On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone.

His-DTrp-Ala-Trp-DPhe-LysNH2, [His1,Lys6] GHRP, is a new synthetic hexapeptide which specifically elicits a dosage-related release of GH in vitro and in vivo without a concomitant release of LH, FSH, TSH, or PRL and, in limited in vivo studies, insulin or glucagon. Our results indicate that this small peptide has the attributes of a hypophysiotropic hormone. In vitro the minimum and maximum active dosages ranged from 1-10 ng/ml in the pituitary incubate assay. It was active in rats, monkeys, lambs, calves, and under special experimental conditions chicks, indicating its lack of species dependency. It was active when administered iv, sc, or ip to rats. After iv injection, GH levels rose within 2 min, peaked at +10-20 min, and by 2 h usually had returned to normal. It was not possible to directly compare the potencies of [His1,Lys6]GHRP, and the GH-releasing factors GHRF-44 and GHRF-40 after a single sc injection in rats because the time course of the GH response of these peptides was different. The GH response of [His1,Lys6]GHRP was longer in duration than either of these larger peptides. Both SRIF-14 and SRIF-28 inhibited the GH response of the hexapeptide; however, SRIF-28 was about four times more active than SRIF-14 in vitro and 7.5 times more active in vivo. When this small peptide was administered sc once or twice daily to immature rats for 9 or 25 days, the BW gain increased above the control. At the end of the weight gain studies the pituitary remained fully responsive to the peptide. Thus, [His1,Lys6] GHRP may be a valuable peptide for investigating the function of the pituitary somatotrophs and, in addition, it has the potential for increasing BW gain of a variety of normal animals by inducing GH release via a direct pituitary site of action.

Variables determining the growth hormone response of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 in the rat.

Previous studies of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GH-RP-6) have shown this synthetic hexapeptide to be a potent and specific stimulator of GH secretion both in vivo and in vitro. In this study the variables determining the in vivo responses were examined in the rat. The magnitude of the GH response to sc GH-RP-6 was dependent on the age and sex of the rat. Animals less than 15 days of age had much larger responses than did rats 21 days and older. At 10 days of age the male rat had a larger GH response than the female. At 21 days of age, bis(4-methyl 1-homo-piperazinyl-thiocarbonyl) disulfide (Fla-63)-pretreated females had larger responses than did Fla-63-pretreated males. In the Fla-63-pretreated adult rat, sc GH-RP-6 stimulated GH release in the female but not in the male. In the 10-day-old male, the ED50 for sc GH-RP-6 was 0.4 micrograms, and the maximal serum GH response was 800 ng/ml. In the 21-day-old female Fla-63-pretreated rat, the ED50 for sc GH-RP-6 was 3.0 micrograms, and the maximal GH response was 200 ng/ml. In the 21-day-old female pentobarbital-anesthetized rat, iv GH-RP-6 had an ED50 of 0.5 micrograms and a maximal serum GH response of 2500 ng/ml. A marked dose- and time-dependent decrease of subsequent GH-RP-6 responses occurred after a single sc GH-RP-6 injection. Decreases in pituitary GH or increases in somatostatin secretion would not explain this decreased response because the GH response of MRZ 2549, an opiate agonist, was unchanged by GH-RP-6 pretreatment. In contrast to the acute effect of GH-RP-6, chronic daily injections of GH-RP-6 resulted in an enhancement of the GH-RP-6 response.

Conformational energy studies and in vitro and in vivo activity data on growth hormone-releasing peptides.

A series of growth hormone-releasing peptides have been designed and tested for both in vitro and in vivo activity. In vitro activity at 1-10 ng/ml was obtained for the pentapeptide, His-DTrp-Ala-Trp-DPhe-NH2 (I) and the hexapeptide, His-DTrp-Ala-Trp-DPhe-Lys-NH2 (II). These peptides, as well as others to be described, are active in releasing GH in vivo at low microgram dosages. In this manuscript, the conformational properties and in vitro and in vivo activity of a series of small peptides are reported. Results of the biological studies are reported in an accompanying paper.

Design, synthesis, and biological activity of peptides which release growth hormone in vitro.

Experimental observations showed that the analogs [D-Trp2]- and [D-Phe2]methionine enkephalin amide were weakly active in releasing GH from rat pituitary in vitro. These observations were used to design more active GH-releasing factors. Conformational energy calculations were carried out, and energetically favored conformations of these polypeptides were found. Structural similarities as well as structural differences between active and inactive analogs were examined, and new sequences were predicted. Progressively more active analogs were designed, then synthesized, and tested. This cycle of steps was repeated, each time using structural and chemical concepts as design guides, until a series of very active analogs resulted. The most active analog to date, Tyr-D-Trp-Ala-Trp-D-Phe-NH2, was shown to release GH in vitro at 10-30 ng/ml medium, which is approximately 10(3) times more active than the two starting enkephalin-based analogs. From the structure-activity data, a mechanism for binding at the receptors is formulated, and a comparison is made between the structural relationships of the GH-releasing peptide analogs and the GH inhibitor, somatostatin.

