Conformational study of eight-membered diazocine turn mimics by two-dimensional NMR spectroscopy.

The eight-membered ring conformations of two diazocine turn mimics, methyl-[2,5-dioxo-3-(S)-(3-omega-tosyl-guanidino-propyl)-4-methyl-octahy dro- 1,4-diazocin-1-yl]acetate (I) and methyl-[2,5-dioxo-3-(S)-(3-omega-tosyl-guanidino-propyl)-octahydro-1,4- diazocin-1-yl]acetate (II), were determined using torsion angle constraints derived from 3J(C,H) coupling constants extracted from 13C-filtered TOCSY spectra with 13C in natural abundance. For I, the torsion angle constraints derived from 3J(C,H) coupling constants were in agreement with torsion angle constraints derived from 3J(H,H) coupling constants extracted from a P.E. COSY spectrum. Similar 3J(C,H) coupling constants were found for I and II, and they shared an identical eight-membered ring conformation characterized by two cis-amide bonds and a staggered conformation of the trimethylene group in which the H3 proton is proximal to both the H6 and H8 protons.

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. In methanol, both peptides show NOE and chemical shift evidence of close contact between the Leu and D-Trp side chains. This interaction is greatly reduced or absent in chloroform, and is stronger in methanol at 203 K than at 298 K.

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. Conformational heterogeneity involving rotation of a peptide bond plane relative to the overall ring plane is identified as a common phenomenon. Nuclear magnetic resonance line-shape studies at temperatures down to 200 K can detect backbone motions with activation free energy barriers down to about 10 kcal/mole, but conformational exchange with lower barriers, though detectable in other ways, will not be obvious from nmr spectra alone.

Investigation of conformational specificity at GPIIb/IIIa: evaluation of conformationally constrained RGD peptides.

RGD-containing proteins and peptides are known to bind to the platelet GPIIb/IIIa receptor and inhibit platelet aggregation. That a conformational component to the specificity exists is suggested by significantly lower activity of linear RGD analogs relative to closely related cyclic peptides and small proteins containing the RGD sequence. Recently, conformations for a suite of RGD containing cyclic peptides have been defined by NMR-based methods and, for one molecule, by X-ray diffraction. We report here the NMR-based conformational analysis of an additional cyclic peptide, cyclo(Pro-Arg-Gly-Asp-D-Pro-Gly), and compare the conformational variations in the suite of peptides and related analogs. Biological activity data for these peptides shows a preference of the platelet GPIIb/IIIa receptor for one conformation of the RGD sequence, but suggests its ability to bind a second, distinct conformation.

Reversible binding of substance P to artificial lipid membranes studied by capacitance minimization techniques.

Interaction of substance P with electrically neutral, planar lipid bilayers prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and with anionic bilayers prepared from mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and brain phosphatidylserine was measured using the capacitance minimization method for monitoring the membrane surface potential caused by the positive charges and electric dipole moment of adsorbed peptide. Substance P bound to the electrically neutral bilayers from 9 mM KCl (buffered to pH 5.5 with 2.0 mM 2-(N-morpholino)ethanesulfonate) with a maximal binding density of about 1 x 10(-2) molecules per nm2 and a dissociation constant of about 2 x 10(-4) M. Measurement of the surface potential at different ionic strengths (shielding of surface charges) allowed distinction between the fixed-charge surface potential and a dipole potential. Ascribing this dipole potential to membrane-bound substance P would imply an effective dipole moment normal to the bilayer surface of about 20 Debye per molecule. Magnitude and polarity are consistent with an alpha-helical domain at the C-terminal end of substance P which is oriented normal to the surface of the membrane, and inserted so as to be inaccessible to the aqueous phase. Consistent measurements were obtained with anionic membranes at low substance P concentrations (10(-7)-10(-6) M; pH 7.2). They indicated electrostatic accumulation of the triply charged peptide on the surface of the membrane followed by hydrophobic interaction with the same parameters as for neutral membranes. The results agree with the membrane structure of substance P determined with infrared attenuated total reflection spectroscopy, circular dichroism measurements, and thermodynamic estimations.

Solvent dependency of rotational barriers in ethamivan and comparison to nikethamide.

