FRET-based cell-based primary high throughput screening assay to identify antagonists of the orexin 1 receptor (OX1R; HCRTR1)
Name: FRET-based cell-based primary high throughput screening assay to identify antagonists of the orexin 1 receptor (OX1R; HCRTR1). ..more
BioActive Compounds: 5748
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Patricia McDonald, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01 DA023915-02
Grant Proposal PI: Patricia McDonald
External Assay ID: OX1R_ANT_FRET_1536_1X%INH PRUN IP-ONE
Name: FRET-based cell-based primary high throughput screening assay to identify antagonists of the orexin 1 receptor (OX1R; HCRTR1).
Heterotrimeric G-protein coupled receptors (GPCRs) are major targets for disease therapeutics, due in part to their broad tissue distribution, structural diversity, varied modes of action, and disease association (1-4). Most non-sensory GPCRs are expressed in the brain and regulate critical neuronal functions involved in feeding, sleep, mood, and addiction (5, 6). For example, in the lateral hypothalamic region of the brain, two orexin neuropeptides (orexin A and orexin B) derived from proteolytic processing of the same orexin precursor (7), signal through the Gq-coupled GPCRs OX1R and OX2R to stimulate food consumption (8, 9). OX1R binds orexin A selectively, while OX2R binds both orexin A and orexin B. Recently, signaling by orexin A through OX1R has been shown to play a critical role in cocaine-seeking behavior (10) and morphine withdrawal (6). Additional studies reveal OX1R involvement in behavioral plasticity (11), the sleep-wake cycle (12, 13), and gastric acid secretion (14), and that OX1R may bind other neuropeptides such as neuropeptide Y and secretin (15). As a result, the identification of a selective OX1R antagonist would serve as a useful tool for exploring orexin biology, and the role of OX1R in drug addiction.
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3. Pan, HL, Wu, ZZ, Zhou, HY, Chen, SR, Zhang, HM and Li, DP, Modulation of pain transmission by G-protein-coupled receptors. Pharmacol Ther, 2008. 117(1): p. 141-61.
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5. Gainetdinov, RR, Premont, RT, Bohn, LM, Lefkowitz, RJ and Caron, MG, Desensitization of G protein-coupled receptors and neuronal functions. Annu Rev Neurosci, 2004. 27: p. 107-44.
6. Sharf, R, Sarhan, M and Dileone, RJ, Orexin mediates the expression of precipitated morphine withdrawal and concurrent activation of the nucleus accumbens shell. Biol Psychiatry, 2008. 64(3): p. 175-83.
7. de Lecea, L, Kilduff, TS, Peyron, C, Gao, X, Foye, PE, Danielson, PE, Fukuhara, C, Battenberg, EL, Gautvik, VT, Bartlett, FS, 2nd, Frankel, WN, van den Pol, AN, Bloom, FE, Gautvik, KM and Sutcliffe, JG, The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci U S A, 1998. 95(1): p. 322-7.
8. Sakurai, T, Amemiya, A, Ishii, M, Matsuzaki, I, Chemelli, RM, Tanaka, H, Williams, SC, Richardson, JA, Kozlowski, GP, Wilson, S, Arch, JR, Buckingham, RE, Haynes, AC, Carr, SA, Annan, RS, McNulty, DE, Liu, WS, Terrett, JA, Elshourbagy, NA, Bergsma, DJ and Yanagisawa, M, Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell, 1998. 92(4): p. 573-85.
9. Hara, J, Beuckmann, CT, Nambu, T, Willie, JT, Chemelli, RM, Sinton, CM, Sugiyama, F, Yagami, K, Goto, K, Yanagisawa, M and Sakurai, T, Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron, 2001. 30(2): p. 345-54.
10. Smith, RJ, See, RE and Aston-Jones, G, Orexin/hypocretin signaling at the orexin 1 receptor regulates cue-elicited cocaine-seeking. Eur J Neurosci, 2009. 30(3): p. 493-503.
11. Winrow, CJ, Tanis, KQ, Reiss, DR, Rigby, AM, Uslaner, JM, Uebele, VN, Doran, SM, Fox, SV, Garson, SL, Gotter, AL, Levine, DM, Roecker, AJ, Coleman, PJ, Koblan, KS and Renger, JJ, Orexin receptor antagonism prevents transcriptional and behavioral plasticity resulting from stimulant exposure. Neuropharmacology, 2009.
12. Dugovic, C, Shelton, JE, Aluisio, LE, Fraser, IC, Jiang, X, Sutton, SW, Bonaventure, P, Yun, S, Li, X, Lord, B, Dvorak, CA, Carruthers, NI and Lovenberg, TW, Blockade of orexin-1 receptors attenuates orexin-2 receptor antagonism-induced sleep promotion in the rat. J Pharmacol Exp Ther, 2009. 330(1): p. 142-51.