Conformational analysis of the molecule luteinizing hormone-releasing hormone. 3. Analogue inhibitors and antagonists.

Conformational energy calculations have been carried out on analogues of luteinizing hormone-releasing hormone which have been shown to be potent inhibitors of the release of luteinizing hormone and follicle-stimulating hormone. The analogues included in this study have D-amino acid substitutions in the 2 and/or 3 positions, such as [D-X2]-LH-RH, [D-X2,D-Y3]-LH-RH, [D-X2,Pro3]-lh-rh, and [D-X2,Leu3]-LH-RH. A configurational property which was common to the low-energy conformers of all the analogues is the directional change of the cis-peptide bond of the pyroglutamate ring. Further, there was no overall structural change in the analogues relative to the conformation of native LH-RH, and the orientation of the aromatic side chains relative to one another remained the same throughout this series of analogues.

Computational studies on carbohydrates: in vacuo studies using a revised AMBER force field, AMB99C, designed for alpha-(1-->4) linkages.

Modifications to the AMBER force field [W.D. Cornell, P. Cieplak, C.I. Bayly, I.R. Gould, K. Merz, D.M. Ferguson, D.C. Spellmeyer, T. Fox, J.W. Caldwell, P.A. Kollman, J. Am. Chem. Soc., 117 (1995) 5179-5197] have been made to improve our ability to reproduce observed molecular properties of alpha-linked carbohydrates when calculated using empirical potential-energy functions. Molecular structures and energies obtained using gradient-optimized density functional methods with ab initio basis sets (B3LYP/6-31G*) on ten minimum-energy conformations of maltose [F.A. Momany, J.L. Willett, J. Comp. Chem., submitted for publication] were used to refine the empirical potentials. Molecular dynamics simulations on beta-maltose (i.e., the beta anomer of maltose), cyclohexamylose (alpha-cyclodextrin), cycloheptamylose (beta-cyclodextrin) and larger cyclomaltooligosaccharide structures were carried out and compared with experimental structural studies to test the new potentials. Ring-puckering potential during dynamics as well as conformational transitions to 'flipped' structures were examined. Results of the tests described here suggest that the revised AMBER parameters (AMB99C) are very good for computational studies of alpha-(1-->4)-linked carbohydrates.

Computational studies on carbohydrates: solvation studies on maltose and cyclomaltooligosaccharides (cyclodextrins) using a DFT/ab initio-derived empirical force field, AMB99C.

An empirical force field, denoted AMB99C, has been used to study molecular properties of alpha-(1-->4)-linked carbohydrates in solution. AMB99C was parameterized using structural and energetic parameters from density functional ab initio methodology. In this work we examine the solution behavior of the beta anomer of maltose and cyclohexa-, cyclohepta-, and cyclooctaamyloses (alpha-, beta-, and gamma-cyclodextrins or alpha-, beta-, and gamma-CDs, respectively), as well as of two larger (DP 10, epsilon-CD; DP 21) cyclomaltooligosaccharides, CA10 and CA21. Experimental data used for comparison purposes include X-ray structures, small-angle scattering radius of gyration values, NMR nuclear Overhauser enhancements (NOEs), and proton coupling constants. Molecular dynamics simulations were carried out using explicit water molecules (TIP3P) to establish equilibrium populations of conformations in solution, and these results are compared with other calculated values and a variety of experimental parameters, such as average H-1-H-4' distances between the rings in beta-maltose, and the primary hydroxyl groups' conformational populations. Medium-to-large cyclomaltooligosaccharide molecules were studied to test for glucose ring puckering and stability of kinked and 'flipped' conformations. The results of the solvation studies are in excellent agreement with experimental structural parameters.

B3LYP/6-311++G** study of alpha- and beta-D-glucopyranose and 1,5-anhydro-D-glucitol: 4C1 and 1C4 chairs, (3,O)B and B(3,O) boats, and skew-boat conformations.

