Review articleChallenges in the design of insulin and relaxin/insulin-like peptide mimetics
Graphical abstract
Introduction
Currently available drugs in the market fall broadly into two categories. There are ‘small molecule’ drugs (molecular weight of <500 Da) with oral bioavailability and much larger ‘biologics’ (molecular weight of typically >5000 Da) with no oral bioavailability. Due to their small size, small molecule drugs often suffer from reduced target specificity and toxicity. Large biologics, on the other hand, are highly target-specific and thus less toxic than small molecules. Therefore, the compounds that fit between these two molecular weights (500–5000 Da) and possess the advantages of both the small molecule (e.g. bioavailability and stability) and larger biologics (e.g. highly target specific) are of great interest. Peptidomimetics are such compounds that fall into this category.
Peptidomimetics are designed to circumvent the problems associated with the native peptide or protein on which they are based (e.g. poor proteolytic stability and bioavailability).1 They can also be improved in terms of potency and target specificity. Hence peptidomimics have great potential as synthetic tools of drug discovery. The design process of mimics starts from developing structure-activity relationships (SAR) of the native ligand-target pair that identify the key residues that are responsible for the biological effect of the native peptide or protein. Then minimization of the structure and introduction of constraints (e.g. cyclization through hydrocarbon stapling, lactam, Disulfide Bridge, disulfide isostere, N-methylation) are applied to create the core active site that can interact with the receptor or enzyme with high affinity and selectivity.1, 2, 3, 4, 5, 6, 7, 8, 9 Developing peptidomimetics is not trivial and often challenging, particularly when peptides’ interaction mechanism with their target is complex. This review will discuss the challenges of developing peptidomimetics of therapeutically important insulin superfamily peptides.
In humans, there are ten insulin superfamily peptides that includes three insulin family peptides (insulin, insulin-like growth factor (IGF) I and II and seven relaxin family peptides. The latter consists of human relaxin 1 (H1 relaxin), human relaxin 2 (H2 relaxin), human relaxin 3 (H3 relaxin), insulin-like peptide 3 (INSL3 also known as relaxin-like factor or Leydig insulin-like peptide), insulin-like peptide 4 (INSL4 or placentin), insulin-like peptide 5 (INSL5) and insulin-like peptide 6 (INSL6).10, 11, 12, 13, 14, 15, 16 Except IGF-I and II, which are single-chain peptides, all others are proven or predicted to be mature hormones with two chains (A and B) linked by two inter-chain disulfide bonds and one intra-A-chain disulfide bond17, 18 [Fig. 1A and B]. Insulin acts on tyrosine kinase receptors [Fig. 1C], the relaxin and INSL peptides, on the other hands, act upon G protein-coupled receptors that are now known as relaxin family peptide (RXFP) receptors [Fig. 1D].19
The native receptors for INSL4 and INSL6 are yet to be identified.20 H2 relaxin is the ortholog of the relaxin-1 gene found with relaxin-3 in most other mammalian species21 and it is presumed that H1 relaxin is also a ligand of RXFP1. While the function of H1 relaxin is unknown, all the family members of relaxin peptides and their target receptors (RXFPs) have wide physiological roles and clinical significance (reviewed in: Bathgate et al.22).
While insulin family peptides share little sequence homology apart from six cysteines that form three disulfide bonds [Fig. 1A], the overall fold of these peptides is similar [Fig. 1B]. However, the differences in their amino acid sequences have resulted in subtle changes in the length of the conserved helices and their functional selectivity towards their receptors. Detailed SAR towards understanding the role of residues in each of these chains have been reported over the years. This review summarizes the current knowledge of the SAR of insulin, H2 relaxin, H3 relaxin, INSL3 and INSL5 and describes how this knowledge has been used to develop peptidomimetic agonists and antagonists of RXFP receptors.
Section snippets
Insulin and insulin receptor
Mature human insulin consists of two chains, an A chain of 21 residues, and a B chain of 30 residues (51 residues in total, molecular weight 5808). They are linked by two inter-chain disulfide bridges and one intra-A-chain bridge [Figs. 1A, 2A, B]. The crystal structure of insulin was solved in 1969.23 Insulin forms dimers and hexamers (3 dimers of insulin) in the presence of zinc in humans.
