A Trifluoromethylphenyl Piperazine Derivative with High Affinity for 5-Hydroxytryptamine-1A Sites in Rat Brain

The inhibition of [3H]5-hydroxytryptamine [( 3H]5-HT) binding in rat brain by 1-[2-(3-bromoacetamidophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine (BrAcTFMPP) and that by spiperone were compared. Spiperone inhibition of [3H]5-HT binding in cortex was consistent with displacement from two sites with dissociation constants (KD) of 24 nM (5-HT-1A site) and 19 microM (5-HT-1B site) for spiperone. BrAcTFMPP also discriminated two subpopulations of [3H]5-HT binding sites with dissociation constants of 0.5 nM and 146 nM for the compound. The proportion of high-affinity sites for each compound represented about 35% of the specific [3H]5-HT binding. In the presence of 1 microM spiperone, a concentration that saturates the 5-HT-1A sites while having a minimal effect on 5-HT-1B sites, BrAcTFMPP displaced [3H]5-HT from a single site with a KD for BrAcTFMPP of 145 nM. The inhibition of [3H]5-HT binding by spiperone in the presence of 30 nM BrAcTFMPP was best fit by a single-site model with a KD of 21 microM for spiperone. In corpus striatum, 5-HT-1A sites, as defined with spiperone, represented 15% of the specific [3H]5-HT binding and 30 nM BrAcTFMPP also blocked about 15% of the binding. A significant difference between spiperone and BrAcTFMPP was their affinity for 5-HT-2 receptors. BrAcTFMPP (KD = 41 nM) had an 80-fold lower affinity for these sites than spiperone (KD = 0.5 nM). Thus, BrAcTFMPP and spiperone discriminate the same two subpopulations of [3H]5-HT binding sites and BrAcTFMPP displays a high affinity and a selectivity for 5-HT-1A sites versus both 5-HT-1B and 5-HT-2 sites.     

Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Characterization of the putative anxiolytic SM-3997 recognition sites in rat brain

In order to clarify the mechanism of action of the putative nonbenzodiazepine anxiolytic SM-3997 [3a alpha,4 beta,7 beta,7a alpha)-Hexahydro-2-(4-(4-(2-pyrimidinyl)-1- piperazinyl)-butyl)-4,7-methano-1H-isoindole-1,3 (2H)-dione dihydrogen citrate), in vitro binding studies with radiolabeled compound were performed. 3H-SM-3997 bound rapidly, reversibly and in a saturable manner with high affinity to rat brain hippocampal membranes (Kd = 9.4 nM, Bmax = 213 fmol/mg protein). This specific binding was displaced by 5-hydroxytryptamine (5-HT) and related compounds. Especially, 8-OH-DPAT, a 5-HT-1A selective agonist, bound with the highest affinity to these binding sites. 3H-SM-3997 binding, however, was not displaced by a variety of other neurotransmitters, neuropeptides and some other drugs. EDTA and physiological concentration of Na+ inhibited this specific binding, but several divalent cations, Mn2+, Ca2+ and Mg2+, enhanced this binding. GTP decreased the affinity of these binding sites for 3H-SM-3997 without changing the number of binding sites, but GMP and ATP did not influence 3H-SM-3997 binding. Furthermore, 3H-SM-3997 bound with marked regional selectivity to hippocampal membranes. These characteristics and the regional distribution of 3H-SM-3997 binding sites were very similar to those of 3H-8-OH-DPAT binding sites (5-HT-1A receptors). Therefore, these results indicate that SM-3997 binds selectively and with high affinity to 5-HT-1A receptors in rat brain and may be an agonist.     

Further evidence that the serotonin receptor in the rat stomach fundus is not 5HT1A or 5HT1B

The nature of the receptor mediating serotonin contraction in the rat stomach fundus has not been clearly associated with either 5HT1 or 5HT2 receptors. We have explored the possibility that such receptors in the rat fundus may better correlate with 5HT1A or 5HT1B receptor subtypes as defined by radiolabeled ligand binding studies with brain cortical membranes. Meta chlorophenylpiperazine (CPP) and meta trifluoromethylphenylpiperazine (TFMPP), selective ligands for the 5HT1B receptor and LY165163, a selective ligand for the 5HT1A receptor, have been evaluated for their agonist and antagonist activity at serotonin receptors in the rat stomach fundus. CPP and TFMPP were partial agonists in the rat stomach fundus whereas LY165163 showed no agonist activity in this smooth muscle in concentrations up to 10(-4)M. All three phenylpiperazines antagonized serotonin-induced contractions in the rat stomach fundus. The affinity for serotonin receptors in the rat fundus was similar for all three phenylpiperazines in spite of the reported selectivity of MCPP and TFMPP for 5HT1B and of LY165163 for 5HT1A receptors. Furthermore, the affinity of these agents for serotonin receptors in the rat stomach fundus did not agree with their reported affinity for either 5HT1A or 5HT1B binding sites in rat cortical membranes. Thus, the similarity in affinities of these phenylpiperazine derivatives for serotonin receptors mediating contraction in the rat fundus along with their different affinities for 5HT1A and 5HT1B binding sites argues against the possibility that the serotonin receptor in the rat fundus is of the 5HT1A or 5HT1B subtype of serotonin receptor.     