Geometry optimization, at the B3LYP/6-311++G** level of theory, was carried out on 4C1 and 1C4 chairs, (3,O)B and B(3,O) boats, and skew-boat conformations of alpha- and beta-D-glucopyranose. Similar calculations on 1,5-anhydro-D-glucitol allowed examination of the effect of removal of the 1-hydroxy group on the energy preference of the hydroxymethyl rotamers. Stable minimum energy boat conformers of glucose were found, as were stable skew boats, all having energies ranging from approximately 4-15 kcal/mol above the global energy 4C1 chair conformation. The 1C4 chair electronic energies were approximately 5-10 kcal/mol higher than the 4C1 chair, with the 1C4 alpha-anomers being lower in energy than the beta-anomers. Zero-point energy, enthalpy, entropy, and relative Gibbs free energies are reported at the harmonic level of theory. The alpha-anomer 4C1 chair conformations were found to be approximately 1 kcal/mol lower in electronic energy than the beta-anomers. The hydroxymethyl gt conformation was of lowest electronic energy for both the alpha- and beta-anomers. The glucose alpha/beta anomer ratio calculated from the relative free energies is 63/37%. From a numerical Hessian calculation, the tg conformations were found to be approximately 0.4-0.7 kcal/mol higher in relative free energy than the gg or gt conformers. Transition-state barriers to rotation about the C-5-C-6 bond were calculated for each glucose anomer with resulting barriers to rotation of approximately 3.7-5.8 kcal/mol. No energy barrier was found for the path between the alpha-gt and alpha-gg B(3,O) boat forms and the equivalent 4C1 chair conformations. The alpha-tg conformation has an energy minimum in the 1S3 twist form. Other boat and skew-boat forms are described. The beta-anomer boats retained their starting conformations, with the exception of the beta-tg-(3,O)B boat that moved to a skew form upon optimization.

B3LYP/6-311++G** study of monohydrates of alpha- and beta-D-glucopyranose: hydrogen bonding, stress energies, and effect of hydration on internal coordinates.

Twenty-six monohydrates of alpha- and beta-D-glucopyranose were studied using gradient methods at the B3LYP/6-311++G** level of theory. Geometry optimization was carried out with the water molecules at different configurations around the glucose molecule. A new nomenclature for hydrated carbohydrates was developed to describe the water configurations. Zero-point vibrational energy, enthalpy, entropy, and relative free energy were obtained using the harmonic approximation. Hydrogen-bond energies for the monohydrates range from approximately -5 to -12 kcal/mol, and the average relative free energy is approximately 5 kcal/mol. The 1-hydroxy position is the most energetically favored site for hydration, and the region between the two and three positions is the next-most favored site. A water molecule approaching alpha-D-glucose between the 1- and 2-hydroxy positions pulls the 2-hydroxyl hydrogen atom away from the 1-hydroxy oxygen atom, thus increasing the hydrogen-bond length and also increasing the alpha-D-glucose energy. The increase in energy that occurs with a similar interaction on the beta-anomer is much less effective since the hydrogen bond is much longer. Using the calculated free energies of all 26 configurations, the anomer population (alpha/beta) increases in the beta-anomer population relative to the in vacuo case by approximately 10% at the expense of the alpha-anomer, giving an (alpha/beta) ratio of approximately 50/50. This result arises from entropy contributions favoring the beta-anomer more than the alpha-anomer. From analysis of donor and acceptor hydrogen-bond lengths, excellent correlation is found between the DFT calculated distances and those taken from carbohydrate structures in the Cambridge Crystallographic Data Bank.

X-ray structure determination and modeling of the cyclic tetrasaccharide cyclo.

The cyclic tetrasaccharide cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] is the major compound obtained by the action of endo-alternases on the alternan polysaccharide. Crystals of this cyclo-tetra-glucose belong to the orthorhombic space group P2(1)2(1)2(1) with a = 7.620(5), b = 12.450(5) and c = 34.800(5) A. The asymmetric unit contains one tetrasaccharide together with five water molecules. The tetrasaccharide adopts a plate-like overall shape with a very shallow depression on one side. The shape is not fully symmetrical and this is clearly apparent on comparing the (phi, psi) torsion angles of the two alpha-(1-->6) linkages. There is almost 10 degrees differences in phi and more than 20 degrees differences in psi. The hydrogen bond network is asymmetric, with a single intramolecular hydrogen bond: O-2 of glucose ring 1 being the donor to O-2 of glucose ring 3. These two hydroxyl groups are located below the ring and their orientation, dictated by this hydrogen bond, makes the floor of the plate. Among the five water molecules, one located above the center of the plate occupies perfectly the shallow depression in the plate shape formed by the tetrasaccharide. Molecular dynamics simulation of the tetrasaccharide in explicit water allows rationalization of the discrepancies observed between the X-ray structures and data obtained previously by NMR.

