Human Corticotropin-releasing Factor Receptor Research Articles

In silico and in vitro evaluation of newly synthesized pyrazolo-pyridine fused tetrazolo-pyrimidines derivatives as potential anticancer and antimicrobial agents

Diversely functionalized pyrazolo-pyridine fused tetrazolo-pyrimidines 10aa-am and 10ba-bn were successfully synthesized via a catalyst-free synthetic protocol with moderate to very good yields. The compounds were evaluated for cytotoxicity against MCF-7 and HEK-293 cells using MTT assay. Among the tested compounds, 10ab (IC50- 23.83 µM) and 10ah (IC50- 23.30 µM) demonstrated the highest potency against MCF-7 cells, while 10bc (IC50- 14.46 µM) and 10bh (IC50- 2.53 µM) exhibited excellent cytotoxicity against HEK-293 cells. Additionally, antibacterial screening was performed against three Gram-negative bacteria (E. coli, P. aeruginosa, and S. enterica) and three Gram-positive bacteria (S. aureus, B. megaterium, and B. subtilis) using broth dilution method, while antifungal activity was assessed against three fungal strains (A. niger, Penicillium, and S. cerevisiae) using agar well diffusion method. In antimicrobial screening, the majority of the compounds demonstrated significant antibacterial efficacy compared to antifungal activity. We also conducted comprehensive computational studies, including DFT calculations, molecular docking and dynamics, and drug-likeness assessments. In the DFT study, compounds 10ac and 10bc displayed stable conformations, indicating their potential for higher therapeutic activity. Molecular docking analyses revealed compelling interactions, with compound 10ah demonstrating docking score −7.42 kcal/mol against catalytical domain PARP1 (PDB ID: 7KK4) and 10bh exhibiting a best docking score −10.77 kcal/mol against human corticotropin-releasing factor receptor 1 (PDB ID: 4Z9G). A 100 ns molecular dynamics (MD) simulation study of compounds 10ah and 10bh revealed the stable conformation and binding energy in a stimulating environment. In drug-likeness assessments, both the compounds 10ah and 10bh adhere all the established guidelines. Communicated by Ramaswamy H. Sarma

Strategy for the Thermostabilization of an Agonist-Bound GPCR Coupled to a G Protein.

Structure determination of G protein-coupled receptors (GPCRs) in the inactive state bound to high-affinity antagonists has been very successful through the implementation of a number of protein engineering and crystallization strategies. However, the structure determination of GPCRs in their fully active state coupled to a G protein is still very challenging. Recently, mini-G proteins were developed, which recapitulate the coupling of a full heterotrimeric G protein to a GPCR despite being less than one-third of the size. This allowed the structure determination of the agonist-bound adenosine A2A receptor (A2AR) coupled to mini-Gs. Although this is extremely encouraging, A2AR is very stable compared with many other GPCRs, particularly when an agonist is bound. In contrast, the agonist-bound conformation of the human corticotropin-releasing factor receptor is considerably less stable, impeding the formation of good quality crystals for structure determination. We have therefore developed a novel strategy for the thermostabilization of a GPCR-mini-G protein complex. In this chapter, we will describe the theoretical and practical principles of the thermostability assay for stabilizing this complex, discuss its strengths and weaknesses, and show some typical results from the thermostabilization process.

An insight into antagonist binding and induced conformational dynamics of class B GPCR corticotropin-releasing factor receptor 1.

