QSAR analysis of Delta(8)-THC analogues: relationship of side-chain conformation to cannabinoid receptor affinity and pharmacological potency.

A novel quantitative structure-activity relationship (QSAR) for the side-chain region of Delta(8)-tetrahydrocannabinol (Delta(8)-THC) analogues is reported. A series of 36 side-chain-substituted Delta(8)-THCs with a wide range of pharmacological potency and CB1 receptor affinity was investigated using computational molecular modeling and QSAR analyses. The conformational mobility of each compound's side chain was characterized using a quenched molecular dynamics approach. The QSAR techniques included a modified active analogue approach (MAA), multiple linear regression analyses (MLR), and comparative molecular field analysis (CoMFA) studies. All three approaches yielded consistent results. The MAA approach applied to a set of alkene/alkyne pairs identified the most active conformers as those with conformational mobility constrained within an approximately 8 A radius. MLR analyses (restricted to 15 hydrocarbon side-chain analogues) identified two variables describing side-chain length and terminus position that were able to fit the pharmacological data for receptor affinity with a correlation coefficient for pK(D) of 0.82. While chain length was found to be directly related to receptor affinity, the angle made by the side chain from its attachment point to its terminus (angle defined by C3-C1'-side-chain terminus carbon, see Figure 1) was found to be inversely related to affinity. These results suggest that increased side-chain length and increased side-chain ability to wrap around the ring system are predicted to increase affinity. Therefore, the side chain's conformational mobility must not restrict the chain straight away from the ring system but must allow the chain to wrap back around toward the ring system. Finally, the CoMFA analyses involved all 36 analogues; they also provided data to support the hypothesis that for optimum affinity and potency the side chain must have conformational freedom that allows its terminus to fold back and come into proximity with the phenolic ring.

Manipulation of the tetrahydrocannabinol side chain delineates agonists, partial agonists, and antagonists.

Structure-activity relation studies have established that the alkyl side chain in tetrahydrocannabinol (THC) plays a crucial role in the activation of the cannabinoid receptor. Unfortunately, the flexible nature of this side chain has hampered efforts to elucidate the precise nature of the interaction of THC with its receptors. Therefore, a series of analogs with structurally restrained side chains of varying length was synthesized and evaluated for pharmacological potency in mice and for receptor affinity. The introduction of cis double bonds inserted rigid angles, whereas triple bonds developed regions of planarity. Receptor affinity for the acetylenic and saturated side chains were the same, whereas double bond substitution increased affinity 10-fold. Moreover, the relationship between receptor affinity and potency was 10-fold less than that of Delta(8)-THC in the case of some acetylenic derivatives, whereas changing the triple bond to a double bond restored the potency/affinity ratio. Additionally, an acetylene at C2-C3 in the octyl and nonyl side chains favored antinociception by as much as 70-fold. Surprisingly, several high-affinity acetylenic derivatives, especially those with cyano substitutions at the terminus of the side chain, were partial agonists or were inactive. Some of these low-efficacy, high-affinity ligands elicited antagonistic activity. The finding that manipulations of the side chain produces high- affinity ligands with either antagonist, partial agonist, or full agonist effects reveals a critical structural feature for receptor activation.

An investigation into the structural determinants of cannabinoid receptor ligand efficacy.

1. A number of side-chain analogues of delta8-THC were tested in GTPgammaS binding assay in rat cerebellar membranes. O-1125, a saturated side-chain compound stimulated GTPgammaS binding with an Emax of 165.0%, and an EC50 of 17.4 nM. 2. O-1236, O-1237 and O-1238, three-enyl derivatives containing a cis carbon-carbon double bond in the side-chain, stimulated GTPgammaS binding, acting as partial agonists with Emax values ranging from 51.3-87.5% and EC50 values between 4.4 and 29.7 nM. 3. The stimulatory effects of O-1125, O-1236, O-1237 and O-1238 on GTPgammaS binding were antagonized by the CB1 receptor antagonist SR 141716A. The K(B) values obtained ranged from 0.11-0.21 mM, suggesting an action at CB1 receptors. 4. Five-ynyl derivatives (O-584, O-806, O-823, O-1176 and O-1184), each containing a carbon-carbon triple bond in the side-chain, did not stimulate GTPgammaS binding and were tested as potential cannabinoid receptor antagonists. 5. Each -ynyl compound antagonized the stimulatory effects of four cannabinoid receptor agonists on GTPgammaS binding. The K(B) values obtained, all found to be in the nanomolar range, did not differ between agonists or from cerebellar binding affinity. 6. In conclusion, alterations of the side-chain of the classical cannabinoid structure may exert a large influence on affinity and efficacy at the CB1 receptor. 7. Furthermore, this study confirms the ability of the GTPgammaS binding assay to assess discrete differences in ligand efficacies which potentially may not be observed using alternative functional assays, thus providing a unique tool for the assessment of the molecular mechanisms underlying ligand efficacies.

