo(Gly-Pro-Phe-Ala-Asn-Ala-Val-Ser).

Stereoisomers of cyclo(Gly-Pro-Phe-Ala-Asn-Ala-Val-Ser) were synthesized. NMR studies of their solution conformations, focusing on peptide N-H solvent exposure, were made. These indicated that a single proline residue in the cyclic octapeptide ring is insufficient constraint to stabilize the backbone conformations that were previously established for cyclo(Gly-Pro-Phe-Ala)2.

Synthesis and backbone conformations of cyclic hexapeptides cyclo-(Xxx-Pro-D-Gln)2.

The solution syntheses of cyclo-(Xxx-Pro-D-Gln)2, where Xxx = Gly, Ala, Leu, Phe and Val are described. Several routes were examined, the most successful involving the intermediate Z-Xxx-Pro-D-Gln-O-tBu and proceeding to cyclization of H-Xxx-Pro-D-Gln-Xxx-Pro-D-Gln-OH using diphenylphosphoryl azide. The N--H regions of the proton magnetic resonance spectra of aqueous solutions of these peptides were examined, and in the Xxx = Leu and Val peptides an unsymmetrical backbone, presumably with one cis Xxx-Pro peptide bond, was found to be important.

Nitroxyl-induced T1 relaxation rates as a probe of conformation in peptide hormones.

Proton spin-lattice relaxation rates in the N-H region of the n.m.r.

Conformation of cyclo-(L-threonine)2 and cyclo-(L-allothreonine)2. A proton and carbon n.m.r study.

The diketopiperazines cyclo-(L-Thr)2 and cyclo-(L-allo Thr)2 in water and in dimethyl sulfoxide were studied by proton and carbon-13 nuclear magnetic resonance, and the dominant conformation were deduced from proton-proton and proton-carbon coupling constants. In cyclo-(L-Thr)2 the chi 1 = 60 degrees, hydroxyl over the ring, side chain conformation is favored; this conformation is also favored for cyclo-(L-Ser)2 and cyclo-(L-Ser-D-Ser). However, the important side chain conformation for cyclo-(L-allo Thr)2 is chi 1 = -60 degrees, methyl group over the diketopiperazine ring.

Peptide--water association in peptide crystals.

The structures of 37 peptide crystals, containing 78 water-peptide hydrogen bonds and 77 other hydrogen bonds involving water, were surveyed to identify the geometry of peptide backbone hydration. In the sample, hydration of peptide carbonyl occurred more frequently than hydration of peptide N--H. The most probable value of the C'=O .

Circular dichroism of beta turns in peptides and proteins.

Circular dichroism (CD) spectra are reported for two groups of cyclic hexapeptides having beta turns whose geometry can be firmly established by X-ray crystallography and by NMR spectroscopy. One series contains the sequence L-Pro-D-Phe in the geometry of the classical type II beta turn, while the second group has the sequence D-Phe-L-Pro in the closely related geometry of the gramicidin S turn. CD data on the hydrogenated peptides show that in neither series do Cotton effects due to the aromatic phenylalanyl chromophore make a significant contribution to the spectra in the 195--240-nm region.

Solution conformation of a retro-D analogue of tocinamide.

The solution conformation of a retro-D analogue of tocinamide H-D-Cys-D-Asn-D-Gln-D-aIle-D-Tyr-NHCH2CH2S was examined using proton magnetic resonance and circular dichroism spectroscopy. The observations support major contributions to the conformational distribution from structures with a type I beta turn in the sequence D-Asp-D-Gln-D-aIle-D-Tyr. This is topologically similar to the beta turn proposed for oxytocin, L-Tyr-L-Ile-L-Gln-L-Asn, but with the polarity of the CONH groups reversed along the chain; the peptide is, however, hormonally inert.