Synthesis and pharmacology of 11-nor-1-methoxy-9-hydroxyhexahydrocannabinols and 11-nor-1-deoxy-9-hydroxyhexahydrocannabinols: new selective ligands for the cannabinoid CB2 receptor

Fourteen novel CB2 receptor selective cannabinoids were synthesized via initial Lewis acid catalyzed rearrangement of resorcinol precursors to obtain the cannabinoid moiety. These are the 1-methoxy-9-hydroxyhexahydrocannabinols and the 1-deoxy-9-hydroxyhexahydrocannabinols, with 1',1'-dimethylalkyl side chains of four to seven carbon atoms at C-3 of the cannabinoid nucleus. The cannabinols synthesized and described in this paper all exhibit greater affinity for the CB2 receptor than for the CB1 receptor. Exceptionally high CB2 affinity was observed for 1-deoxy-9beta-hydroxy-dimethylhexylhexahydrocannabinol (JWH-361, 9, n = 3) K(i) = 2.7 nM and 1-deoxy-9beta-hydroxydimethylpentylhexahydrocannabinol (JWH-300, 9, n = 2) K(i) = 5.3 nM. In general, the stereochemistry of the 9-hydroxy group is important and the beta-orientation enhances both CB2 receptor affinity and selectivity.     

Kinetic and structural characterisation of Escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate CB1954

Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme-prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.     

Arachidonylsulfonyl derivatives as cannabinoid CB1 receptor and fatty acid amide hydrolase inhibitors

Arachidonylsulfonyl fluoride (3), reported here for the first time, is similar in potency to its known methyl arachidonylfluorophosphonate (2) analogue as an inhibitor of mouse brain fatty acid amide hydrolase activity (IC(50) 0.1 nM) and cannabinoid CB1 agonist [3H]CP 55,940 binding (IC(50) 304-530 nM). Interestingly, 3 is much more selective than 2 as an inhibitor for fatty acid amide hydrolase relative to acetylcholinesterase, butyrylcholinesterase and neuropathy target esterase. N-(2-Hydroxyethyl)arachidonylsulfonamide (4) is at least 2500-fold less potent than N-(2-hydroxyethyl)arachidonamide (anandamide) (1) at the CB1 agonist site.     

Nucleotide-binding sites in the functional unit of sarcoplasmic reticulum Ca2+-ATPase as studied by photoaffinity spin-labeled 2-N3-SL-ATP

2-N3-SL-ATP [2-azido-2',3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl-pyrroline) adenosine triphosphate], a photoaffinity spin-labeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotide-binding site stoichiometry of sarcoplasmic reticulum (SR) Ca2+-ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found. The ESR spectra showed signals from spin-spin interactions between two radicals corresponding to a distance of about 15 A between labels bound to adjacent sites on the enzyme. This indicates that the minimal functional unit of the Ca2+-ATPase is a dimer with the nucleotide-binding sites in close proximity.     

Synthesis and pharmacology of 1-alkyl-3-(1-naphthoyl)indoles: steric and electronic effects of 4- and 8-halogenated naphthoyl substituents

To develop SAR at both the cannabinoid CB(1) and CB(2) receptors for 3-(1-naphthoyl)indoles bearing moderately electron withdrawing substituents at C-4 of the naphthoyl moiety, 1-propyl and 1-pentyl-3-(4-fluoro, chloro, bromo and iodo-1-naphthoyl) derivatives were prepared. To study the steric and electronic effects of substituents at the 8-position of the naphthoyl group, the 3-(4-chloro, bromo and iodo-1-naphthoyl)indoles were also synthesized. The affinities of both groups of compounds for the CB(1) and CB(2) receptors were determined and several of them were evaluated in vivo in the mouse. The effects of these substituents on receptor affinities and in vivo activity are discussed and structure-activity relationships are presented. Although many of these compounds are selective for the CB(2) receptor, only three JWH-423, 1-propyl-3-(4-iodo-1-naphthoyl)indole, JWH-422, 2-methyl-1-propyl-3-(4-iodo-1-naphthoyl)indole, the 2-methyl analog of JWH-423 and JWH-417, 1-pentyl-3-(8-iodo-1-naphthoyl)indole, possess the desirable combination of low CB(1) affinity and good CB(2) affinity.     