The effects of calcium site occupancy and reagent length on reactivity of calmodulin lysyl residues with heterobifunctional aryl azides. Mapping interaction domains with specific calmodulin photoprobe derivatives.

The relationship of structural and functional moieties on calmodulin is important in all venues of cell activity. In this study, we investigate the effect of lysine modification on calmodulin function. Azidosalicylate reagents containing different "linker arm" lengths, between the photoactive terminus and an amine-reactive N-hydroxysuccinimidyl ester moiety were used to modify calmodulin lysines at three different positions in a calcium-dependent manner. The short cross-linker, (ASNE-2 (where ASNE represents azidosalicylate N-hydroxysuccinimidyl ester), modifies Lys-75, whereas the longer reagent, ASNE-6, modifies lysines 21, 75, and 94. The modification of these different lysines is shown to be calcium-dependent. At 1-100 microM levels of calcium, only Lys-94 is modified, suggesting that modification of this residue is directed by both the binding of calcium to calcium-binding loops III and IV and the hydrophobic pocket exposed between these two loops as a result of calcium binding. At higher calcium concentrations (> 200 microM), where sites I and II become filled, modification of Lys-21 or Lys-75 also was observed. All the modified calmodulins were able to stimulate 3',5'-cyclic-nucleotide phosphodiesterase fully although the Kact for the Lys-75 and Lys-21 derivatives increased 10- and 50-fold, respectively. None of the modifications affected the activation of erythrocyte plasma membrane Ca(2+)-ATPase. Only the ASNE-6 Lys-75 derivative showed efficient (40%) photocross-linking to the Ca(2+)-ATPase. The ASNE-2 Lys-75 derivative as well as the ASNE-6 Lys-21 and Lys-94 derivatives did not show efficient calcium-dependent photocross-linking to this enzyme.

Application of an N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosine-substituted peptide as a heterobifunctional cross-linking agent in a study of protein O-glycosylation in yeast.

In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, [125I]-N-(4-azido-2,3,5,6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-T hr-Ser-Val ([125I]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosine, and resin-bound Asn-Pro-Thr(tBu)-Ser(tBu)-Val. When this modified hexapeptide substrate was incubated with O-mannosyltransferase preparations, the hexapeptide was an acceptor of [14C]-mannose from dolichol phosphate-[14C]mannose. After partially purifying the O-mannosyltransferase and photolabeling these enzyme preparations with [125I]azidoTyr-peptide, a ca. 82-kDa protein was shown to be the only apparent photolabeled protein that was protected by unmodified hexapeptide. This ca. 82-kDa protein may be the catalytic subunit of the O-mannosyltransferase. The susceptibility of the N-(4-azido-2,3,5,6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.

Photolysis of 3-aryl-3-(trifluoromethyl)diazirines: a caveat regarding their use in photoaffinity probes.