Specific vasopressin binding to rat adrenal glomerulosa cells. Relationship to inositol lipid breakdown.

Cells from the zona glomerulosa of rat adrenals were isolated and maintained for 3 days in primary culture. Specific vasopressin binding was determined by using [3H]vasopressin. [3H]Vasopressin binding was time-dependent (half-time of about 2 min for 6 nM free ligand) and reversible on addition of unlabelled vasopressin (80% dissociation within 30 min). Dose-dependent [3H]vasopressin binding at equilibrium indicated that vasopressin interacted with two populations of sites: high-affinity sites (dissociation constant, Kd = 1.8 nM; maximal binding capacity = 10 fmol/10(6) cells) and low-affinity sites. Vasopressin increased the cellular content of labelled inositol mono-, bis- and tris-phosphate in cells prelabelled with myo-[3H]inositol. The vasopressin concentration eliciting half-maximal inositol phosphate accumulation was very close to the Kd value for vasopressin binding to high-affinity sites. Competition experiments using agonists and antagonists with enhanced selectivity for previously characterized vasopressin receptors indicated that vasopressin receptors from rat glomerulosa cells are V1 receptors of the vascular or hepatic subtype. The detected specific vasopressin-binding sites might represent the specific receptors mediating the mitogenic and steroidogenic effects of vasopressin on glomerulosa cells from rat adrenals.     

[3H]1-[2-(4-Aminophenyl)Ethyl]-4-(3-Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5-HT1A Receptors in Rat Brain

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.     

Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein

The interactions between purified rat hepatic microsomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b5 have been studied in the presence of phospholipid using difference spectrophotometry. Cytochrome b5 was shown to interact with cytochrome P-450 to form a tight 1:1 complex (Kd = 275 nM), in which the proportion of high spin cytochrome P-450 was increased from 7 to 30%. The presence of saturating cytochrome b5 was shown to cause a decrease in the apparent Kd for benzphetamine binding from 111 microM to 40 microM. Likewise, the presence of benzphetamine was shown to cause a decrease in the apparent dissociation constant for cytochrome b5 binding to cytochrome P-450 (Kd = 90 nM). The above interactions were resolved into the basic equilibria inter-relating the various ligation states of the hemoprotein in an energetically closed eight-state free energy coupling model and the relative magnitudes of the microequilibria were analyzed to determine the degree of coupling of the interactions between cytochrome P-450 and both benzphetamine and cytochrome b5. Consequently, the spin state changes in cytochrome P-450 induced by benzphetamine and cytochrome b5 binding were shown to arise because these ligands interact 7 and 4 times more tightly with high spin cytochrome P-450, respectively. Furthermore, the data revealed that these ligands interact at independent sites on cytochrome P-450. Thus the effects of cytochrome b5 upon benzphetamine binding and vice versa were rationalized simply in terms of an increase in the proportion of a high spin (high affinity) conformation of cytochrome P-450 brought about by pre-equilibration with the effector ligand, with the intrinsic binding affinities of the two ligands for the low or high spin states remaining relatively unaltered. The thermodynamic parameters associated with the interactions between cytochrome P-450 and cytochrome b5, determined from the temperature dependence of these interactions, revealed that these protein interactions are entropy driven and probably occur by a hydrophobic mechanism.     

Multiple species of estrogen binding sites in the nuclear fraction of the rat prostate

Specific estrogen-binding sites have been demonstrated in purified nuclear fractions of prostates from intact rats. Saturation analysis of nuclei over a wide range of [³H]-estradiol concentrations (0.15 to 90 nM) has shown two different types of binding sites: a) one with high affinity (Kd of 0.5–0.8 nM) and low capacity for estradiol (approximately 162 fmole/mg DNA); b) a second with a lower affinity (Kd of 30–40 nM), which shows a higher capacity (approximately 860 fmole/mg DNA), and displays a saturation curve that is sigmoidal and that appears to be similar to those for Type II estrogen-binding sites in rat uterus. These results suggest that the actions of estradiol in the prostate are mediated by specific nuclear binding sites.     