The human corticotropin-releasing factor receptor type 1 (CRF1R) is a class B G-protein-coupled receptor (GPCR), which mediates the response to stress and has been considered as a drug target for depression and anxiety. Based on the CRF1R-antagonist crystal structure, we study the binding mechanism of two distinct antagonists, CP-376395 and MTIP, and the dynamics behaviors of CRF1R induced by an antagonist binding. Key residues interacting with both antagonists and residues specifically binding to one of them are identified. Both antagonists interact with Asn283, Phe203, Met206, Leu280, Tyr316, Leu323, Leu287, Phe284, Val279, Leu319, Phe207, Gly210 and Phe362. CP-376395 specifically binds to Glu209 and Phe160, while MTIP specifically binds to Leu320, Leu213, Ile290, Phe162 and Val313. The total binding free energy of MTIP is lower than that of CP-376395; this is consistent with the experimental observation that MTIP shows higher binding affinity than CP-376395. The conformational dynamic behaviors of antagonist bound holo-CRF1R were found to be different from those of apo-CRF1R in three aspects: (i) the "ionic lock" between side chains of Arg151 in TM2 and Glu209 in TM3 was broken in apo-CRF1R, but was formed in holo-CRF1Rs; (ii) Phe203 in TM3 and Tyr327 in TM6 were in close proximity to each other in apo-CRF1R, while they were far apart resulting from the shift of TM6 in holo-CRF1Rs; and (iii) the "rotamer toggle switch", Tyr327/Leu323/Phe284, adopted different rotameric conformations in apo-CRF1R and holo-CRF1Rs. We hope that our results could be helpful in further development of the drug design of CRF1R.

Meet the B family

The first crystal structures of class B G-protein-coupled receptors have been solved. They reveal features that might inform drug-development strategies for diseases ranging from osteoporosis to diabetes. See Articles p.438 & p.444 G-protein-coupled receptors (GPCRs) are membrane proteins that act as sensors for a broad range of extracellular signals, including photons, ions, small organic molecules and even entire proteins. Approximately a third of known drugs target GPCRs. Until now all the published structures of GPCRs have been from class A GPCRs. In this issue of Nature two groups independently report the crystal structures of two receptors of the B family, the second largest of four family divisions based on primary sequence and pharmacology. Hollenstein et al. solved the structure of human corticotropin-releasing factor receptor 1. This GPCR binds to corticotropin-releasing hormone, a potent mediator of endocrine, autonomic, behavioral and immune responses to stress. In all known class A GPCRs, the ligand-binding sites are close to the extracellular boundaries of the receptors; in this GPCR, the antagonist (CP-376395) binds in a hydrophobic pocket located in the cytoplasmic half of the V-shaped receptor. Siu et al. solved the X-ray crystal structure of the human glucagon receptor. This GPCR binds to the glucagon peptide, which triggers the release of glucose from the liver, making it a potential drug target for type 2 diabetes. The structure reveals a larger ligand-binding pocket than that seen in class A GPCRs.

Structure of class B GPCR corticotropin-releasing factor receptor 1

Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.

High-Affinity CRF1 Receptor Antagonist NBI-34041: Preclinical and Clinical Data Suggest Safety and Efficacy in Attenuating Elevated Stress Response

There is an extensive evidence that corticotropin releasing factor (CRF) is hypersecreted in depression and anxiety, and blockade of CRF could have therapeutic benefit. We report preclinical data and the results of a clinical Phase I study with the novel nonpeptide CRF(1) antagonist NBI-34041/SB723620. Preclinical data conducted with different cell lines expressing human CRF receptors and in Wistar and Sprague-Dawley rats indicate that NBI-34041 is effective in reducing endocrine responses to pharmacological and behavioral challenge mediated by CRF(1) receptors. These specific properties and its well-documented safety profile enabled a clinical Phase I study with 24 healthy male subjects receiving NBI-34041 (10, 50, or 100 mg) or placebo for 14 days. Regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis was evaluated by intravenous stimulation with 100 microg of human CRF. Psychosocial stress response was investigated with the Trier Social Stress Test (TSST). Treatment with NBI-34041 did not impair diurnal adrenocorticotropic hormone (ACTH) and cortisol secretion or CRF evoked ACTH and cortisol responses but attenuated the neuroendocrine response to psychosocial stress. These results suggest that NBI-34041 is safe and does not impair basal regulation of the HPA system but improves resistance against psychosocial stress. NBI-34041 demonstrates that inhibition of the CRF system is a promising target for drug development against depression and anxiety disorders.