Photochemical probes of the active site of myosin. Irradiation of trapped 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate labels the 50-kilodalton heavy chain tryptic peptide

3'-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP), an analog of ATP containing a photoreactive benzophenone moiety, was used as a probe of the ATP binding site of myosin subfragment 1 (SF1). The inactivation of SF1 NH+4-EDTA ATPase by the bifunctional thiol crosslinking system cobalt(II)/cobalt(III) phenanthroline complexes was enhanced by Bz2ATP to the same degree as by ATP. This treatment resulted in the stable trapping of Bz2ATP at the active site in nearly stoichiometric amounts in a manner exactly analogous to ATP (Wells, J.A., and Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970). Irradiation of SF1 containing trapped [3H]Bz2ATP gave approximately 50% covalent incorporation of the trapped nucleotide into the enzyme. Analysis of photolabeled SF1 by gel electrophoresis showed that all of the [3H]Bz2ATP was attached to the 95-kDa heavy chain fragment. No label was found in the light chains. Similar analysis of the same protein after limited trypsin treatment demonstrated that approximately 75% of the [3H]Bz2ATP was bound to the central 50-kDa peptide and its 75-kDa precursor from the heavy chain. The N-terminal 25-kDa tryptic peptide, shown to be photolabeled by other ATP analogs (Szilagyi, L., Balint, M., Sreter, F.A., and Gergely, J. (1979) Biochem. Biophys. Res. Commun. 87, 936-945; Okamoto, Y., and Yount, R.G. (1983) Biophys. J. 41, 298a), was not labeled (less than 1%) by Bz2ATP. These results demonstrate that portions of the 50 kDa-peptide of the heavy chain are within 6-7 A of the ATP binding site on SF1 and possibly contribute to nucleotide binding.     

Molecular modeling for the design of a biomimetic chimeric ligand. Application to the purification of bovine heart L-lactate dehydrogenase

Molecular modeling was employed for the design of a biomimetic chimeric ligand for L-lactate dehydrogenase (LDH). This ligand is an anthraquinone monochlorotriazinyl dye comprising two moieties: (a) the ketocarboxyl biomimetic moiety, 2-(4-aminophenyl)-ethyloxamic acid, linked on the monochlorotriazine ring, mimicking the natural substrate of LDH, and (b) the anthraquinone chromophore moiety, linked also on the same monochlorotriazine ring via a diaminobenzenesulfonate group, acting as pseudomimetic of the cofactor NAD+. The positioning of the dye in the enzyme's binding site is primarily achieved by the recognition and positioning of the pseudomimetic anthraquinone moiety. The positioning of the biomimetic ketocarboxylic moiety is based on a match between the polar and hydrophobic regions of the enzyme's binding site with those of the biomimetic moiety of the ligand. The length of the biomimetic moiety is predetermined for the ketoacid to approach the enzyme catalytic site and form charge-charge interactions. The biomimetic chimeric ligand and the commercial nonbiomimetic ligand Cibacron(R) blue 3GA (CB3GA), were immobilized on crosslinked beaded agarose gel via their chlorotriazine ring. The two affinity adsorbents were evaluated for their purifying ability for LDH from six sources (bovine heart and pancreas, porcine muscle, chicken liver and muscle, and pea seeds). The biomimetic adsorbent exhibited approximately twofold higher purifying ability for LDH compared to the CB3GA adsorbent; therefore, the former was integrated in the purification procedure of LDH from bovine heart extract. The LDH afforded by this two-step purification procedure shows specific activity equal to 600 U/mg (25 degrees C) and a single band after SDS-PAGE analysis.     

An affinity label for alpha 2-adrenergic receptors in rat brain

Clonidine, a potent and highly selective alpha 2-adrenergic agonist of the central nervous system, was modified. Insertion of the strong alkylating isothiocyanate group (NCS) group, at its aromatic residue, makes clonidine a potential affinity label of the alpha 2-adrenergic receptors. In displacement of [3H]clonidine and p-[3H]aminoclonidine from rat brain membrane preparations, clonidine-NCS demonstrates high affinity for the alpha 2-adrenergic receptors (Kd = 50 mM). The covalent labelling of the central alpha 2-receptors requires higher concentrations of the irreversible ligand (1-70 microM), thus indicating possible non-productive interactions at the environment of the receptor site. Only partial protection of the receptors is observed with a reversible alpha 2-agonist. The new clonidine analog appears to be a general ligand for the alpha 2-adrenergic receptors and might serve as a potential affinity probe for these receptors.     

