Biomedical subjects
Publications and source records attributed to L Toll.
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Ketomethylene containing peptide analogs, modeled after a snake venom pentapeptide, have been shown to be potent angiotensin converting enzyme inhibitors. Although the most potent compounds are up to five times more potent than captopril in inhibiting angiotensin converting enzyme activity, they are relatively weak inhibitors of [3H]captopril binding to membrane bound angiotensin converting enzyme. This indicates that inhibition of [3H]captopril binding and enzymatic activity is due to binding to distinct sites. These results suggest that the inhibitors bind to an additional site on the enzyme distinct from the captopril binding site.
A series of 3-methyl-3-(m-hydroxyphenyl)piperidines with N-substituent variations have been synthesized and resolved, and an X-ray crystal structure of one analogue was determined. The compounds have been characterized, pharmacologically, by detailed opiate receptor binding studies and determination of in vivo analgesia and opiate antagonism. The results indicate that all compounds bind with high selectivity and moderate affinity to mu-receptors with no qualitative difference between enantiomeric pairs. By contrast a striking difference in activities is found, with the (-) enantiomers being pure agonists and the (+) enantiomers having both agonist and antagonist activity. The effect of N-substituents on relative agonist and antagonist potency does not mimic that of fused ring opiates with the N-phenethyl compound, the most potent antagonist. These results together with the X-ray structure obtained suggest that agonist and antagonist activity is initiated by a bimodel binding of the compounds in two different orientations at the mu-receptor site.
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Detailed energy-conformation studies of a linear (DTLET) and four cyclic (DPDPE, DPLPE, DPLCE,DCLPE), delta-selective opioid peptides were combined with computer assisted detailed receptor binding studies. The results of these studies have allowed the identification of a low energy conformer common to all of these analogs which could be responsible for their high affinity delta-receptor binding. This conformer contains multiple intramolecular H-bonds and is very different from the beta-II type structure previously postulated to lead to high affinity mu-receptor binding. This mu-binding conformer was found either to have higher energies or be greatly distorted in these delta selective analogs.
In this study we report the systematic investigation of conformational profiles and electronic properties of a series of analogs of the mu-selective opioid peptide, morphiceptin, together with receptor-binding studies of some of these analogs. In particular, we have investigated the effect of: substitution in the second position, substitution of D-Pro for L-Pro in the second and fourth positions, the addition of an N-methyl group at the third position, and variations in the carboxyl end group. The binding studies confirm the preference of these analogs for mu- versus delta-receptor-binding sites and also indicate differences in mu-receptor affinity among them. The theoretical analyses allow identification of a preferred conformation leading to high mu-receptor affinity and two reliable indicators of relative mu-receptor affinities. These properties are the energy required to obtain the candidate mu-binding conformer and the extent to which each compound overlaps with the highest affinity compound in this conformation. In addition, electronic interactions deleterious to high affinity mu-binding are identified.
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A series of 4-(m-OH phenyl)-piperidine analogs with R4 = methyl or t-butyl and NR = methyl, allyl and phenethyl have been synthesized; and their receptor affinities, in vivo analgetic agonism and antagonism, and energy-conformational profiles determined. These analogs bind selectively and with moderate to high affinity to opioid mu-receptors. Binding in their preferred phenyl axial conformation appears to lead to meperidine-like agonism. In addition, for some R4 = methyl but not t-butyl compounds, binding of an energy-accessible phenyl equatorial conformation produces antagonism.
The techniques of theoretical chemistry have been used to elucidate the molecular properties and modes of receptor binding that modulate receptor affinity and antagonist activity of the beta-carbolines, a class of potent benzodiazepine antagonists. Six analogs were chosen in order to investigate the role of the amine (NH) group, the aromatic nitrogen, and the C3-substituent in determining receptor affinities. Electrostatic potential mapping and characterization of explicit drug-receptor interactions have led to the hypothesis that simultaneous interaction of a model cationic arginine site with the N2 and C3-substituents could play a key role in determining receptor affinities. The electron-withdrawing effects of C3-substituents on the amine nitrogen appear less important, though interactions of these groups with an anionic glutamate or aspartate site could also occur at the receptor. Similarly, stacking interactions with neutral or cationic aromatic residues such as tryptophan or protonated histidine could occur, but do not appear to be determinants of the relative receptor affinity of the beta-carbolines.
A number of beta-carboline analogs have been obtained or synthesized, and their in vitro receptor affinities and in vivo antagonist activities determined. The choice of analogs was made in order to explore the importance of the N9 -H, the aromatic nitrogen and the C3-ester moiety for high-receptor affinity and antagonist activity of this class of benzodiazepine antagonist. Among the analogs investigated, we describe the properties of 3-cyano-beta-carboline (1h), the first potent beta-carboline antagonist without a carbonyl at the C3-position. The results obtained indicate: (1) Specific interactions of the C3-substituent with key cationic receptor sites rather than electron-withdrawing properties are important for high-receptor affinity and antagonist activity. (2) Specific in-plane interactions of the aromatic nitrogen with a cationic receptor site, rather than stacking with neutral aromatic residues of the receptor are also important for high affinity and antagonist activity. (3) While the presence of an N9 -H enhances receptor affinity, interaction with an anionic receptor site does not appear essential for antagonist activity.
