Imidazo[4,5-c]pyridines as corticotropin releasing factor receptor ligands

A series of high affinity CRF receptor ligands with an imidazo[4,5-c]pyridine core is described. Individual analogues were synthesized and tested in vitro in rat brain receptors to determine binding affinity. The best compound was further tested in the dog N-in-1 pharmacokinetic model to assess oral bioavailability at 1 mg/kg po.     

Differential responsiveness of CRF receptor subtypes to N-terminal truncation of peptidic ligands

The role of the N-terminal domains of corticotropin-releasing factor (CRF) and CRF-like peptides in receptor subtype selectivity, ligand affinity and biological potency was investigated. Therefore, human CRF(12-41), human URP(12-38) and antisauvagine-30 (aSvg) were N-terminally prolonged by consecutive addition of one or two amino acids. The peptides obtained were tested for their binding affinities to rat CRF1 and murine CRF(2beta) receptor, and their capability to stimulate cAMP-release by HEK cells producing either receptor.It was observed that human CRF N-terminally truncated by eight residues was bound with high affinity to CRF2 receptor (Ki=5.4nM), whereas affinity for CRF1 receptor was decreased (Ki=250 nM). A similar shift of affinity was found with sauvagine (Svg) analogs. Truncation of human URP analogs did not affect their preference for CRF(2beta) receptor, but reduced their affinity. Changes in affinity were positively correlated with changes in potency. These results indicated that CRF1 receptor was more stringent in its structural requirements for ligands to exhibit high affinity binding than CRF(2beta) receptor.     

Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: biochemical characterization and autoradiographic localization

CRF receptors were characterized using radioligand binding and chemical affinity cross-linking techniques and localized using autoradiographic techniques in porcine, bovine and rat pituitaries. The binding of 125I-[Tyr0]-ovine CRF (125I-oCRF) to porcine anterior and neurointermediate lobe membranes was saturable and of high affinity with comparable KD values (200-600 pM) and receptor densities (100-200 fmoles/mg protein). The pharmacological rank order of potencies for various analogs and fragments of CRF in inhibiting 125I-oCRF binding in neurointermediate lobe was characteristic of the well-established CRF receptor in anterior pituitary. Furthermore, the binding of 125I-oCRF to both anterior and neurointermediate lobes of the pituitary was guanine nucleotide-sensitive. Affinity cross-linking studies revealed that the molecular weight of the CRF binding protein in rat intermediate lobe was identical to that in rat anterior lobe (Mr = 75,000). While the CRF binding protein in the anterior lobes of porcine and bovine pituitaries had identical molecular weights to CRF receptors in rat pituitary (Mr = 75,000), the molecular weight of the CRF binding protein in porcine and bovine intermediate lobe was slightly higher (Mr = 78,000). Pituitary autoradiograms from the three species showed specific binding sites for 125I-oCRF in anterior and intermediate lobes, with none being apparent in the posterior pituitary. The identification of CRF receptors in the intermediate lobe with comparable characteristics to those previously identified in the anterior pituitary substantiate further the physiological role of CRF in regulating intermediate lobe hormone secretion.     

A Screening Strategy Based on Differential Binding of Ligand to Receptor and to Binding Proteins: Screening for Compounds Interacting with Corticotrophin-Releasing Factor-Binding Protein

The ligand-receptor interaction has been commonly used in development of high throughput screening assays for new drugs. In some cases, an endogenous ligand interacts not only with membrane receptors but also with soluble binding proteins. Corticotrophin-releasing factor (CRF) is an important stress neurotransmitter/hormone involved in both the central and peripheral nervous systems. CRF exerts its function by interacting with CRFR1 and CRFR2 receptors. In addition, CRF-binding protein (CRF-BP) binds CRF with high affinity. Accordingly, CRF-BP has been suggested to play an important role in modulating CRF function. Based on the potential involvement of CRF-BP in many neurological disorders, it is desirable to develop a screening assay to look for drugs that either mimic or interfere with CRF binding to CRF-BP. An assay was developed to monitor the interactions of radiolabeled CRF with human/rat CRF-BP and the mouse CRFR1 (mCRFR1) receptor. By carefully examining the binding characteristics of radiolabeled CRF to mCRFR1, the assay was able to identify compounds that bind to CRF-BP with high affinity and have little or no affinity for mCRFR1 receptors. Based on a mathematical model, we have verified the screening system with several well-characterized CRF ligands that all have different affinities for CRF receptors and CRF-BP.     