The photolysis of 3-(4-tolyl)-3-(trifluoromethyl)diazirine in the presence of benzene, methanol, carbon tetrachloride, cyclohexane, triethylsilane, or diethylamine led to photoproducts consistent with the intermediacy of a singlet carbene. In the case of diethylamine, the photoinsertion into the N-H bond of diethylamine produced the expected adduct, 1-(diethylamino)-2,2,2-trifluoro-1-(4-tolyl)ethane. However, the base-catalyzed elimination of hydrogen fluoride from this adduct afforded an enamine, alpha-(diethylamino)-beta,beta-difluoro-4-methylstyrene, and the subsequent hydrolysis of this enamine furnished diethylamine and 2,2-difluoro-1-(4-tolyl)ethanone. This elimination and hydrolysis sequence effectively reversed the photoinsertion process. A similar photoinsertion and hydrolysis process using 3-(4-n-octylphenyl)-3-(trifluoromethyl)diazirine also produced 2,2-difluoro-1-(4-n-octylphenyl)ethanone in modest yield. These results suggest that the photoinsertion products from 3-aryl-3-(trifluoromethyl)-diazirines in biological systems may suffer similar fates limiting, in part, their utility in obtaining primary sequence data.

The photochemistry of iodo, methyl and thiomethyl substituted aryl azides in toluene solution and frozen polycrystals.

The photolysis of para-methyl and para-thiomethylphenylazide at 77 K produces the corresponding triplet nitrenes which can be detected by electron paramagnetic resonance (EPR) spectroscopy. Photolysis of these azides in frozen toluene at 77 K leads to insertion of the nitrene into a benzylic C-H bond of the matrix in modest yields. Photolysis of iodinated aryl azides under these conditions does not produce triplet nitrenes that can be detected by EPR spectroscopy. In contrast to the para-methyl and para-thiomethyl substituted phenyl nitrenes, photo-induced coupling of iodo-substituted phenyl nitrenes to toluene proceeds in very poor yield.

Heterobifunctional cross-linking agents incorporating perfluorinated aryl azides.

New heterobifunctional cross-linking reagents were developed that possess a photoactive tetrafluorinated phenyl azide as the photoactive terminus and a chemically reactive succinimidyl ester as the electrophilic terminus. These reagents, succinimidyl N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosinate (9) and succinimidyl 2-(4-azido-2,3,5,6-tetrafluorophenyl)thiazole-4-carboxylate (15), were designed to possess either an 125I or 35S radiolabel, respectively. In a biochemical study, the latter reagent was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was photochemically cross-linked, in a calcium-dependent manner, to the porcine erythrocyte plasma membrane Ca2+, Mg2(+)-ATPase. Densitometry scans of the gel indicated a reproducible 22% cross-linking of the CaM with one of the Ca2+,Mg2(+)-ATPase bands. Since the purification of the Ca2+,Mg2(+)-ATPase results in micelles having Ca2+,Mg2(+)-ATPase with its CaM binding site oriented both to the inside and outside of the micelle, the amount of Ca2+,Mg2(+)-ATPase available for cross-linking was reduced by approximately half, suggesting that the actual cross-linking efficiency was on the order of 40%.

Photoaffinity heterobifunctional cross-linking reagents based on N-(azidobenzoyl)tyrosines.

New heterobifunctional cross-linking reagents that possessed a photoactive terminus, an electrophilic terminus, and a linking arm between the two termini that had a radiolabeled, enzymatically cleavable bond were synthesized. In a model study, succinimidyl N-[N'-(4-azidobenzoyl)tyrosyl]-beta-alanate (16A) was coupled to n-butylamine (a Lys surrogate), iodinated, and cleaved with chymotrypsin in the presence of tyrosylamide to afford the desired adduct (N-(N'-(4-azidobenzoyl)-3-iodotyrosyl)tyrosinamide, thereby demonstrating the feasibility of the enzymatic cleavage. In a biochemical study, succinimidyl N-[N'-(3-azido-5-nitrobenzoyl)tyrosyl]-beta-alanate (16C) was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was successfully photo-cross-linked, in a calcium-dependent manner, to the human erythrocyte plasma membrane Ca2+,Mg2(+)-ATPase and to a synthetic fragment (M13) containing the CaM-binding region of myosin light-chain kinase. In the latter case, densitometry readings indicated 20% cross-linking efficiency.

Occult bacteremia in the emergency department: diagnostic criteria for the young febrile child.

