Operation Poop Chute

JPET #150193

Supplemental Table 1.

Intrinsic activity and pharmacokinetic properties for a structurally related series of GhrR agonist compounds.

Binding affinities of compounds were determined by 12-point concentration-response curve analysis using membranes from Rapid Amplification of Gene Expression (RAGE)-based GhrR-expressing H4 glioma cells. Typically, 0.5 µg of membrane was incubated in the presence of 0.02 nM [35S]-MK-0677 in 25 mM HEPES (pH 7.2) with 10 mM MgCl2, 2 mM EGTA, and 0.1% BSA, in a total volume of 0.2 ml, for 1 hour in the presence of increasing concentrations of compounds. Reactions were terminated by rapid vacuum filtration over GFB filter plates coated with 0.1% polyethylenimine, and plates were then washed with 0.8 ml of binding buffer. Radioactivity was measured by scintillation spectroscopy on a TopCount (Perkin Elmer) scintillation counter. Binding displacement data were fit by nonlinear regression analysis using a four parameter logistic equation, and Ki values were determined using the Cheng-Prusoff equation. Compounds were tested in three separate experiments. A prototype of the class of molecules examined here, EX-1315 bound with high affinity to the human GhrR (Ki = 55 nM) while other related compounds in this class (EX-1314, EX-1966, EX-1968) showed higher affinities with Ki values of 2.1-2.5 nM.

Functional agonist activity of compounds was assessed by determining agonist-induced intracellular calcium mobilization using a FLIPR assay, as described previously (Li et al., 2005). Briefly, expression of human GhrR was enforced using RAGE in H4 glioma cells (Harrington et al., 2001). The EC50 of compounds (concentration at which 50% of maximal activation was achieved) was measured in triplicate in the FLIPR assay and intrinsic activity was compared to that of ghrelin, where the ghrelin signal represented 100% intrinsic functional activity. EX-1315 showed potent activity with an EC50 value of 1.5 nM. As with binding assays EX-1314, EX-1966 and EX-1968 showed higher intrinsic affinities with EC50 values of 0.3-0.4 nM.

Selectivity of compounds for the ghrelin receptor was determined by assessing their ability to stimulate the motilin receptor, which shares ~50% amino acid identity to the GhrR. Human motilin receptor-expressing cells were used to calculate EC50 values for intracellular calcium concentrations by a FLIPR assay. Human motilin and human ghrelin were used as positive and negative controls, respectively. None of the compounds tested showed agonist activity in HeLa cells expressing the human motilin receptor. In these assays, motilin showed potent activity (EC50 ~0.1 nM) whereas ghrelin lacked any stimulatory activity at this receptor.

For initial oral bioavailability and exposure (AUC) determinations, compounds were dissolved in 10% ethanol: 90% distilled water. Male Sprague-Dawley rats (250-300g) or C57Bl/6 mice (20g) were dosed either orally (5 mg/kg) or IV (1 mg/kg) with ghrelin agonists. Plasma samples were harvested in rats and mice over a 24 hour period and levels of each compound were determined using a liquid chromatography/mass spectrometry assay. In general, the compounds achieved low, but detectable, plasma levels that exceeded their EC50 values and thus activation of receptors in vivo was expected.

Supplemental References:

Harrington JJ, Sherf B, Rundlett S, Jackson PD, Perry R, Cain S, Leventhal C, Thornton M, Ramachandran R, Whittington J, Lerner L, Costanzo D, McElligott K, Boozer S, Mays R, Smith E, Veloso N, Klika A, Hess J, Cothren K, Lo K, Offenbacher J, Danzig J and Ducar M (2001) Creation of genome-wide protein expression libraries using random activation of gene expression. Nat Biotechnol19:440-445.

Li JJ, Wang H, Qu F, Musial C, Tino JA, Robl JA, Slusarchyk D, Golla R, Seethala R, Dickinson K, Giupponi L, Grover G, Sleph P, Flynn N, Murphy BJ, Gordon D, Kung M and Stoffel R (2005) Tetrahydroisoquinoline 1-carboxamides as growth hormone secretagogues. Bioorg Med Chem Lett15:1799-1802.