Peptide ligand binding properties of the corticotropin-releasing factor (CRF) type 2 receptor: pharmacology of endogenously expressed receptors, G-protein-coupling sensitivity and determinants of CRF2 receptor selectivity

The CRF2 receptor is involved in stress responses, cardiovascular function and gastric motility. Endogenous agonists (urocortin (UCN) 2, UCN 3) and synthetic antagonists (astressin2-B, antisauvagine-30) are selective for CRF2 over the CRF1 receptor. Peptide ligand binding properties of the CRF2 receptor require further investigation, including ligand affinity for endogenously expressed receptors, the effect of receptor-G-protein coupling on ligand affinity, and the molecular basis of ligand selectivity. Ligand affinity for rat CRF(2a) in olfactory bulb and CRF(2b) in A7r5 cells was similar to that for the cloned human CRF(2a) receptor (within three-fold), except for oCRF (9.4- and 5.4-fold higher affinity in olfactory bulb and A7r5 cells, respectively). Receptor-G-protein uncoupling reduced agonist affinity only 1.2- to 6.5-fold (compared with 92-1300-fold for the CRF1 receptor). Ligand selectivity mechanisms were investigated using chimeric CRF2/CRF1 receptors. The juxtamembrane receptor domain determined selectivity of antisauvagine-30, the N-terminal-extracellular domain contributed to selectivity of UCN 3, and both domains contributed to selectivity of UCN 2 and astressin2-B. Therefore ligands differ in the contribution of receptor domains to their selectivity, and CRF2-selective antagonists bind the juxtamembrane domain. These findings will be important for identifying the CRF2 receptor in tissues and for developing ligands targeting the receptor, both of which will be useful in identifying the emerging physiological functions of the CRF2 receptor.     

Pharmacological characterization of recombinant rat corticotropin releasing factor binding protein using different sauvagine analogs

Little is known on the structural ligand requirements for corticotropin-releasing factor binding protein (CRFBP) of the rat used as an important experimental animal. To obtain such information recombinant rat CRFBP was produced in stably transfected HEK 293 cells. The primary structure and posttranslational processing of purified rat CRFBP was established by peptide mapping using HPLC combined with mass spectrometric analysis. Rat CRFBP was pharmacologically characterized employing a competition binding assay with tritium-labeled rat urocortin. The rank order of declining affinity of various CRF analogs was urotensin-I, human/rat CRF (h/rCRF), rat urocortin, sauvagine (Svg), and ovine CRF in agreement with the rank order found for human CRFBP. In contrast to astressin, the CRF receptor 2-selective antagonist anti-sauvagine-30 did not show any detectable specific binding to rat CRFBP. The significance of residues 10 to 12 and 21 to 24 of Svg for its low affinity binding was established by changing these residues of Svg to those of h/rCRF. The corresponding residues 22 to 25 of h/rCRF represented the ARAE motif determined to be crucial for binding in agreement with reported data on human CRFBP. Residues 11 to 13 of CRF introduced into Svg also enhanced the affinity to rat CRFBP.     

Identification of Amino Acids in the N-Terminal Domain of Corticotropin-Releasing Factor Receptor 1 that Are Important Determinants of High-Affinity Ligand Binding

Abstract : The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists : one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76-84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg⁷⁶ or Asn⁸¹ but not Gly⁸³ of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100-1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding.     

The discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1 ,5-a]-pyrimidine: a corticotropin-releasing factor (hCRF1) antagonist