We studied a selected series of febrile infants (N = 201) in an attempt to prospectively identify risk factors for bacteremia. Infants with fever less than 39.4 C, vomiting and diarrhea, croup, or viral exanthem or enanthem were not included. Twenty-one infants (9.5%) had positive blood cultures despite the initial judgment of their physician that only viral illness or localized bacterial infection existed. WBC count of more than 15,000 correlated with bacteremia, with a sensitivity of 0.71 and a specificity of 0.73. Extensive multivariate linear regression analysis attempting to increase predictive values was completed. The combination of fever higher than 39.4 C present for more than 12 hours and absolute polymorphonuclear count of more than 9,000 cells/mm3 had a sensitivity of 0.62 and a specificity of 0.78 for bacteremia. Descriptive statistics for groups with and without bacteremia are summarized. We have defined prospectively a population of infants with a high probability of bacteremia and a lower probability of viral illness. Identification of such a group is useful to the emergency physician because early antibiotic therapy may lessen morbidity and mortality. We conclude that an easily obtained data base may be useful in the prospective identification of those at risk for bacteremia.

Pediatric carbon monoxide toxicity.

We report a series of 28 pediatric carbon monoxide exposures. Sixteen patients (57%) were judged to have potentially toxic carboxyhemeglobin (COHb) levels (greater than 15%). In this group the range of COHb blood levels was 16.7% to 44.0% (mean = 26.5%). An unusually high incidence of syncope (56.3%, 9/16) and lethargy (68.7%, 11/16) was observed. Every patient with a COHb level greater than or equal to 24% experienced syncope; a marked departure from published adult values. Lethargy was reported at a mean COHb concentration of 25.9%. All patients with levels over 25% COHb, neurologic findings, acidosis, or syncope were considered candidates for hyperbaric oxygen therapy (HBOT). No morbidity from HBOT was encountered. Eighty-nine percent (25/28) of the patients are reportedly doing well, with no late sequelae identified. Delayed neurologic sequelae in three patients include chronic headaches, memory difficulties, or decline in school performance.

Occult bacteremia in the emergency department.

The problem of occult bacteremia in infants and children has been reviewed. The condition is not uncommon and may occur in 3% to 10% of infants and children six months to two years of age who present with temperature greater than or equal to 38.3 C. Meningitis may complicate bacteremia in 4.5% to 8.5%, regardless of identification of a focus of infection. Early identification and proper therapy are essential and often incumbent on the emergency physician.

Effects of the unsaturation of dietary fat and of arachidonate supplementation on cholesterol pool expansion in the guinea pigs.

We have studied the effects of methyl arachidonate supplementation on the lipid metabolism of guinea pigs fed cholesterol. Four groups of guinea pigs were fed a purified diet containing 9.5% hydrogenated coconut oil (HCNO), a highly saturated fat with or without the addition of 1% cholesterol, for 15 weeks. One half of the animals fed the control and the cholesterol-containing diets were supplemented with 15 mg methyl arachidonate three times per week. Supplementation with methyl arachidonate did not alter the concentration of plasma total (TC) or unesterified (FC) cholesterol, erythrocyte cholesterol and plasma phospholipid or the ratio of plasma FC/TC. Accumulation of cholesterol in the major organs of the cholesterol-fed groups was also unchanged. In both control and cholesterol-fed groups, methyl arachidonate decreased the proportion of oleic (18:1) and linoleic acids (18:2) and increased arachidonic acid (20:4) content of plasma and liver phospholipid. A comparison between the results of this study and studies using cottonseed oil showed that the type of dietary fat modifies the effects of cholesterol: plasma cholesterol levels were higher and liver cholesterol storage was lower in animals fed the saturated fat than in those fed the fat rich in polyunsaturated fatty acids (PUFA). Furthermore, in spite of similar changes in erythrocyte cholesterol content and shape abnormalities, no overt hemolytic anemia was observed in the groups fed cholesterol and saturated fat, in contrast to those fed cholesterol + PUFA-containing fat. We conclude that in guinea pigs supplementary methyl arachidonate had no hypocholesterolemic effect at the levels we fed, that circulating cholesterol levels are not a measure of cholesterol accumulation by organs and that the decrease of serum cholesterol in response of PUFA is due in part to an increase of cholesterol storage in the liver.