Recombinant domains of mouse nidogen-1 and their binding to basement membrane proteins and monoclonal antibodies.

The basement membrane protein, nidogen-1, was previously shown to consist of three globular domains, G1 to G3, and two connecting segments. Nidogen-1 is a major mediator in the formation of ternary complexes with laminins, collagen IV, perlecan and fibulins. In the present study, we have produced recombinant proteins of these predicted domains in mammalian cells and used these proteins for crystallographic and binding epitope analyses. These fragments included G1, G2, the rod domain and a slightly larger G3 structure; all were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high affinity binding (Kd = 3-9 nM) of domain G2 for collagen IV, perlecan domain IV-1 and fibulin-2, and a more moderate Kd for fibulin-1C. Domain G3 contained high affinity binding sites for the laminin gamma1 chain and collagen IV (Kd = 1 nM) and weaker binding sites for fibulin-1C and fibulin-2. A moderate binding affinity was also observed between domain G1 and fibulin-2, while no activity could be detected for the nidogen rod domain. Together, these data indicate the potential of nidogen-1 for multiple interactions within basement membranes. A similar binding repertoire was also identified for seven rat monoclonal antibodies that bound with Kd = 2-30 nM to either G1, G1-G2, G2, the rod domain or G3. Three of the antibodies showed strongly reduced binding to G2 and G3 after complex formation with either a perlecan domain or laminin-1.     

Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine derivatives as 5-HT1A serotonin receptor ligands

A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT1A receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and alpha1-, alpha2-adrenergic receptors. A o-OCH3 derivative (2e) bound at 5-HT1A sites with subnanomolar affinity (IC50 = 0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents.     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

4-Methyl-3-oxo-4-aza-5α-androst-1-ene-17β-N-aryl-carboxamides: an approach to combined androgen blockade [5α-reductase inhibition with androgen receptor binding in vitro]

4-Aza-5α-androstan-3-one 17β-(N-substituted carboxamides) are potent human type 2 5α-reductase (5aR) inhibitors with generally poor binding to the human androgen receptor (hAR). When the 17-amide N-substituent included an aromatic residue, potent dual inhibitors of both type 1 and 2 5aR are produced, but hAR binding remained poor. Tertiary-substituted-17-amides have reduced inhibition of both 5aR isozymes. The addition of an N⁴-methyl substitutent to the A-ring profoundly increased hAR affinity and the addition of unsaturation to the A-ring (Δ¹) modestly augmented hAR binding. The unsubstituted carbanilides in the Δ¹-N⁴-methyl series show some selectivity for type 1 5aR over the type 2 isozyme, whereas addition of aryl substituents, particularly at the 2-position, increased type 2 5aR binding to provide dual inhibitors with excellent hAR binding, e.g. N-(2-chlorophenyl)-3-oxo-4-methyl-4-aza-5α-androst-1-ene-17β-carboxamide (9c). Compounds of this type exhibit low nanomolar IC₅₀s for both human 5aR isozymes as well as the human androgen receptor. Kinetic analysis confirms that the prototype 9c displays reversible, competitive inhibition of both human isozymes of 5aR with K_i values of less than 10 nM. Furthermore, this compound binds to the androgen receptor with an IC₅₀ equal to 8 nM. Compounds in this series are projected to be powerful antagonists of testosterone and dihydrotestosterone action in vivo, with potential utility in the treatment of prostatic carcinoma (PC).     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

6-substituted tricyclic partial ergoline compounds are selective and potent 5-hydroxytryptamine1A receptor agents

A series of 6 tricyclic partial ergoline derivatives was analyzed using radioligand binding assays. Four agents (LY 178210, LY 254089, LY 197205, and LY 197206) display high affinity (Ki less than or equal to 1.3 nM) for 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy- 2-(di-n-propylamino) tetralin (8-OH-DPAT) and display greater than or equal to 150 fold selectivity for the 5-HT1A over the 5-HT1D receptor binding site. The most potent agent investigated, LY 178210, is essentially inactive (Ki greater than 1500 nM) at a total of 12 other neurotransmitter receptor binding sites in the brain. Using a forskolin-stimulated adenylate cyclase assay as a model of 5-HT1A receptor function, LY 178210 was found to display partial agonist activity which was blocked by 10(-5) M (-)pindolol. These data indicate that LY 178210 is a potent and selective 5-HT1A receptor partial agonist.     

Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.     