Design and synthesis of novel celecoxib analogues as selective cyclooxygenase-2 (COX-2) inhibitors: replacement of the sulfonamide pharmacophore by a sulfonylazide bioisostere

A group of celecoxib analogues in which the para-SO(2)NH(2) substituent on the N(1)-phenyl ring was replaced by a para-sulfonylazido (SO(2)N(3)) 4, or a meta-SO(2)N(3) 8, substituent were designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1 and COX-2 inhibition studies showed that 4-[5-(4-methylphenyl)-3-trifluoromethyl-1H-pyrazol-1-yl]benzenesulfonyl azide (4) with a para-SO(2)N(3) substituent was a selective COX-1 inhibitor. In contrast, 3-[5-(4-methylphenyl)-3-trifluoromethylpyrazol-1-yl]benzenesulfonyl azide (8a) having a meta-SO(2)N(3) substituent (COX-1 IC(50) >100microM; COX-2 IC(50)=5.16microM; COX-2 selectivity index >19.3) is a selective COX-2 inhibitor. A molecular modeling (docking) study showed that the SO(2)N(3) group of 8a inserts deep inside the secondary pocket of the COX-2 binding site. The SO(2)N(3) moiety of 8a can undergo a dual H-bonding interaction via one of its SO(2) oxygen-atoms, and an electrostatic (ion-ion) interaction via the terminal azido (N(3)) nitrogen-atom, to the guanidino NH(2) of Arg(513) in the secondary pocket of COX-2. These observations indicate that an appropriately positioned SO(2)N(3) moiety is a novel alternative bioisostere to the traditional SO(2)NH(2) and SO(2)Me pharmacophores present in selective COX-2 inhibitors, that are only capable of H-bonding interactions with the COX-2 isozyme, for use in drug design.     

Modification of bovine pancreatic ribonuclease A with the site-specific reagent 4-arsono-2-nitrofluorobenzene. Spectrophotometric titration of arsononitrophenyl ribonuclease A derivatives

The 4-arsono-2-nitrophenyl chromophore can serve as a versatile spectrophotometric probe of the surface structure of proteins. Values of pK1' and pK2' for the arsonic acid ionizations are near 3 and 8, respectively, and the presence of nearby positive and negative charges produces substantial alterations in the spectral response of the probe. Changes in the extinction at the wavelength of maximum difference are 30-50% of the extinction coefficients, epsilonmax, for each ionization of the arsonic acid moiety. The titration of 41-(4-arsono-2-nitrophenyl)ribonuclease A indicates that the arsonate dianion binds near the active-site histidine residues. With protonation of a carboxylate side chain in the acidic region, presumably aspartic acid-121, the active site is disrupted. The 41-(4-arsono-2-nitrophenyl) group interacts to a greater degree with the histidine-119 side chain than it does with the histidine-12 residue. Interactions of uridine or 3'-cytidylic acid with the ligand-binding region of 41-(4-arsono-2-nitrophenyl) ribonuclease A modify the spectrophotometric response extensively. 3'-Cytidylic acid binds 41-(4-arsono-2-nitrophenyl) ribonuclease A with an affinity 300 times less than that for native ribonuclease A and 17 times lower than that for 41-(2,4-dinitrophenyl) ribonuclease A. The arsononitrophenyl chromophore is responsive to changes in the active site of ribonuclease A induced by such perturbants as ligand binding, chemical modification, and both acid and thermal denaturation.     

