An NMR study of conformations of substituted dipeptides in dodecylphosphocholine micelles: implications for drug transport.

Efficient transport of intact drug (solute) across the intestinal epithelium is typically a requirement for good oral activity. In general, the membrane permeability of a solute is a complex function of its size, lipophilicity, hydrogen bond potential, charge, and conformation. In conjunction with theoretical/computational and in vitro drug transport studies, seven dipeptide (R(1)-D-Xaa-D-Phe-NHMe) homologues were each dissolved in a micellar d(38)-dodecylphosphocholine solvent system. In this homologous dipeptide series, factors such as size, lipophilicity, hydrogen-bond potential, and charge were either tightly controlled or well-characterized by other methods in order to investigate by nmr how conformational factors relate to transport. Nuclear Overhauser effect spectroscopy experiments and amide-NH-H(2)O chemical exchange rates showed that the five more lipophilic dipeptides were predominately associated with micelle, whereas the two less lipophilic analogues were not. Rotating frame nuclear Overhauser effect spectroscopy derived interproton distance restraints for each analogue, along with (3)J(HH)-derived dihedral restraints, were used in molecular dynamics/simulated annealing computations. Our results suggest that-other factors being equal-flexible dipeptides having a propensity to fold together nonpolar N- and C-terminal moieties allow greater segregation of polar and nonpolar domains and may possess enhanced transport characteristics. Dipeptides that were less flexible or that retained a less amphiphilic conformation did not have comparably enhanced transport characteristics. We suggest that these conformational/transport correlations may hold true for small, highly functionalized solutes (drugs) in general.

Field-based similarity forcing in energy minimization and molecular matching.

A new field-based similarity forcing procedure for matching conformationally-flexible molecules is presented. The method extends earlier work on similarity matching of molecules based upon the program MIMIC, by directly coupling a similarity function to a molecular mechanics force field. In this way conformational energetics are fully accounted for in the similarity matching process. Simultaneous similarity/conformational searches can then be undertaken within a Monte Carlo or molecular dynamics framework. Here, a Monte Carlo approach is used to provide a simple example of two HIV-1 reverse transcriptase inhibitors, nevirapine and alpha APA, that illustrates the basic characteristics of the method and suggests areas for further investigation.

Classification and prediction of beta-turn types.

Although a beta-turn consists of only four amino acids, it assumes many different types in proteins. Is this basically dependent on the tetrapeptide sequence alone or is it due to a variety of interactions with the other part of a protein? To answer this question, a residue-coupled model is proposed that can reflect the sequence-coupling effect for a tetrapeptide in not only a beta-turn or non-beta-turn, but also different types of a beta-turn. The predicted results by the model for 6022 tetrapeptides indicate that the rates of correct prediction for beta-turn types I, I', II, II', VI, and VIII and non-beta-turns are 68.54%, 93.60%, 85.19%, 97.75%, 100%, 88.75%, and 61.02%, respectively. Each of these seven rates is significantly higher than 1/7 = 14.29%, the completely randomized rate, implying that the formation of different beta-turn types or non-beta-turns is considerably correlated with the sequences of a tetrapeptide.

Conformationally constrained renin inhibitory peptides: cyclic (3-1)-1-(carboxymethyl)-L-prolyl-L-phenylalanyl-L-histidinamide as a conformational restriction at the P2-P4 tripeptide portion of the angiotensinogen template.

Interest in conformationally constrained peptides as potential inhibitors of renin led us to examine an N-terminal cycle of linear renin inhibitory peptides. A cyclic structure was prepared by joining the N-terminal proline at the P4 site to the imidazole ring of histidine at the P2 site via a carboxymethylene fragment. An efficient synthetic route to this 14-membered macrocycle was developed and this N-terminal cyclic tripeptide could be readily incorporated into renin inhibitory peptides. Monte Carlo molecular modeling methods were used to generate bound conformations of a representative inhibitor in a model of the renin active site, suggesting possible modes of binding of these inhibitors to renin. Two representative compounds that contain this 14-membered macrocycle were evaluated for their inhibitory activities against human plasma renin and they were found to exhibit very high binding affinity with IC50 values in the nanomolar and subnanomolar range.

Synthesis, metabolism, and pharmacological activity of 3 alpha-hydroxy steroids which potentiate GABA-receptor-mediated chloride ion uptake in rat cerebral cortical synaptoneurosomes.

Certain 3 alpha-hydroxy steroids have recently been shown to bind to the gamma-aminobutyric acid (GABA) receptor gated chloride ion channel with high affinity and to potentiate the inhibitory effects of GABA when measured both in vitro and in vivo. In the present study, a series of natural and synthetic 3 alpha-hydroxy steroids were tested for their ability to potentiate GABA-receptor-mediated chloride ion (Cl-) uptake into cerebral cortical synaptoneurosomes. The naturally occurring metabolites 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydroDOC) were found to be the most active in augmenting GABAA-receptor-mediated Cl- uptake. Pharmacological activity was reduced in the corresponding isomers with the 5 beta-pregnane configuration and by some, but not all, modifications of the side chain. The ability of these steroids to potentiate muscimol-stimulated Cl- uptake is lost by acetylation at C3, introduction of unsaturation at C9(11), inversion to the 3 beta-hydroxy isomer, or inversion of configuration at C17. A facile procedure is reported for the synthesis of unlabeled and tritium-labeled allopregnanolone and allotetrahydroDOC. The 9 alpha,11 alpha,12 alpha-3H-labeled derivatives of allopregnanolone and allotetrahydroDOC were used to identify the distribution and metabolic products of these active steroids. Uptake of the more hydrophobic [3H]allopregnanolone into brain was significantly greater than that of [3H]allotetrahydroDOC. The principal 3H-labeled metabolites recovered from brain were the 3-ketone derivatives of allopregnanolone and allotetrahydroDOC, which are both inactive on GABA-receptor-mediated Cl- flux. Molecular modeling of the active steroids based on quantitative structure-activity relationships provides evidence to support the stereospecificity of the binding interactions and suggests that there may be more than one type of steroid binding site associated with the GABAA-receptor-mediated chloride ionophore.

Analgesic activities of spinal cord substance P antagonists implicate substance P as a neurotransmitter of pain sensation.

Substance P (SP) injected into intraspinal (i.s.) spaces caused mice to vigorously scratch and bite their skins in an apparent reaction to a perceived cutaneous sensation. The scratching behavior was similar to the reciprocal hindlimb-scratching syndrome (RHS) described for intracranial (i.c.) SP injections. Radiotracer experiments, as well as potency and latency measurements, demonstrated that SP-induced scratching, whether induced by the i.c. or i.s. route, was due to SP receptor stimulation in the cervicothoracic cord. Similarly, biting was due to SP stimulation of the lumbosacral spinal cord. Mice coated with capsaicin, an irritant chemical, scratched and bit the coated areas in a manner similar to animals injected with i.s. SP. Standard analgesics depressed this scratching behavior elicited by topical capsaicin. Non-analgesic drugs, with the exception of amphetamine, did not affect capsaicin-induced pain. It is concluded that i.s. SP induces a painful sensory experience. Some piperazinone derivatives of substance P's C-terminal hexapeptide are shown to specifically antagonize the scratching induced by i.s. SP with little or no effect on motor behavior. These antagonists depressed scratching elicited by topical capsaicin and were analgesic on the hot-plate test. It is concluded that SP is a natural neurotransmitter for pain and that antagonism of endogenous SP systems causes analgesia.