Design and Synthesis of Tricyclic Imidazo[4,5-b]pyridin-2-ones as Corticotropin-Releasing Factor-1 Antagonists

The synthesis and SAR studies of tricyclic imidazo[4,5-b]pyridin-2-ones as human corticotropin-releasing factor receptor (CRF(1)) antagonists are discussed herein. Compound 16g was identified as a functional antagonist that inhibited CRF-stimulated cyclic adenosine monophosphate production and CRF-induced adrenocorticotrophic hormone release. Pharmacokinetics studies in rats showed that 16g was orally bioavailable, had good brain penetration, and had a moderate half-life. In our effort to identify CRF(1) antagonists with improved pharmacokinetics properties, 16g exhibited a favorably lower volume of distribution.

Structure-activity studies on the corticotropin releasing factor antagonist astressin, leading to a minimal sequence necessary for antagonistic activity.

Corticotropin Releasing Factor (CRF) antagonists are considered promising for treatment of stress-related illnesses such as major depression and anxiety-related disorders. We report here the design, synthesis and biological evaluation of 91 truncated astressin analogues in order to deduce the pharmacophoric amino acid residues. Such truncated peptides may serve as valuable lead structures for the development of new small, non-peptide-based CRF antagonists. N-Terminal truncation of astressin led to active CRF antagonists that are substantially reduced in size and are selectively active at the human CRF receptor type 1 in vitro and in vivo. Subsequently, an alanine scan in combination with further truncated derivatives led to the proposal of a new pharmacophoric model of peptide-based CRF antagonists. It was found that the astressin(27-41)C sequence is the shortest active CRF antagonist. The first eight N-terminal amino acid residues were found to be an important structural determinant and were replaceable by alanine residues, thus enhancing the alpha-helical propensity. A covalent structural constraint is of utmost importance for the preorganization of the C-terminal amino acid residues. The C-terminal heptapeptide sequence, however, was found to be crucial for the antagonistic activity, since substitution or deletion of any residue led to inactive compounds.

Human corticotropin-releasing factor type 1 receptor analysis with white blood cells mRNAs and DNA

The human corticotropin-releasing factor receptor type 1 (hCRF-R1) functional transcript is mainly expressed in the anterior pituitary corticotrophs, a tissue usually not available for clinical investigation. Splice variants translated into defective isoforms of the receptor have been described in few peripheral tissues. The aim of this work was to determine whether peripheral white blood cells from healthy individuals, an accessible tissue for clinical investigation, were suitable for the analysis of the hCRF-R1 transcript and gene. We report that: (i) specific amplification of the hCRF-R1 transcript from peripheral white blood cells mRNAs is feasible; (ii) this transcript is similar to the functional transcript; (iii) the draft sequence of chromosome 17 and unrelated sequences allow direct sequencing of all 14 exons of the gene, adjacent splice sites and related branch points. In conclusion, these approaches would be suitable for studies in patients having isolated secondary glucocorticoids deficiency to implicate the hCRH-R1 in the etiology of the disease.

Mapping of the ligand-selective domain of the Xenopus laevis corticotropin-releasing factor receptor 1: implications for the ligand-binding site.

The nonselective human corticotropin-releasing factor receptor 1 (hCRF-R1) and the ligand-selective Xenopus CRF-R1 (xCRF-R1) were compared. To understand the interactions of sauvagine and ovine CRF, both high-affinity ligands for hCRF-R1 but surprisingly weak ligands for xCRF-R1, chimeric receptors of hCRF-R1 and xCRF-R1 followed by double or multiple point mutations were constructed. Binding studies and cAMP assays demonstrated that the N-terminal domain exhibited the complete ligand selectivity of xCRF-R1. The important region was mapped between amino acids 70 and 89; replacement of amino acids Arg76, Asn81, Gly83, Leu88, and Ala89 in hCRF-R1 with the corresponding amino acids of xCRF-R1 (Gln76, Gly81, Val83, His88, and Leu89) resulted in a receptor that had approximately 30-fold higher affinity for human/rat CRF than for sauvagine. Mutagenesis of these amino acids in xCRF-R1 to the human sequence completely abolished the ligand selectivity of xCRF-R1. Mutagenesis of amino acids 88 and 89 in hCRF-R1 or xCRF-R1 had only a minor (approximately 2.5-fold) effect on the ligand selectivity of the mutant receptor. Substitution of Arg76, Asn81, and Gly83 in hCRF-R1 with the corresponding sequence of xCRF-R1 (Gln76, Gly81, and Val83) resulted in a receptor with approximately 11-fold higher affinity for human/rat CRF compared with ovine CRF or sauvagine. When only two of these three amino acids were mutated, no effect on the ligand selectivity was observed. On the basis of these data, it is suggested that amino acids 70-89 of CRF-R1 are important for the ligand binding site.