Structure activity relationship studies led to the discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazo lo-[1,5-a]-pyrimidine 11-31 (DMP904), whose pharmacological profile strongly supports the hypothesis that hCRF1 antagonists may be potent anxiolytic drugs. Compound 11-31 (hCRF1 Ki = 1.0+/-0.2 nM (n = 8)) was a potent antagonist of hCRF1-coupled adenylate cyclase activity in HEK293 cells (IC50= 10.0+/-0.01 nM versus 10 nM r/hCRF, n = 8); alpha-helical CRF(9-41) had weaker potency (IC50 = 286+/-63 nM, n = 3). Analogue 11-31 had good oral activity in the rat situational anxiety test; the minimum effective dose for 11-31 was 0.3 mg/kg (po). Maximal efficacy (approximately 57% reduction in latency time in the dark compartment) was observed at this dose. Chlordiazepoxide caused a 72% reduction in latency at 20 mg/kg (po). The literature compound 1 (CP154526-1, 30 mg/kg (po)) was inactive in this test. Compound 11-31 did not inhibit open-field locomotor activity at 10, 30, and 100 mg/kg (po) in rats. In beagle dogs, this compound (5 mg/kg, iv, po) afforded good plasma levels. The key iv pharmacokinetic parameters were t1/2, CL and Vd,ss values equal to 46.4+/-7.6 h. 0.49+/-0.08 L/kg/h and 23.0+/-4.2 L/kg, respectively. After oral dosing, the mean Cmax, Tmax t1/2 and bioavailability values were equal to 1260+/-290 nM, 0.75+/-0.25 h. 45.1+/-10.2 h and 33.1%, respectively. The overall rat behavioral profile of this compound suggests that it may be an anxiolytic drug with a low motor side effect liability.     

Secondary structure of antisauvagine analogues is important for CRF receptor antagonism: development of antagonists with increased potency and receptor selectivity

Antisauvagine-30 (aSVG) is the only high-affinity antagonist for the corticotropin-releasing factor (CRF) type 2 (CRF(2)) receptor. A structure-activity relationship study was performed to pinpoint residues conferring aSVG's selectivity. The aSVG-analogues being N-terminally extended by one or two residues or containing the Ala(22)Arg(23)Ala(24) (ARA-motif) of CRF, were synthesized. Additionally, a lactam bridge between positions 29 and 32 was introduced. The modified peptides were analyzed for alpha-helicity properties, binding affinities and antagonistic potencies at the rat CRF(1) and mouse CRF(2B) receptors. While N-terminal prolongation and replacement of D-Phe(11) by Tyr(11) increased the affinity for the CRF(2) receptor, the introduction of the ARA motif resulted in a loss of CRF(2) receptor selectivity. These data show that aSVG(10-40) analogues are more potent CRF(2) receptor antagonists than aSVG(11-40) peptides, while introduction of the ARA-motif or a cyclic constraint between residues 29 and 32 favors binding to the CRF(1) receptor.     

High-affinity binding of urocortin and astressin but not CRF to G protein-uncoupled CRFR1

The structure-activity relationship (SAR) between the recently identified neuropeptide urocortin (Ucn) and corticotropin-releasing factor (CRF) receptor, type 1 (CRFR1), has been investigated. To this end, rat Ucn (rUcn), ovine CRF (oCRF) and chimeric peptides of rUcn and oCRF were synthesized and tested for their binding affinity and potency to stimulate cAMP production in human embryonic kidney (HEK) 293 cells stably transfected with cDNA encoding rat CRFR1 (rCRFR1). In binding studies with [125I-TyrO]oCRF or [3H-Leu9]rUcn as radioligand, it was observed that rUcn but not oCRF bound in a similar fashion as the CRF antagonist astressin with high affinity to rCRFR1 coupled to G protein or uncoupled from G protein by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Consequently, rUcn was found to exert a significantly lower potency than oCRF to stimulate cAMP accumulation in transfected cells. CD spectroscopic investigations and reverse-phase HPLC (RPHPLC) retention behavior of the peptides suggested a more pronounced amphipatic alpha-helical character of rUcn when compared to oCRF and the chimeric peptides.     

Design of a potent, soluble glucokinase activator with excellent in vivo efficacy

The optimisation of a series of glucokinase activators is described, including attempts to uncouple the relationship between potency and plasma protein binding, and to better understand the key pharmacokinetic properties of the series. The use of unbound clearance as an optimisation parameter facilitated the identification of GKA50, a compound which combines excellent potency and pharmacokinetics with good free drug levels and solubility, and exhibits in vivo efficacy at 1mg/kg po in an acute rat OGTT model.     