Design, synthesis, and biological evaluation of 1,3-diarylprop-2-en-1-ones : a novel class of cyclooxygenase-2 inhibitors

A group of regioisomeric (E)-1,3-diarylprop-2-en-1-one derivatives possessing a COX-2 SO2Me pharmacophore at the para position of the C-1 or C-3 phenyl ring, in conjunction with a C-3 or C-1 phenyl (4-H) or substituted-phenyl ring (4-F, 4-OMe and 4-Me), were designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. These target (E)-1,3-diarylprop-2-en-1-ones were synthesized via a Claisen-Schmidt condensation reaction. In vitro COX-1/COX-2 isozyme inhibition structure-activity studies identified (E)-1-(4-methanesulfonylphenyl)-3-(4-methylphenyl)prop-2-en-1-one (9f) as a potent COX-2 inhibitor (IC50=0.3 microM) with a high COX-2 selectivity index (SI=106) comparable to that of the reference drug rofecoxib (COX-2 IC50=0.5 microM; COX-2 SI>200). A molecular modeling study where 9f was docked in the binding site of COX-2 showed that the para-SO2Me substituent on the C-1 phenyl ring is oriented in the vicinity of the secondary COX-2 binding site near Val523. The structure-activity data acquired indicate that the propenone moiety constitutes a suitable scaffold to design novel acyclic 1,3-diarylprop-2-en-1-ones with selective COX-2 inhibitory activity.     

Affinity labeling of steroid binding sites. Study of the active site of 20beta-hydroxysteroid dehydrogenase with 2alpha-bromoacetoxyprogesterone and 11alpha-bromacetoxyprogesterone.

To further characterize the active site of 20beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) from Streptomyced hydrogenans we synthesized 2alpha-bromoacetoxyprogesterone, a substrate for the enzyme in 0.05 M phosphate buffer at 25 degrees, pH 7.0, with Km and Vmax values of 1.90 X 10(-5) M and 6.09 nmol/min/mg of enzyme, respectively. This affinity labeling steroid inactivates 20beta-hydroxysteroid dehydrogenase in an irreversible and time-dependent manner which follows pseudo-first order kinetics with a t1/2 value of 4.6 hours. 2alpha-[2-3H]Bromoacetoxyprogesterone was synthesized and used to radiolabel the enzyme active site. Amino acid analysis of the acid hydrolysate of the radiolabeled enzyme supports a mechanism whereby the steroid moiety delivers the alkylating group to the steroid binding site of the enzyme where it reacts with a methionyl residue. Both 2alpha- and 11alpha-bromoacetoxyprogesterone alkylate a methionyl residue at the active site of 20beta-hydroxysteroid dehydrogenase. The enzyme was inactivated with a mixture containing both 2alpha-[2-3H]Bromoacetoxyprogesterone and 11alpha-2[2-14C]bromoacetoxyprogesterone. Following degradation of separate aliquots of the radiolabeled enzyme by cyanogen bromide or trypsin, the protein fragments were separated by gel filtration and ion exchange chromatography. Resolution of peptides carrying the 3H label from those possessing the 14C label demonstrates that 2alpha-bromoacetoxyprogesterone and 11alpha-bromoacetoxyprogesterone each label a different methionine at the steroid binding site of 20beta-hydroxysteroid dehydrogenase.     

Detecting precatalytic conformational changes in F1-ATPase with 4-benzoyl(benzoyl)-1-amidofluorescein, a novel fluorescent nucleotide site-specific photoaffinity label

A novel photoaffinity label for studies with the F1-ATPase has been synthesized and found to be an effective reporter of subunit conformational changes that occur in this enzyme upon multiple nucleotide-binding site occupancy. The new probe, 4-benzoyl(benzoyl)-1-amidofluorescein (BzAF), which possesses structural similarity to purine nucleotides, exhibits bifunctional characteristics that enable it to bind covalently to the exchangeable nucleotide sites on beef heart F1 (via photoactivation of the benzophenone moiety) and, once covalently linked, emit environmentally sensitive fluorescence (via selective excitation of the fluorescein moiety). BzAF binds competitively with ATP in the absence of illumination, with a KI of 50 microM. Under actinic irradiation necessary for generating the covalently reacting diradical triplet state of benzophenone, BzAF behaves as a nucleotide site-directed photoaffinity label of exchangeable (catalytic) sites, and the resulting photoinhibition of ATPase activity displays pseudo first-order rate-saturation kinetics that support formation of a dissociable BzAF.F1 complex (k-1/k1 = 58 microM) prior to covalent binding. The BzAF-induced photoinactivation is protectable with native nucleotide ligand (e.g. MgADP, Kprotect = 0.4 mM). Added corroboration of a catalytic cooperativity mechanism for F1 was obtained by finding a molar stoichiometric ratio [( 3H]BzAF:F1) of 1 required for complete inhibition of ATPase activity. Steady-state fluorescence studies with a unisite-labeled BzAF.F1 complex (a catalytically inactive species on which at least one exchangeable nucleotide-binding site remains unoccupied) display a saturable fluorescence quenching of the bound fluorescein upon titration with MgADP, but no change with MgAMP. These data imply that the filling of more than one of the catalytic binding sites/mol of F1 with nucleotide signals a precatalytic conformational adjustment that is transmitted between catalytic sites and across the beta-alpha-beta domain of the enzyme's subunit structure.     

