Identification of tetrapeptides from a mixture based positional scanning library that can restore nM full agonist function of the L106P, I69T, I102S, A219V, C271Y, and C271R human melanocortin-4 polymorphic receptors (hMC4Rs)

Abstract

Human obesity has been linked to genetic factors and single nucleotide polymorphisms (SNPs). Melanocortin-4 receptor (MC4R) SNPs have been associated with up to 6% frequency in morbidly obese children and adults. A potential therapy for individuals possessing such genetic modifications is the identification of molecules that can restore proper receptor signaling and function. These compounds could serve as personalized medications improving quality of life issues as well as alleviating diseases symptoms associated with obesity including type 2 diabetes. Several hMC4 SNP receptors have been pharmacologically characterized in vitro to have a decreased, or a lack of response, to endogenous agonists such as α-, β-, and γ2-melanocyte stimulating hormones (MSH) and adrenocorticotropin hormone (ACTH). Herein we report the use of a mixture based positional scanning combinatorial tetrapeptide library to discover molecules with nM full agonist potency and efficacy to the L106P, I69T, I102S, A219V, C271Y, and C271R hMC4Rs. The most potent compounds at all these hMC4R SNPs include Ac-His-(pI)DPhe-Tic-(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Tic-(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Arg-(pI)Phe-NH2, and Ac-Arg-(pCl)DPhe-Tic-(pNO2)DPhe-NH2, revealing new ligand pharmacophore models for melanocortin receptor drug design strategies.

Figure 1

Putative locations of the I69T, I102S, L106P, A219V, C271Y, and C271R SNPs within the serpentine structure of the hMC4R.

Figure 2

Summary of the previously reported in vitro pharmacological characterization of the L106P hMC4R SNP as compared with the wild-type (WT) hMC4R control stably expressed in HEK293 cells.^, (A) Fluorescence activated cell sorting (FACS) demonstrating that both the WT and L106P hMC4R proteins are expressed within the cell at approximately the same levels (white box) yet reduced cell surface expression is observed for the L106P hMC4R (black box). (B) Represents the total specific binding counts per minute (cpm) of radiolabeled I^125-NDP-MSH agonist binding to the cells stably expressing the WT and L106P hMC4Rs. (C) Illustrates the ligand binding affinity curves of the WT and L106P hMC4Rs competing I¹²⁵-NDP-MSH and unlabeled NDP-MSH in a dose–response fashion that result in the same IC₅₀ values, within experimental error. (D) Illustrates the pharmacological agonist dose–response curves for the endogenous melanocortin agonists α-, β-, and γ₂-MSH and ACTH at the L106P hMC4R.

Figure 3

Illustration of the primary screening results of the TPI 924 tetrapeptide library at the L106P hMC4R. The primary screen was assayed using a rough approximation at 100 μg/mL concentrations. The X-axis represents the amino acid residue that was held constant at that position (O) of the tetrapeptide library with the three remaining positions composed of mixtures of the 60 amino acids (X), and the Y-axis represents the functional agonist activity observed. The agonist activity (average of duplicate wells) was determined using a β-galactosidase colorimetric reporter gene bioassay that has been normalized to both relative protein content as well as the maximal value observed for the nonreceptor dependent forskolin control (average of four wells). A value of 1 indicates a result that is able to generate the same maximal stimulation level observed for the forskolin control. The dotted line for each position indicates the criteria of >0.7 that was used as the cut-off point for classification as deconvolution “hits”.

Figure 4

Summary of the key amino acid structures used in this study.

Figure 5

Summary of the Pipeline Pilot experimental design modular approach that was utilized in this study.

Figure 6

Electrostatic surface area for the control Ac-His-DPhe-Arg-Trp-NH₂ (JRH887-9 1), Ac-His-(pI)DPhe-Arg-Trp-NH₂ (EMH4-105 3), and Ac-His-(pCl)DPhe-Arg-Trp-NH₂ (EMH4-104 12) control tetrapeptides as well as the 8 tetrapeptides (4–11) that were tested at all the polymorphic hMC4Rs and mouse MCRs examined in this study (Tables 5 and 6). The His/Arg residue at the first position is oriented at the top of the molecule, and the Arg/amino acid side chain at the third position is oriented down. The electrostatic surfaces were calculated using Maestro 9.5 (Schr̈odinger) with red (−5.0) to blue (+5.0) using the solute dielectric constant of 10 and the solvent dielectric constant of 80.

Figure 7

Comparison of the single substitution (Table 3) and positional scanning deconvolution approaches (Table 4) using the L106P hMC4R pharmacological data. (A) When considering only improvements on the activity of the known ligand Ac-His- DPhe-Arg-Trp-NH₂ (1, EC₅₀ < 215 nM), there is no statistical difference between single substitution and positional scanning deconvolution; 5 of the 20 single substitution compounds had an EC₅₀ < 215, comparable to the 8 of the 36 positional scanning deconvolution compounds (Fisher’s Exact Test, 1-tail, p = 0.718). (B) When considering improvements an order of magnitude or greater over the known ligand (EC₅₀ < 21.5 nM), the difference is readily apparent and statistically significant; none of the 20 single substitution compounds had an EC₅₀ <21.5 nM, but 8 of the 36 positional scanning deconvolution compounds did (Fisher’s Exact Test, 1-tail, p = 0.021).