Corticotropin-releasing factor inhibits the acute inflammatory response of rat pawskin to cold injury

The anti-inflammatory effects of human/rat corticotropin-releasing factor (CRF), a 41-residue peptide hormone, on an experimental model of cold injury were examined. Male albino rats were anesthetized with sodium pentobarbital 60 mg/kg ip and the paws immersed for 1 or 2 min in a 22% NaCl solution maintained at -20 +/- 0.5 degrees C. Swelling in response to cold was measured by changes in paw volume using the fluid displacement method, and protein leakages from blood vessels were measured using Evans blue and Monastral blue dyes. Thirty minutes after cold exposure the paw volume increased from 1.5 +/- 0.1 to 2.4 +/- 0.1 ml/paw and the Evans blue content increased from 4 +/- 1 to 178 +/- 9 micrograms/pawskin. These responses to cold were inhibited by 40 to 60% after CRF was injected 56 micrograms/kg sc 30 min before or 28 micrograms/kg iv 10 min before or 5 min after cold exposures. Microscopic studies of the skin showed that CRF reduced leakage of Monastral blue pigment from the vascular compartment into the walls of capillaries and venules. The anti-inflammatory effects of CRF were blocked by alpha-helical CRF(9-41), a CRF receptor antagonist, injected 92 micrograms/kg iv 5 min before and 15 min before cold exposure.     

The brain corticotropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. Evidence from chemical cross-linking studies

The ligand binding subunits of the corticotropin-releasing factor (CRF) receptors in brain and anterior pituitary of a number of species have been identified by chemical affinity cross-linking using the homobifunctional cross-linking agent disuccinimidyl suberate and 125I-Tyr0-oCRF (ovine CRF). In homogenates of rat, monkey, and human cerebral cortex, 125I-Tyr0-oCRF was covalently incorporated into a protein of Mr = 58,000. Under identical conditions in the anterior pituitary of rat, monkey, cow, and pig, 125I-Tyr0-oCRF was incorporated into a protein of apparent Mr = 75,000. The specificity of the labeling was typical of the CRF binding site since both the cerebral cortex- and pituitary-labeled proteins exhibited the appropriate pharmacological rank order profile characteristic of the CRF receptor (Nle21,Tyr32-oCRF approximately equal to rat/human CRF approximately equal to ovine CRF approximately equal to alpha-helical CRF(6-41) greater than alpha-helical oCRF(9-41) greater than or equal to oCRF(7-41) greater than rat/human CRF(1-20) approximately equal to vasoactive intestinal peptide). In addition to the major labeled proteins, 125I-Tyr0-oCRF was incorporated into higher molecular weight peptides which may represent precursors and into lower molecular weight components which may represent fragments of the major labeled proteins or altered forms of the CRF binding subunit. In summary, these data indicate a heterogeneity between brain and pituitary CRF receptors with the ligand binding subunit of the brain CRF receptor residing on a Mr = 58,000 protein, while in the anterior pituitary, the identical binding subunit resides on a protein of apparent Mr = 75,000.     

NKP608: a selective NK-1 receptor antagonist with anxiolytic-like effects in the social interaction and social exploration test in rats

NKP608 is a non-peptidic derivative of 4-aminopiperidine which acts as a selective, specific and potent antagonist at the neurokinin-1 (NK-1) receptor both in vitro and in vivo. In vitro, the binding of NKP608 to bovine retina was characterized by an IC50 of 2.6+/-0.4 nM, whereas the compound's affinity to other receptor binding sites, including NK-2 and NK-3, was much lower. Species differences in IC(50) values with NKP608 were less pronounced than with previously described NK-1 receptor antagonists, being 13+/-2 and 27+/-2 nM in gerbil midbrain and rat striatum, respectively. In vivo, using the hind foot thumping model in gerbils, NKP608 exhibited a potent NK-1 antagonistic activity following oral administration (ID(50)=0.23 mg/kg; 2 h pretreatment), supporting a central activity of NKP608. The compound had a long duration of action with an ID(50) value of 0. 15 mg/kg p.o. and 0.38 mg/kg p.o. following a pretreatment of 5 and 24 h, respectively. Following a subchronic administration for 7 consecutive days (once daily) there was no evidence for the development of tolerance or accumulation. In the social interaction test performed in a highly illuminated, unfamiliar test arena, NKP608 specifically increased the time the two rats spent in social contact, and there was no concomitant increase in parameters reflecting general activity, i.e. ambulation (number of square entries) or the number of rearings. Active social time was maximally increased at a dose range of 0.01-1 mg/kg p.o. NKP608, the effect being weaker or absent at both lower (0.001 mg/kg p.o.) and higher (10 mg/kg p.o.) doses. A comparable bell-shaped dose-response relation was seen in the social exploration test in rats. In this modified resident/intruder paradigm, maximal increase in social contact of the intruder rat directed towards the resident rat was seen at a similar dose range (0.03-3 mg/kg p.o.) The effects observed following an acute oral administration of NKP608 were comparable to those seen following a treatment with the well-known benzodiazepine, chlordiazepoxide, in both these tests. These findings indicate that NKP608 exhibits an anxiolytic-like effect and that this effect, as concluded from the observed antagonism of the hind foot thumping induced by i.c.v. administration of the NK-1 receptor agonist SPOMe, is centrally mediated. This makes this compound a potentially promising candidate for treating anxiety-related disorders in humans.     