A new simpler photoaffinity analogue of peptidyl tRNA

The synthesis of the n-hydroxysuccinimide ester of N-(2-nitro-4-azidophenyl)glycine (NAG) is described. This reacts with E. coli phe-tRNA(Phe) to yield the photoaffinity label NAG-Phe-tRNA(Phe). This peptidyl tRNA analogue binds correctly to the peptidyl site of the E. coli ribosome. The only significant covalent products found after irradiation of a peptidyl site bound NAG-Phe-tRNA(Phe)-70S-poly(U) complex are 50S proteins L11 and L18. After irradiation the complex can still bind [(3)H]Phe-tRNA to the amino acyl site and participate in peptide bond formation with the covalently attached NAG-Phe moiety. Alternatively, one can allow peptide bond formation to occur first, prior to photolysis. The reaction products are still L11 and L18. Hence, both of these two proteins appear to be centrally located at the peptidyl transferase center.     

A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.     

Synthesis and biological activity of novel 4,4-difluorobenzazepine derivatives as non-peptide antagonists of the arginine vasopressin V1A receptor

To find potent and selective antagonists of the arginine vasopressin (AVP) V1A receptor, optimization studies of compounds structurally related to (Z)-N-{4'-[(4,4-difluoro-5-carbamoylmethylidene-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl)carbonyl]phenyl}carboxamide were performed. The synthesis and pharmacological properties of these compounds are described. We first investigated the effect of the carboxamide moiety, and found that a 2-methylfuran-3-carbonyl group at this position increased V1A binding affinity and selectivity for the V1A receptor versus the V2 receptor. The amino group of the 5-carbamoylmethylidene moiety was also examined, and a 4-piperidinopiperidino group was found to be optimal at this position. The hemifumarate of compound 12l (YM218) was shown to exhibit potent binding affinity, V1A receptor selectivity, and in vivo antagonist activity.     

Affinity probes for the GABA-gated chloride channel: Selection of 5e-tert-Butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes and optimization of linker moiety

The noncompetitive blocker (NCB) site of the γ-aminobutyric acid (GABA)-gated chloride channel is the target for many important insecticides and potent convulsants. This site is specifically blocked by ³H ethynylbicycloorthobenzoate (³H EBOB) and other trioxabicyclooctane radioligands and might be suitable for affinity probes with an appropriate heterocyclic substituent and linker moiety. Optimal potency at the NCB site is achieved with 5e-tert-butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes compared with analogs in which the butyldithiane portion is replaced with butyldithiane-sulfoxide or -sulfone, n-propyltrioxabicyclooctane or dioxatricyclododecene. Three positions were examined for coupling the linker and dithiane: C-2 of the dithiane; a branched substituent within the alkynyl moiety; the terminus of a straight chain extension from the ethynyl group, which proved to be the best. Optimized linkers for addition to the ethynylphenyldithiane to achieve appropriate length and fit within the active site, i.e. receptor potency, are CH₂OCH₂C(O)SCH₂CH₂(SH or NH₂) and the corresponding thiolates and amides. Several compounds with these spacers block the chloride channel, measured as inhibition of ³H EBOB binding, at 4–50 nM.     