Molecular dynamics simulations on the mycotoxin fumonisin B1.

The solution conformational properties of the mycotoxin fumonisin B(1) have been studied using molecular dynamics methodology. Fumonisins have been shown to inhibit sphinganine (sphingosine) N-acyltransferase (ceramide synthase) and show a wide range of toxic effects in many animals. This study of the solution properties of fumonisin B(1) attempts to add to the structural models necessary for the understanding of the binding and activity properties. The computational method uses a box with periodic boundaries, filled with explicit TIP3P water molecules, the substrate fumonisin B(1), and selected counterions for charge neutrality. The starting structure of fumonisin B(1) is added to the box by excluding water molecules. The explicit image method using 12-A cutoffs is applied to the system and molecular dynamics are carried out on different starting conformations at 300 K in 100-picosecond (ps) steps. Examination of the resulting equilibrated conformations suggests that the structure is relatively extended and that previous computational studies in vacuo, showing a compact folded structure, may not be consistent with the solution structure.

DFTMD studies of ß-cellobiose: conformational preference using implicit solvent.

Previous DFT in vacuo studies on the conformational preferences for cellobiose showed that upon optimization the φ(H)-anti conformations were of lower energy than the syn forms. Upon optimization using an implicit solvation method, COSMO, the syn or observed form was still not predicted to be of lower energy than the φ(H)-anti form, even though optimization after addition of several explicit water molecules did show a relative energy difference favoring the syn form. In order to examine the predictive ability of COSMO on this carbohydrate, constant energy dynamics, DFTMD, simulations were carried out on low energy syn and φ(H)-anti conformations with and without COSMO included during the dynamics. The resulting analysis confirmed that when COSMO is included in the dynamics, the syn conformations become energetically favored over the φ(H)-anti forms suggesting that both solvent and entropy play roles in dictating the solution conformation of cellobiose. Analysis of the dynamic runs includes distributions of selected dihedral angles versus time, conformational transitions, and populations of some quasi-planar, boat, skew forms during the simulations.

DFTMD studies of glucose and epimers: anomeric ratios, rotamer populations, and hydration energies.

Results are presented from density functional molecular dynamics (DFTMD) simulations, based on constant energy dynamics, of glucose and its cyclic form of 6-carbon epimers. Both in vacuo and an implicit solvent method (COSMO) were examined, including simulations of low-energy conformations of each molecule. Analysis of the DFTMD results includes the following: energies averaged over the simulation time, calculated anomeric ratios, hydroxyl and hydroxymethyl rotamer populations, and hydration energies. Hydrogen-bonding networks persistence times were examined, and the effects of solvation on rotamer populations were described. Anomeric ratios calculated from energy optimization of an ensemble of low-energy conformers are compared to those obtained from ensemble averages from molecular dynamics, with dynamics simulations giving populations in best agreement with experimental anomeric ratios. Ensemble results in vacuo were not in agreement with experimental anomeric ratios or hydroxymethyl populations, producing in some cases reversal of the alpha:beta ratios. The difficulty in obtaining correct alpha:beta ratios increases with the number of axial groups; the mono-axial epimers being best represented, epimers with two axial groups being more difficult, and the epimers with three axial hydroxyl groups being most difficult to analyze, the result of a large number of very strong hydrogen-bonding networks that form the ensemble of low-energy conformations in the multi-axial structures.

Conformational energy calculations on enkephalins and enkephalin analogs. Classification of conformations to different configurational types.

Conformational energy calculations were carried out on the peptide enkaphalins (ENK) and selected analogs to find those conformers of low energy. The analogs studied include [D-Ala2]Enk-NH2, [D-Ala2]Enk, [D-Met2, Pro5]Enk-NH2, [D-Ala2, D-Phe5]Enk, [D-Ala2, D-Leu5]Enk, [D-Ala2, (N-Me)Phe4, Met5] Enk-NH2 and [D-Ala2, (N-Me)Met5]Enk-NH2. When the low-energy conformers for all the analogs are compared, different allowed backbone conformations are found which orient the functional side-chains such that three classifications of structures appear. Each classification shows a unique configuration of side-chain positions in space even though different backbone conformations are found within each classification.

Conformational analysis of thyrotropin releasing factor.

Conformational energy calculations on thyrotropin releasing factor and on several of its analogues indicate that the central histidyl residue of the native molecule is in an extended conformation. Some derivatives (with a reduced biological activity) have an altered conformation. Since some substitutions leave the conformation unchanged but alter the biological activity, these substitutions must involve the sites responsible for binding of the hormone to its receptor.