Localization of Agonist- and Antagonist-Binding Domains of Human Corticotropin-Releasing Factor Receptors

Localization of Agonist- and Antagonist-Binding Domains of Human Corticotropin-Releasing Factor Receptors

Localization of agonist- and antagonist-binding domains of human corticotropin-releasing factor receptors.

The CRF receptors, CRFR1 and CRFR2, are members of the G protein-coupled receptor superfamily. Despite their considerable sequence similarity, CRFR1 and CRFR2 have quite different affinities for the peptide ligand rat/human CRF. Previous studies using chimeric receptors between human CRFR1 and CRFR2 have identified three potentially important regions in the second and third extracellular domains of CRF receptor for the binding of rat/human CRF. The present report further demonstrates that these same three regions also affect the binding of urocortin and sauvagine, two other members of the CRF peptide family, albeit to different extents. We also show that a fourth region in the third extracellular domain, Asp254, has been identified to be important for sauvagine but not CRF or urocortin binding. Thus, the three peptide ligands not only interact with a different set of regions on CRFR1 and CRFR2 but also differentially interact with some of the same regions. These data could, at least in part, account for the much higher affinity of CRFR2 for urocortin and sauvagine compared with rat/human CRF. We have also identified two amino acid residues, His199 in the third transmembrane domain and Met276 in the fifth transmembrane domain, that are important for binding the non-peptide high-affinity CRFR1 antagonist NBI 27914. Mutations of His199 and Met276 to the corresponding amino acids in CRFR2 each decreased the binding affinity of NBI 27914 for CRFR1 by 40- and 200-fold, respectively. This suggests that the transmembrane regions are critically important in forming the binding pocket for the nonpeptide antagonist.

Molecular cloning and function expression of a diuretic hormone receptor from the house cricket, Acheta domesticus

Insect diuretic hormones regulate fluid and ion secretion and the receptors with which they interact are attractive targets for new insect control agents. Recently, a diuretic hormone receptor from the moth Manduca sexta was isolated by expression cloning and found to be a member of the calcitonin/secretin/corticotropin releasing factor family of G-protein coupled receptors [Reagan J. D. (1994) J. Biol. Chem. 269, 9–12]. Degenerate oligonucleotides were designed based upon conserved regions in this receptor family and used to isolate a diuretic hormone receptor from the house cricket, Acheta domesticus. The complementary DNA isolated encodes a protein consisting of 441 amino acids with seven putative membrane spanning regions. Interestingly, unlike the M. sexta diuretic hormone receptor, the cricket diuretic hormone receptor contains a putative signal sequence. The receptor shares 53% and 38% sequence identity with the M. sexta diuretic hormone and human corticotropin releasing factor receptors respectively. When expressed in COS-7 cells, the receptor binds A. domesticus diuretic hormone with high affinity and stimulates adenylate cyclase with high potency. Four other insect diuretic hormones are considerably less effective at stimulating adenylate cyclase in COS-7 cells transfected with the receptor. This is in contrast to the M. sexta diuretic hormone receptor which is stimulated by all five insect diuretic hormones with high potency.

A Variant of the Human Corticotropin-Releasing Factor (CRF) Receptor: Cloning, Expression, and Pharmacology

A variant of the human corticotropin-releasing factor receptor has been characterized. The variant has a 40-amino-acid deletion in the amino-terminal domain of the receptor and is the only form of this receptor detectable in the individual from which it was isolated. In contrast to the high affinity expressed by the normal human CRF receptor, the receptor variant fails to bind [ 125I]oCRF With high affinity in transfected COS-1 cells. High concentrations of human CRF are required to elevate intracellular cAMP levels in the cells transfected with the variant receptor; EC 50 values of 301 and 0.55 nM were obtained in cells expressing the receptor variant and normal CRF receptors, respectively.