Solid phase synthesis and structural analysis of novel A-chain dicarba analogs of human relaxin-3 (INSL7) that exhibit full biological activity

Abstract

Replacement of disulfide bonds with non-reducible isosteres can be a useful means of increasing the in vivo stability of a protein. We describe the replacement of the A-chain intramolecular disulfide bond of human relaxin-3 (H3 relaxin, INSL7), an insulin-like peptide that has potential applications in the treatment of stress and obesity, with the physiologically stable dicarba bond. Solid phase peptide synthesis was used to prepare an A-chain analogue in which the two cysteine residues that form the intramolecular bond were replaced with allylglycine. On-resin microwave-mediated ring closing metathesis was then employed to generate the dicarba bridge. Subsequent cleavage of the peptide from the solid support, purification of two isomers and their combination with the B-chain via two intermolecular disulfide bonds, then furnished two isomers of dicarba-H3 relaxin. These were characterized by CD spectroscopy, which suggested a structural similarity to the native peptide. Additional analysis by solution NMR spectroscopy also identified the likely cis/trans form of the analogs. Both peptides demonstrated binding affinities that were equivalent to native H3 relaxin on RXFP1 and RXFP3 expressing cells. However, although the cAMP activity of the analogs on RXFP3 expressing cells was similar to the native peptide, the potency on RXFP1 expressing cells was slightly lower. The data confirmed the use of a dicarba bond as a useful isosteric replacement of the disulfide bond.

The Minimal Active Structure of Human Relaxin-2

“…Competition binding experiments were performed with either europium-labeled H2 relaxin (26) or Eu 3ϩ -labeled INSL3 (27) in the absence or presence of increasing concentrations of unlabeled hormones. Nonspecific binding was determined with an excess of unlabeled peptides (500 nM H2 relaxin or INSL3).…”

“…All H2 relaxin analogues synthesized in this study contain two chains and three disulfide bonds. Both regioselectively S-protected A-and B-chains were synthesized separately by the continuous flow Fmoc solidphase method (24) using an automatic PerSeptive Biosystems Pioneer peptide synthesizer (Framingham, MA) or by using microwave-assisted synthesis on a Liberty system (CEM Corp.) (21,23,25,26).…”

Synthetic relaxins

“…The residues in the B-chain central a-helix that are involved in RXFP3 binding have also been identified, and it was shown that activation is mediated via two critical C-terminal residues, ArgB26 and TrpB27 [47]. Replacement of the intra-A-chain disulfide bond with a dicarba bond did not cause loss of biological activity and also increased in vitro serum stability [48]. Furthermore, replacement of the A-chain with that from INSL5 (R3/I5) confers selectivity to RXFP3 and RXFP4 by substantially reducing affinity for the relaxin receptor, RXFP1 [47,49].…”

“…A significant advance in the solid phase synthesis of INSLs was heralded by the successful synthesis of H2 relaxin via regioselective disulfide bond formation, in which each of the three disulfide bonds were formed sequentially by stepwise removal of pairs of orthogonal S-protecting groups followed by oxidation of the resulting liberated thiol groups [17]. A complex eight-step strategy was followed in which the A-chain was assembled by Fmoc chemistry SPPS and contained three different S-protecting groups, each removable by a different chemical 48 …”

Chemical Biology: What is Its Role in Drug Discovery?

“…Cyclotides, disulfide rich cyclic peptides, have been isolated for the first time from the Melicytus family of plants (Trabi et al, 2009). Using solid phase chemistry, a novel A-chain analogue of human relaxin-3 (INSL7) has been synthesised in which the intramolecular disulfide bond is replaced with a non-reducable carbon-carbon bond (Hossain et al, 2009). A novel class of peptidomimetics, Ψ[CH(CF 3 )NH]Gly peptides, have been reported, which utilises a stereogenic trifluoroethylamine group as a peptide bond mimic (Molteni et al, 2009).…”