New 1,2,3,4-tetrahydropyrrolo[3,4-b]indole derivatives as selective CB2 receptor agonists

The preparation and evaluation of a novel class of CB2 agonists based on a 1,2,3,4-tetrahydropyrrolo[3,4-b]indole moiety are reported. They showed binding affinities up to 4.2 nM toward CB2 with sub-nanomolar EC(50) values. They also showed moderate to good (>350-fold) selectivity over the CB1 receptor.     

A base substitution at a splice site in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers-Danlos syndrome type IV

The dermis of a child with Ehlers-Danlos syndrome type IV (EDS-IV) contained about 11% of the normal amount of type III collagen and cultured dermal fibroblasts produced a reduced amount of type III procollagen which was secreted poorly. Type III collagen produced by these cells contained normal and abnormal alpha-chains and cyanogen bromide peptides. The site of the structural defect in the abnormal alpha 1 (III) chains was localized to the region of Met797, which is at the junction of the two carboxyl-terminal CB5 and CB9 cyanogen bromide peptides. Chemical cleavage of heteroduplexes formed between EDS-IV mRNA and a normal cDNA clone covering the CB5 and CB9 region showed that about 100 nucleotides were mismatched. Sequencing of amplified and cloned cDNA spanning the mutant region revealed a 108 nucleotide deletion corresponding to amino acid residues Gly775 to Lys810. The deleted nucleotide sequence corresponded to sequences that, by analogy to the organization of the type I collagen genes, should be precisely encoded by exon 41 of the COL3A1 gene. Sequencing of amplified genomic DNA, prepared using disimilar amounts of primers specific for exons 41 and 42, displayed a base substitution (G-to-A) in the highly conserved GT dinucleotide of the 5' splice site of intron 41. Normal sequences were also obtained from the normal allele. It is likely that the GT-to-AT transition at the splice donor site of intron 41 generated an abnormally spliced mRNA in which sequences of exon 40 and 42 were joined together with maintenance of the reading frame. The corresponding peptide deletion included the cyanogen bromide cleavage site Met797-Pro798 and the mammalian collagenase cleavage site at Gly781-Ile782. These losses account for the resistance of EDS-IV collagen to cyanogen bromide and mammalian collagenase digestion. Cultured fibroblasts produced normal homotrimer, mutant homotrimer, and mixed heterotrimer type III collagen molecules. The mutant homotrimer molecules were the major pepsin-resistant species and about 69% of the alpha 1(III) mRNA was in the mutant form.     

Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase

The specificity of the two intrasubunit cGMP binding sites of cGMP-dependent protein kinase was determined by measuring the ability of 46 cGMP analogs to compete with [3H]cGMP. Both sites of the enzyme exhibited high specificity for the ribose cyclic phosphate moiety, and lower specificity for the guanine moiety. Effects of modifications in the ribose cyclic phosphate moiety suggested that cGMP is bound at both sites by three hydrogen bonds at 2'-OH, 3'-O, and 5'-O. A negative charge in the cyclic phosphate is apparently required. Modifications of the pyrimidine part of guanine, particularly at C-1, generally caused selectivity for the rapidly exchanging site while modifications of the imidazole part of guanine at C-7 and C-8 caused selectivity for the slowly exchanging site. These increases in selectivity for a site were mainly due to losses in affinity of the other site. There was an apparent requirement of the intact amino group at C-2, particularly for the slowly exchanging site. Comparison of the molecular interactions of cAMP and cGMP with their specific protein kinases showed that both nucleotides are bound by similar forces in the 2', 3' and 5' region, both bases may be bound in syn conformation, but that each base moiety is bound by different molecular interaction, thus leading to the selectivity of the two enzymes. cGMP analogs which possessed strong selectivity for the rapidly exchanging site, but not those selective for the slowly exchanging site, stimulated the binding of [3H]cGMP. Only a few cGMP analogs were more potent than cGMP in stimulating protein kinase activity. The potency of cGMP analogs as stimulators of kinase activity correlated better with the mean binding affinity for both binding sites than with the affinity for either site alone. Two analogs added in combination were synergistic in kinase activation, particularly if one analog was selective for the slowly exchanging site and the other for the rapidly exchanging site. These observations are suggestive that cGMP binding at the rapidly exchanging site stimulates cGMP binding at the slowly exchanging site and that both sites are involved in the activation process.