Constipation and diarrhea are common gastrointestinal motility disorders that affect the general population and are associated with several neurological disorders. Infectious diarrheas are the fifth most common cause of death worldwide. We recently showed that a peptide analogue of insulin-like peptide 5 (INSL5), INSL5-A13, an agonist of RXFP4, stimulates colorectal propulsion. Thus, INSL5 may have a physiological role in the control of colorectal motility. Because bacterial products are stimuli for diarrhea, we hypothesized that excess intraluminal bacterial products (short-chain fatty acids; SCFA) would stimulate the release of INSL5, act on RXFP4 and, if endogenous INSL5 has a role, there would be an increase in colorectal propulsion that would be inhibited by an antagonist of RXFP4.
We designed and developed a novel high affinity RXFP4-specific antagonist, INSL5-A13NR. We substituted the Arg23 in the B-chain of INSL5-A13 with an arginine mimetic, norarginine (NR). We synthesised the A and B-chains and formed two disulfide bridges regioselectively that resulted in the target compound, INSL5-A13NR. The new compound (INSL5-A13NR) (100nM to 10µM) reduced inhibition of cAMP caused by INSL5-A13. A b-Arrestin2 recruitment assay showed that INSL5-A13NR did not induce RXFP4/β-arrestin2 interactions, but it completely blocked the response triggered by an RXFP4-agonist. INSL5-A13NR blocked RXFP4 agonist-induced increased colon motility in mice of both genders that express the receptor, RXFP4. The antagonist reduced the acceleration of colorectal propulsion and fecal pellet output caused by a mixture of SCFA that was introduced into the lumen of the colon.
Our results demonstrate that INSL5 released in response to excess bacterial products acts on RXFP4 to cause propulsive reflexes of the colon, and suggest that RXFP4 is a potential target for the treatment of colon motility disorders, including constipation and diarrhea.