The presence of benzodiazepine receptor heterogeneity was investigated on whole rat brain membranes, at 0 degree C, using computerized, weighted nonlinear least-squares regression analysis. Data from [3H]flunitrazepam and [3H] beta-carboline ethyl ester self and cross-competition studies were analyzed simultaneously with data from the inhibition of both labeled ligands by various known and novel benzodiazepine receptor ligands. The binding model which best fits the data indicated the presence of at least two independent binding sites. The benzodiazepines flunitrazepam and 2'-Cl-diazepam showed a small difference in affinity at the two sites. The beta-carbolines and Cl 218,872 showed a larger difference in affinity, and had higher affinities at the lower affinity site for the benzodiazepines. Analogous experiments could be useful in the determination of the effect of GABA on benzodiazepine receptor affinities.
Pyrazolo[1,5-a]pyrimidines (PZP) have been reported to be specific anxiolytic agents which do not potentiate ethanol or barbiturates. To further investigate these compounds, three of the most promising analogs were synthesized and a tritium-labeled analog of one of them prepared by a new synthetic procedure. These analogs did not compete with [3H]flunitrazepam or [3H]beta-carboline ethyl ester binding nor did they potentiate the [3H]flunitrazepam binding. Receptor binding studies with the [3H]PZP revealed a low affinity receptor site, distinct from that of the benzodiazepines, but with only a small fraction (20%) of specific binding. Behavioral tests using three different animal models for anxiety: muricide, approach/avoidance conflict and two-chamber exploration tests gave conflicting results, positive in the first and negative in the latter two. Furthermore, these compounds were found not to be antagonists of diazepam's anticonvulsant activity. Taken together, these results, while provocative, do not support evidence that these analogs are promising specific anxiolytic agents.
The computer curve fitting program LIGAND has been used to simultaneously analyze 50 competition experiments using five labeled opioid ligands. The results describe four or five distinct sites, although the maximum number of sites cannot be determined. A site with high affinity for all the compounds tested, similar to the mu 1 site described by Pasternak, was apparent in 4 and 5-site models.
A peptide analogue of Leu-enkephalin was synthesized in which the amide linkages between Tyr-Gly and Gly-Gly were replaced by ketomethylene groups. The resulting analogue, 12, had 1/4000th and 1/2400th the opiate receptor binding activity of Leu-enkephalin when (3H) [D-Ala2,D-Leu5]enkephalin and (3H)naloxone, respectively, were used as tritiated ligands. When tested for analgesia in mice by the tail-flick assay, 12 produced analgesia in 50% of the mice tested at a dose of 24.3 micrograms/mouse (icv), while the ED50 of Leu-enkephalin is 240 micrograms/mouse (icv). At a dose of 40 micrograms/mouse (icv) or higher, 12 caused convulsions in a dose-dependent manner. No analgesia was observed after intravenous (iv) administration of 240 micrograms/mouse of 12.
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Computer-assisted analysis of competitive binding studies of [3H]-naloxone, [3H]-DADL, morphine and meperidine in the presence and absence of 100 mM NaCl have been made. The results are most consistent with a 3-site model of opiate receptors and indicate that in the presence of Na+ naloxone changes from a mu-specific (81%) to a more kappa-specific (63%) ligand. The large IC50 shift in inhibition of [3H]-naloxone by three agonists, DADL, morphine and meperidine is found not to be due to a decrease in affinity at any receptor site but rather to a low affinity of these agonists at "kappa" in the presence of Na+.
Receptor-binding studies with [3H]-naloxone and [3H]-DADL have been made for a series of 3-arylpiperidines known to have moderate analgesic agonist and antagonist activities. All analogues were found to bind selectively to the opiate "mu" receptor with affinities consistent with their in vivo activity. However, Na+ did not appreciably alter their IC50 values in competition with [3H]-naloxone, independent of their relative agonist/antagonist activity. This anomaly is consistent with our previous theoretical studies of these 3-phi piperidines which postulated different modes of agonist and antagonist receptor binding than that for fused ring opiates. Computer-assisted analysis of the binding shows that this low IC50 ratio is probably due to a greatly enhanced affinity of these analogues at a third site in the presence of Na+, behavior parallel to that of naloxone.
PC12 cells, a clonal rat pheochromocytoma cell line, possess voltage-dependent calcium channels that bind the high affinity dihydropyridine calcium antagonist [3H]nitrendipine and other calcium channel blockers. The binding is temperature-dependent and saturable, and shows no cooperativity. The calcium antagonists inhibit potassium-induced 45Ca uptake into the cells with approximately the same potencies as those needed to inhibit [3H]nitrendipine binding to cell membranes. The affinity of these compounds for the PC12 cell calcium channel is slightly lower than that reported for binding to brain and heart. Potassium-stimulated 45Ca uptake into PC12 cells is rapid, being half-maximal within 30 s at 20 degrees C. Different classes of calcium antagonists seem to block calcium flux at different sites on the calcium channel. A lower limit of the rate of calcium movement through a single channel is given. PC12 cells seem to be a suitable model system for the study of the pharmacology and biochemistry of the voltage-dependent calcium channel.