Discovery and Characterization of the First Nonpeptide Antagonists for the Relaxin-3/RXFP3 System

Abstract

The neuropeptide relaxin-3/RXFP3 system is involved in many important physiological processes such as stress responses, appetite control, and motivation for reward. To date, pharmacological studies of RXFP3 have been limited to peptide ligands. In this study, we report the discovery of the first small-molecule antagonists of RXFP3 through a high-throughput screening campaign. Focused structure-activity relationship studies of the hit compound resulted in RLX-33 (33) that was able to inhibit relaxin-3 activity in a battery of functional assays. RLX-33 is selective for RXFP3 over RXFP1 and RXFP4, two related members in the relaxin/insulin superfamily, and has favorable pharmacokinetic properties for behavioral assessment. When administered to rats intraperitoneally, RLX-33 blocked food intake induced by the RXFP3-selective agonist R3/I5. Collectively, our findings demonstrated that RLX-33 represents a promising antagonist scaffold for the development of drugs targeting the relaxin-3/RXFP3 system.

Figure 1.

Structure of hit compound 1 and sites for preliminary SAR

Figure 2.

Antagonist activity of R3(B1-22)R and hit compound 1 in CHO-hRXFP3 cAMP accumulation (A) and [³⁵S]GTPγS binding (B) assays. Data are expressed as the percentage of relaxin-3 peptide response. cAMP data are the mean ± SEM of eight to ten independent experiments conducted in duplicate or quadruplicate. [³⁵S]GTPγS binding data are the mean ± SEM of three to four independent experiments conducted in duplicate.

Figure 3.

Competitive binding studies on relaxin-3, R3(B1-22)R, 30, and 33 at RXFP3. Data are expressed as the percentage of specific binding of [¹²⁵I]R3/I5 peptide, and curves were generated using a one-site non-linear regression model. Data are the mean ± SEM of three independent experiments conducted in duplicate.

Figure 4.

Insurmountable antagonism of 33 in CHO-hRXFP3 cAMP accumulation (A) and [³⁵S]GTPγS binding (B) assays. Data are the means of duplicate measurements with standard deviation shown as error bars and are representative of four independent experiments for cAMP accumulation and two independent experiments for [³⁵S]GTPγS binding.

Figure 5.

Antagonist activity of R3(B1-22)R, 30, and 33 in CHO-hRXFP3 ERK1/2 phosphorylation assay. Data are expressed as the percentage of the relaxin-3 peptide response. Data are the mean ± SEM of three independent experiments conducted in duplicate.

Figure 6.

Agonist activity (A) and antagonist activity (B) of 30 and 33 in CHO-hRXFP1 cells. Agonist data are expressed as the percentage of the maximal relaxin-2 peptide response, while antagonist data are expressed as the percentage of relaxin-2 peptide response at 50 pM. Data are the mean ± SEM of two independent experiments conducted in duplicate.

Figure 7.

Agonist activity (A) and antagonist activity (B) of 33 in CHO-hRXFP4 cells. Agonist data are expressed as the percentage of the maximal relaxin-3 peptide response, while antagonist data are expressed as the percentage of relaxin-3 peptide response at 3 nM. Data are the mean ± SEM of three independent experiments conducted in duplicate.

Figure 8.

Plasma and brain PK profile of 33 in male Wistar rats (i.p., 10 mg/kg)

Figure 9.

(A) Compound 33 (10 mg/kg, i.p.) attenuated the RXFP3-selective agonist R3/I5-induced increase in feeding in male Wistar rats during the first 2 h of the feeding test. (B) There were no treatment effects on water intake during the first 2 h of the feeding test. *p < 0.05

Scheme 1.. Synthesis of Compounds 1 and 6–33^a

^a Reagents and conditions: (a) NH₄OH, NaOH, EtOH/H₂O (4:1), reflux, 36 h; (b) 4-nitrobenzoyl chloride, K₂CO₃, toluene, reflux, overnight; (c) Na₂S, 1,4-dioxane/H₂O (1:1), 80 °C, 1 h; (d) 2/SOCl₂, CH₂Cl₂, reflux, then 5/Et₃N, CH₂Cl₂, rt, overnight; (e) TCFH, NMI, MeCN, rt, 3 h.