3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 and [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6(O-tert-butyl). Two new enkephalin analogs with both a good selectivity and a high affinity toward delta-opioid binding sites

Insertion of bulky tertiobutyl groups into the sequence of [D-Ser2,Leu5]enkephalyl-Thr6 leads to a conformationally induced large increase in selectivity toward rat brain delta-opioid binding sites, as shown by the ratio of apparent affinities for mu and delta receptors of [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6,KI(mu)/KI(delta) = 130, and [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 (O-tert-butyl),KI(mu)/KI(delta) = 280. In addition to a selectivity similar to that of the cyclic compounds [D-Pen2, D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin, the affinity of [3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 for the delta sites of rat brain membranes is significantly better (KD = 2.2 nM) than that of [3H][D-Pen2,D-Pen5]enkephalin (KD approximately 8.5 nM). Therefore, [3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 seems to be the most appropriate delta-probe currently available for binding studies. Moreover, the lipophilic and protected peptide [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6(O-tert-butyl) behaves as the most specific ligand for the delta-opioid binding sites and appears appropriate for in vivo investigations. The inactive analogue [D-Thr2(O-tert-butyl),Leu5]enkephalyl-Thr6 might serve as a negative control in biochemical or pharmacological studies.     

Novel aromatase and 5α-reductase inhibitors

Inhibitors of aromatase and 5α-reductase may be of use for the therapy of postmenopausal breast cancer and benign prostatic hyperplasia, respectively. FCE 27993 is a novel steroidal irreversible aromatase inhibitor structurally related to exemestane (FCE 24304). The compound was found to be a very potent competitive inhibitor of human placental aromatase, with a K_i of 7.2 nM (4.3 nM for exemestane). In preincubation studies with placental aromatase FCE 27993, like exemestane, was found to cause time-dependent inhibition with a higher rate of inactivation ( ) and a similar K_i(inact) (56 vs 66 nM). The compound was found to have a very low binding affinity to the androgen receptor (RBA 0.09% of dihydrotestosterone) and, in contrast to exemestane, no androgenic activity up to 100 mg/kg/day s.c. in immature castrated rats. Among a series of novel 4-azasteroids with fluoro-substituted-17β-amidic side chains, three compounds, namely FCE 28260, FCE 28175 and FCE 27837, were identified as potent in vitro and in vivo inhibitors of prostatic 5α-reductase. Their IC₅₀ values were found to be 16, 38 and 51 nM for the inhibition of the human enzyme, and 15, 20 and 60 nM for the inhibition of the rat enzyme, respectively. When given orally for 7 days in castrated and testosterone (Silastic implants) supplemented rats, the new compounds were very effective in reducing prostate growth. At a dose of 0.3 mg/kg/day inhibitions of 42, 36 and 41% were caused by FCE 28260, FCE 28175 and FCE 27837, respectively.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.     

The GCN4 bZIP targets noncognate gene regulatory sequences: quantitative investigation of binding at full and half sites

We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5'-TGACTCA-3') and cAMP-response element (CRE) (5'-TGACGTCA-3') but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and E-box (5'-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full- and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with Kd values of 13 and 12 nM, respectively, to noncognate sites with Kd values of 120 nM to low microM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (Kd = 84 nM) to noncognate half sites (all Kd values > 2 microM). CD shows that comparable levels of alpha-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, alpha-helical proteins with desired DNA recognition properties.     

Synthesis and benzodiazepine receptor (omega receptor) affinities of 3-substituted derivatives of pyrrolo[2,3-c]pyridine-5-carboxylate, a novel class of omega1 selective ligands.

Based on the structure of ZK91296 (4d), a high affinity partial agonist of the central benzodiazepine (omega) receptor, a series of pyrrolo[2,3-c]pyridine-5-carboxylate derivatives having mainly aralkyl and aralkyloxy substituents at C-3 was synthesized. The in vitro binding affinities of these compounds for three subclasses of the omega receptor (omega1, omega2, omega5) were determined using rat brain tissue. Practically all of these compounds (except the diethyl ester derivative 22c) showed an approximately twofold selectivity for omega1 (IC50's in the 200-500 nM range) compared to omega2 receptors and practically no affinity for omega5 receptors. Compound 22c showed the highest affinity of all the compounds synthesized (IC50 = 70 nM for omega1 receptors) as well as a fivefold selectivity for omega1 versus omega2 receptors but also displayed significant binding to omega5 receptors (IC50 = 250 nM). The absence of appreciable binding of 4-methyl and 4-methoxymethyl derivatives to omega receptors, in contrast to beta-carbolines having these similarly located substituents, suggests that the pyrrolo[2,3-c]pyridine-5-carboxylates may be considered an entirely novel class of selective omega receptor ligands.