Optimization of 3-phenylpyrazolo[1,5-a]pyrimidines as potent corticotropin-releasing factor-1 antagonists with adequate lipophilicity and water solubility

In our efforts to identify potent CRF(1) antagonists with proper physicochemical properties, a series of 3-phenylpyrazolo[1,5-a]pyrimidines bearing polar groups, such as amino, hydroxyl, methoxy, sulfoxide, were designed and synthesized. Several positions of the core structure were identified, where a polar group was tolerated with slight reduction in receptor binding. NBI 30545 (18n) was found to have good binding affinity and potent antagonistic activity at the human CRF(1) receptor. Moreover, this compound had proper lipophilicity (log D = 2.78) and good solubility in water (>10mg/mL), and exhibited good plasma and brain exposure when given orally.     

Effect of epostane, ZK 98299, and ZK 98734 on the interruption of pregnancy in the rat

The objective of these studies was to determine whether treatment of 10-day pregnant rats with a combination of epostane (a progesterone biosynthesis inhibitor) and either ZK 98299 or ZK 98734 (progesterone receptor antagonists) would result in additive or synergistic effects on the interruption of pregnancy. When these compounds were tested individually, the order of potency in interrupting pregnancy was ZK 98734 greater than ZK 98299 greater than epostane (50% effective doses 1.3, 4.0, and 35 mg/kg, respectively). Epostane and ZK 98299 were then tested in combination. When epostane was given either 4 h prior to or concurrently with ZK 98299, the combined drug treatment resulted in a significant additive increase in interceptive activity compared to when ZK 98299 was administered alone. In vitro binding studies showed that ZK 98299 and ZK 98734 bound to the rat uterine progesterone receptor in vitro with approximately equal affinity. ZK 98734 bound to the rat thymus glucocorticoid receptor and to the rat ventral prostate androgen receptor with a greater affinity than ZK 98299. The affinity of ZK 98299 for the rat uterine estrogen receptor was weak while the binding of ZK 98734 was not detectable. Thus, the in vitro receptor binding profiles observed were consistent with the known progesterone and glucocorticoid antagonist activities of ZK 98299 and ZK 98734. Overall these findings show that the interceptive activity of epostane and ZK 98299, agents that exert their interceptive activity via different molecular mechanisms, is additive in the 10-day pregnant rat.     

Pyrazolo-[1,5-a]-1,3,5-triazine corticotropin-releasing factor (CRF) receptor ligands

The syntheses and rat CRF receptor binding affinities of 'retro-pyrazolotriazine' corticotropin-releasing factor (CRF) ligands 4 are reported. Some have high affinity for rat CRF receptors (K(i)< or =10 nM). The data provide additional support for the hypothesis that it is possible to interchange isosteric cores with similar electronic properties in the design of high-affinity CRF receptor ligands, provided the peripheral pharmacophore elements are maintained in the same three-dimensional array.     

Novel high-affinity photoactivatable antagonists of corticotropin-releasing factor (CRF) photoaffinity labeling studies on CRF receptor, type 1 (CRFR1).