Carbon-13 nuclear magnetic resonance (NMR) techniques were employed to examine the effects of solvent environment on rotational barriers in two drugs known to cause widespread stimulation in the mammalian central nervous system: ethamivan and nikethamide. Total NMR bandshape analysis was performed for the exchanging alkyl carbon resonances of these compounds as a function of temperature in six solvent systems: D2O, CH3OD, CH3CH2OD, CDCl3, C6D6 and CF3CH2OH. The rate constants for rotation about the amide bond obtained in this way were used to calculate free energy (delta G++), enthalpy (delta H++) and entropy (delta S++) of activation parameters for this process. Our results indicate that the magnitude of rotational barriers is affected markedly by (1) the size and polarity of the solvent molecules, and (2) the nature of the aromatic ring system attached to the amide grouping. Comparative interpretation of the thermodynamic parameters in light of the structures of nikethamide and ethamivan (in the various solvent systems examined) has further clarified the manner in which hydrogen bonding interactions between solvent molecules and the carbonyl oxygen of these analogues stabilize transition state conformers.

Conformation and orientation of regulatory peptides on lipid membranes. Key to the molecular mechanism of receptor selection.

The reaction of regulatory peptides with their membrane-bound receptors often occurs via a membrane-associated state of the peptide. From infrared studies on thin lipid films, we have shown that several ligands of the opioid kappa receptor and the neurokinin NK-1 receptor insert their message segments as an alpha-helix, more or less perpendicularly, into the membrane. The binding parameters for these membrane-associated states were determined from the capacitance minimization potential of lipid bilayers. A theory has been developed to account for the observed binding constants and the preferred conformation and orientation of these peptides. In contrast to the kappa and NK-1 receptors, ligands of the opioid mu and delta, and the neurokinin NK-2 and NK-3 receptors, are predicted not to form the inserted alpha-helical structure. A selection between the mu and delta (or NK-2 and NK-3) receptors appears to be made on the basis of an electrostatic gradient near the membrane surface. The molecular mechanism of receptor selection thus appears to be based to a large extent on the membrane-induced compartmentalization of ligands for the different receptors.

Cation dependence of opioid receptor binding supports theory on membrane-mediated receptor selectivity.

A quantitative analysis of the binding of dynorphin A-(1-9)-nonapeptide to the opioid Kappa-receptors of the guinea pig cerebellum [Paterson et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6216-6220] shows that changes in electrostatic surface accumulation of the ligand fully account for the observed suppression of binding by a series of univalent and divalent salts. Binding to mu- and delta-receptors, on the other hand, is subject to additional ion-specific effects. These observations support the membrane locations for the receptor sites proposed by the "membrane compartments" theory for opioid receptor selection.

Ligand/receptor interactions--the influence of the microenvironment on macroscopic properties. Electrostatic interactions with the membrane phase.

The heterogeneous environment in which ligand/receptor interactions occur often leads to complex binding behaviour. We consider here the ligand/membrane interaction, emphasizing the possibilities of electrostatic modulation of the overall binding characteristics. The binding of Substance P to neutral or negatively charged planar lipid bilayers was monitored using the capacitance minimization technique. The electrostatic attraction to the charged bilayer potentiates the interaction by more than two orders of magnitude and leads to a nonlinearity in the Scatchard plot of bound vs. bulk concentrations. The Boltzmann accumulation factor, along with the direct measurement of the surface potential, provides an easy explanation of the effect. The general importance of electrostatic accumulation (or repulsion) at surfaces is discussed and the concept applied to examples from the literature.

Transscaphoid fracture/dislocations treated with open reduction and Herbert screw internal fixation.

Six cases of dorsal transscaphoid perilunate fracture/dislocations and two cases of palmar transscaphoid lunate fracture/dislocations were treated by open reduction and internal fixation of the scaphoid with a Herbert screw. Supplemental Kirschner wire stabilization of the carpus was also used in the two cases of palmar transscaphoid lunate fracture/dislocation. All patients were male, with an average age of 23.6 years. The follow-up averaged 18.9 months. A clinical evaluation scoring system assessing pain, ability to function in an occupation, range of motion, grip strength, and radiographic appearance of the wrist was used. Based on this system the scoring of the six cases of dorsal transscaphoid perilunate fracture/dislocations that were treated was three excellent, one good, and one fair result, while the scoring of both palmar transscaphoid lunate fracture/dislocations showed poor results.

Solvent interactions with N, N-dialkylnicotinamides and their effects on rotational barriers.