13. Hagan, JJ, Leslie, RA, Patel, S, Evans, ML, Wattam, TA, Holmes, S, Benham, CD, Taylor, SG, Routledge, C, Hemmati, P, Munton, RP, Ashmeade, TE, Shah, AS, Hatcher, JP, Hatcher, PD, Jones, DN, Smith, MI, Piper, DC, Hunter, AJ, Porter, RA and Upton, N, Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci U S A, 1999. 96(19): p. 10911-6.
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orexin, Orexin 1 receptor, OX1, OX1R, OXR1, hypocretin-1 receptor, hypocretin, Hcrtr1, Hcrtr-1, GPCR, antagonism, inhibition, inhibit, inhibitor, addiction, relapse, cocaine, substance abuse, brain, CHO cells, inositol phosphate, IP, IP1, IP-One, IP one, IPOne, FRET, HTRF, TR-FRET, fluorescence, fluorescent, primary, primary screen, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that inhibit orexin 1 receptor (OX1R; Hcrtr1) activity. This assay employs the IP-One competitive immunoassay kit (Cisbio International) and CHO cells that stably express the Gq-coupled human receptor OX1R/Hcrtr1 (CHO-OX1R). This receptor signals through phospholipase C (PLC) to trigger the inositol phosphate (IP) cascade and IP1 production. Cryptate-labeled anti-IP1 antibody and D2-labeled IP1 are included for HTRF-based detection of receptor activity. In this assay, CHO-OX1R cells are incubated with test compounds, labeled IP1 antibody, and D2-IP1. Binding of the labeled IP1 antibody to D2-IP1 causes energy transfer from the antibody to the D2-IP1 molecule, increasing well FRET. Ligand-mediated OX1R activation stimulates endogenous cellular IP1 production by the cells, which competes with the labeled IP1 for binding to the labeled IP1-antibody, thereby reducing FRET. As designed, compounds that act as OX1R/Hcrtr1 antagonists will prevent receptor activation, reduce cellular IP1 production, reduce levels of IP1 available to compete with labeled D2-IP1, leading to increased interactions between D2-IP1 and the labeled anti-IP1 antibody, resulting in increased well FRET. Compounds are tested in singlicate at a nominal concentration of 6.6 uM.
The CHO/OX1R cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH) up to a confluency of approximately 30 million cells per flask. The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 25 mM HEPES, 350 ug/mL Geneticin, , and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
The day of the assay, 3000 cells in 3 uL of growth media were seeded into each well of 1536-well microtiter plates. Then, 20 nL of test compound in DMSO and antagonist controls in DMSO were added to the appropriate wells and plates were incubated at room temperature for 15 minutes. Next, 15 nL of Orexin-A agonist in DMSO (EC80 average response), or DMSO alone were dispensed to the appropriate wells. After incubation for 1 hour at 37 C, 5% CO2, and 95 % RH, 1 uL of D2-labeled IP1 (prepared in lysis buffer according to the manufacturer's protocol) was added to each well and centrifuged for 30 seconds. Next, 1 uL of Cryptate-labeled anti-IP1 antibody (prepared in lysis buffer according to the manufacturer's protocol) was added to each well, centrifuged for 30 seconds and plates were incubated at room temperature for 1 hour before measurement of well FRET. After excitation at 340 nm (with 30 nm bandwidth), well fluorescence was monitored at 618 nm (with 4 nm bandwith) for Cryptate and 671 nm (with 4 nm bandwidth) for D2-IP1, using the ViewLux microplate reader (Perkin Elmer).
For each well, a fluorescence ratio was calculated to normalize assay data, according to the following mathematical expression:
Ratio = I671nm / I618nm
I671nm represents the measured fluorescence emission at 671nm.
I618nm represents the measured fluorescence emission at 618nm.
Percent inhibition was calculated from the median ratio as follows:
% Inhibition = ( 1 -( ( Ratio_Test_Compound - Median_Ratio_High_Control ) / ( Median_Ratio_Low_Control - Median_Ratio_High_Control ) ) ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing Orexin A peptide challenge.
High_Control is defined as wells containing the antagonist SB-334867-A.
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-9, and for inactive compounds 36-0.
List of Reagents:
CHO/OX1R cells (supplied by Assay Provider)
IP-One Tb Kit (Cisbio, part 62IPAPEJ)
Hams F-12 media (Invitrogen, part 11765-054)
Geneticin (Invitrogen, part 10131-027)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140-071)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: Orexin A peptide (Tocris, part 1455)
Antagonist: SB-334867-A (Tocris, part 1960)
1536-well plates (Greiner, part 789072)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned Active/Inactive status based upon that campaigns specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)