Novel photoactivatable antagonists of human/rat corticotropin-releasing factor (h/rCRF) have been synthesized and characterized. The N-terminal amino acid D-phenylalanine in astressin ?cyclo(30-33) [D-Phe12, Nle21,38, Glu30, Lys33]h/rCRF-(12-41)?, a potent CRF peptide antagonist, was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. Additionally, His32 of astressin was substituted by either alanine or tyrosine for specific radioactive labeling with 125I at either His13 or Tyr32, respectively. The photoactivatable CRF antagonists were tested for their ability to displace 125I-labeled Tyr0 ovine CRF ([125I-labeled Tyr0]oCRF) in binding experiments and to inhibit oCRF-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1) or human Y-79 retinoblastoma cells known to carry endogenous functional human CRFR1 (hCRFR1). ATB-cyclo(30-33)[Nle21,38, Glu30, Ala32, Lys33]h/rCRF-(13-41) (compound 1) was found to bind with higher affinity to rat or human CRFR1 when compared with ATB-cyclo(30-33)[Nle21,38, Glu30, Tyr32, Lys33]h/rCRF-(13-41) (compound 2) and exhibited higher inhibition of oCRF-stimulated cAMP accumulation in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK-rCRFR1 cells) or Y-79 cells. A highly glycosylated, 66-kDa protein was identified with SDS/PAGE, when the radioactively iodinated compounds 1 or 2 were covalently linked to rCRFR1. The specificity of the photoactivatable 125I-labeled CRF antagonists was demonstrated with SDS/PAGE by the finding that these analogs could be displaced from the receptor by their corresponding nonlabeled form, but not other unrelated peptides such as vasoactive intestinal peptide. The observed molecular size of the receptor was in agreement with the size of CRFR1 found in rat pituitary (66 kDa), but was significantly larger than the size of CRFR1 found in rat cerebellum and olfactory bulb (53 kDa).     

Brain neurotransmitter receptor-binding characteristics in rats after oral administration of haloperidol, risperidone and olanzapine

The present study was conducted to characterize the binding of neurotransmitter receptors (dopamine D(2), serotonin 5-HT(2), histamine H(1), adrenaline alpha(1) and muscarine M(l) receptors) in the rat's brain after the oral administration of haloperidol, risperidone, and olanzapine. Haloperidol at 1 and 3 mg/kg displayed significant activity to bind the D(2) receptor (increase in the Kd value for [(3)H]raclopride binding) in the corpus striatum with little change in the activity toward the 5-HT(2) receptor (binding parameters for [(3)H]ketanserin). In contrast, risperidone (0.1-3 mg/kg) showed roughly 30 times more affinity for the 5-HT(2) receptor than D(2) receptor. Also, olanzapine (1-10 mg/kg) was most active toward the H(1) receptor in the cerebral cortex, corpus striatum, and hippocampus, was less active in binding 5-HT(2) and D(2) receptors, and showed the least affinity for alpha(1) and M(1) receptors. In conclusion, haloperidol and risperidone administered orally selectively bind D(2) and 5-HT(2) receptors, respectively, in the rat brain, while olanzapine binds H(1), 5-HT(2), and D(2) receptors more than alpha(1) and M(1) receptors.     

Residues of corticotropin releasing factor-binding protein (CRF-BP) that selectively abrogate binding to CRF but not to urocortin 1

Corticotropin releasing factor-binding protein (CRF-BP) binds CRF and urocortin 1 (Ucn 1) with high affinity, thus preventing CRF receptor (CRFR) activation. Despite recent progress on the molecular details that govern interactions between CRF family neuropeptides and their cognate receptors, little is known concerning the mechanisms that allow CRF-BP to bind CRF and Ucn 1 with picomolar affinity. We conducted a comprehensive alanine scan of 76 evolutionarily conserved residues of CRF-BP and identified several residues that differentially affected the affinity for CRF over Ucn 1. We determined that both neuropeptides derive their similarly high affinity from distinct binding surfaces on CRF-BP. Alanine substitutions of arginine 56 (R56A) and aspartic acid 62 (D62A) reduce the affinity for CRF by approximately 100-fold, while only marginally affecting the affinity for Ucn 1. The selective reduction in affinity for CRF depends on glutamic acid 25 in the CRF peptide, as substitution of Glu(25) reduces the affinity for CRF-BP by approximately 2 orders of magnitude, but only in the presence of both Arg(56) and Asp(62) in human CRF-BP. We show that CRF-BP(R56A) and CRF-BP(D62A) have lost the ability to inhibit CRFR1-mediated responses to CRF that activate luciferase induction in HEK293T cells and ACTH release from cultured rat anterior pituitary cells. In contrast, both CRF-BP mutants retain the ability to inhibit Ucn 1-induced CRFR1 activation. Collectively our findings demonstrate that CRF-BP has distinct and separable binding surfaces for CRF and Ucn 1, opening new avenues for the design of ligand-specific antagonists based on CRF-BP.