Carbon-13 nuclear magnetic resonance techniques were employed to examine the effects of solvent environment on rotational barriers in a series of molecules structurally-related to the analeptic, nikethamide: N,N-dimethylnicotinamide, N,N-di-n-propylnicotinamide, and 1-nicotinoyl piperidine. Total bandshape analysis was performed for the exchanging alkyl carbon resonances of these compounds as a function of temperature in four solvent systems: D2O, CH3OD, CH3CH2OD and CDCl3. The rate constants for rotation about the amide bond obtained in this way were used to calculate free energy (delta G), enthalpy (delta H) and entropy (delta S) of activation parameters for this process. Our results indicate that rotational barriers are less affected by the nature of the alkyl chain attached to the amide nitrogen than by the size and polarity of the solvent molecules. Interpretation of the thermodynamic parameters in light of both nikethamide analogue structure and solvent type has further clarified the manner in which hydrogen bonding interactions between solvent molecules and the carbonyl oxygen of these analogues stabilize transition state conformers.

The effect of solvent polarity upon rotational barriers in nikethamide.

In summary, dynamic nuclear magnetic resonance techniques were used to study the hindered internal rotation of the amide bond of the analeptic nikethamide. The rotatory motion of this bond was studied in a series of solvents of increasing polarity: CDCl3, CH3(CH2)3OD, CH3CH2OD, CH3OD and D2O. Motion about the amide bond was increasingly hindered in direct proportion to solvent polarity, correlating with enhanced hydrogen bond formation between nikethamide and the more polar solvent molecules. Diethylamide group motion would be expected to affect binding of the carbonyl oxygen to cholinergic receptor sites. The degree to which association to a receptor site can be affected by this rotatory motion may vary from 0 to 4 kcal/mole, the variability being entirely dependent upon the polarity of the binding site. An increase in rotamer lifetime, corresponding to a more polar environment, would be expected to enhance the kinetics of nikethamide association to the receptor site.

The caloric and intoxicating properties of fluid intake as components of stress-induced ethanol consumption in rats.

Rats housed continuously in a test environment for 25 days were offered water, 5% v/v ethanol, 10% v/v ethanol, and propylene glycol at 7.5% w/v. The propylene glycol concentration represented a caloric midpoint between the 5 and 10% ethanol. After 10 baseline sessions, during which preference for the four solutions was shown to be statistically equal, shock schedules were introduced. The consumption of ethanol at both concentrations showed significant peaks for the interval immediately following 12 min of shock each hour. Intake peaks were not observed for the water or propylene glycol. Baseline blood alcohol levels were negligible, but blood levels under shock averaged 143 mg/dl and ranged from 45.0 mg/dl to 295.0 mg/dl. After the shock sessions were terminated, baseline drinking indicated no significant change in preference relative to pre-shock baselines. but there was an elevation in preference for 5% ethanol relative to the other fluids. The preference for propylene glycol or water did not change.

Shock induced ethanol consumption in rats.

Two experiments are presented which describe the temporal and volumetric changes in ethanol consumption by rats exposed to recurring schedules of inescapable random shock. The animals in Experiment 1, which had a choice between ethanol and water, increased their voluntary ethanol consumption immediately after the shock schedule. The postshock changes occurred with both 5% and 10% V/V ethanol, were specific to the presence of shock and were not reflected by measures of total daily ethanol intake. Experiment 2 exposed rats to extended 22 hr stress sessions, during which each animal had four simultaneous fluid choices available: water, saccharin 0.1% W/V, ethanol 5% V/V, and ethanol 10% V/V. Temporal intake patterns for both 5% and 10% ethanol showed pronounced peaks for the interval immediately following the shock schedule. A shift of intake from 5% to 10% ethanol was also demonstrated with increasing time under shock, while saccharin and water intake decreased. The results are interpreted as a relationship between voluntary ethanol intake and escape from the consequences of stress.

The temporal and volumetric components of stress induced drinking in rats.

Two experiments are presented which describe the temporal and volumetric changes in ethanol consumption with rats exposed to recurring schedules of inescapable random shock stress. The animals in Experiment I had an ethanol and water choice and the data demonstrated cumulative increases in voluntary ethanol consumption which occurred immediately after the shock schedule. The post shock changes occurred with both 5% and 10% V/V ethanol, were specific to the appearance of shock and were not reflected by measures of total daily ethanol intake. Experiment II exposed rats to extended 22 hr. stress sessions and each animal had four simultaneous fluid choices available: water, saccharin. 1% W/V, ethanol 5% V/V, and ethanol 10% V/V. Temporal intake patterns for both 5% and 10% ethanol showed pronounced peaks for the interval immediately following the shock schedule. A progressive shift of intake from 5% to 10% ethanol was also evidenced with increasing time under shock, while saccharin and water intake decreased. Blood ethanol levels during shock increased significantly from baseline for all animals and ranged from 30 to 162 mg/100 ml. The results are interpreted as a relationship between voluntary ethanol intake and escape from the consequences of stress.