The insulin receptor (IR) is a transmembrane tyrosine kinase protein with momodimeric (αβ)2 structure
3.1. Binding sites within insulin molecule
Extensive biochemical and structural characterization of insulin demonstrated two binding sites within the molecule. Site 1 (classical or primary binding site) utilizes 13 residues (GA1, IA2, VA3, QA5, TA8, YA19, NA21, VB12, YB16, GB23, FB24, FB25, and YB26) which forms the surface of the insulin dimer [Fig. 2A].24, 25, 26 Site 2 (secondary binding site) comprises 6 residues (SA12, LA13, EA17, HB10, EB13, and LB17) which forms the surface of the insulin hexamer24, 25, 26 [Fig. 2B]. The
Insulin mimetics
Despite decades of endeavour and the recent elucidation of the structure of the insulin-bound IR site 1,37 the mechanism of insulin-induced signal transduction remains largely elusive. The extremely complex interaction between insulin and IR made it almost impossible to develop peptidomimetics as truncating or minimising the structure of insulin significantly alters its biological activity. Hence no small molecule agonist of the IR has been discovered to date. Thus all of the insulin analogues
Human relaxin 2 (H2 relaxin) and relaxin family peptide receptor 1 (RXFP1)
The native form of the H2 relaxin peptide has been isolated from human ovary49 and prostate50 and demonstrated to contain a 24 residue A-chain and a 29 residue B-chain (ie. 53 residues, molecular weight 5963)24, 51 [Fig. 1A]. Although this is a major circulating form of H2 relaxin, there are two other equally active isoforms found in vivo with longer B-chains, B1–31 and B1–33.52 The X-ray crystallography and solution structure of H2 relaxin were solved in 199153 and 2016 [Fig. 4A]54
Binding and activation mechanism
Studies on the mode of binding and activation of RXFP1 by H2 relaxin have demonstrated that both the A- and B-chain seem to be necessary for full activity. Interaction with RXFP1 requires several interactions involving the LRR domain,58 the linker between the LRR and LDLa and the extracellular TM loops.59, 60, 61 Importantly the linker and N-terminal LDLa module are essential for activation and may act together as a tethered agonist61, 62, 63, 64, 65 [Fig. 4D]. The specific aspects of binding
H2 relaxin peptidomimetics
The complex mechanism of H2-RXFP1 interaction, as discussed above, suggests considerable challenges for developing peptidomimetics of the H2 relaxin without altering its biological activity. However, as the A-chain is thought to possesses low affinity secondary binding residues74 and the mid-region of the B-chain contains the high affinity primary binding residues69 it was hypothesized that it might be possible to develop a minimised peptidomimetic or even a B-chain-only mimetic of H2 relaxin
Human relaxin 3 (H3 relaxin) and relaxin family peptide receptor 3 (RXFP3)
Mature H3 relaxin is predicted to consist of two chains, an A chain of 24 residues, and a B chain of 27 residues (51 residues, molecular weight 5500)19, 51, 81 [Figs. 1A, 6A] based on the native porcine relaxin-3 sequence isolated from pig brain.18 The chains are linked by two inter-chain disulfide bridges and one intra-A-chain bridge [Figs. 1A, 6A]. The solution structure of H3 relaxin was solved in 2008 [Fig. 6A].82 The cognate receptor for H3 relaxin is the GPCR, RXFP3 [Fig. 1E].18 However
Binding and activation mechanism
There are distinct differences in the residues and modes of binding and activation of the different receptors – RXFP1, RXFP3 and RXFP4 by H3 relaxin. Our studies demonstrate that deletion of the N-terminal residues of the B-chain does not affect the ligand binding activity for RXFP1, RXFP3 or RXFP4.85, 86, 87 H3 relaxin contains an RXXXRXXI motif (RB12, RB16 and IB19) and mutation studies85, 86 confirm that, like H2 relaxin, this is the driver of high affinity RXFP1 binding.88 The binding of H3
Receptor-specific H3 relaxin mimetics
From SAR studies it is clear that the H3 relaxin B-chain is responsible for binding and activation of RXFP3 and RXFP4. Other studies suggest that the A-chain of H3 relaxin may play a role in interacting with RXFP1. Therefore, in an attempt to develop a receptor-specific agonist, the A-chain of H3 relaxin was replaced with that from INSL5 and the resulting chimeric R3/I5 analogue exhibited selectivity for RXFP3 and RXFP4 over RXFP1.92 Additionally, by truncating the C-terminus of the B-chain of
Human insulin 3 (INSL3) and relaxin family peptide receptor 2 (RXFP2)
The predicted structure of human INSL3 involves an A-chain of 26 residues and a B-chain of 31 residues (57 residues, molecular weight 6292). Both chains are connected by three disulfide bridges [Figs. 1A, 7A]. The solution structure of INSL3 determined by our lab in 2006 [Fig. 7A]97 suggests insulin like folding of the peptide. The cognate receptor for INSL3 is the G protein-coupled receptor, RXFP2.57 The receptor is closely related to RXFP1 with a complex structure containing the 7 TMD, 10
Primary binding site
Like H2 relaxin, INSL3 binds to the LRR domain of RXFP2 using B-chain-specific residues: HB12, RB16, VB19, RB20 and WB2798, 99 [Figs. 1A, 7A]. The complementary binding site in the LRR of RXFP2 that interact with the B-chain of INSL3 was also determined and characterised by receptor mutation studies and demonstrated to be distinct from the H2 relaxin-RXFP1 site.100
Secondary binding site
While no single amino acid within the INSL3 A-chain was found to be critical for RXFP2 binding and activity, the potency of the
Minimised INSL3 mimetics
As part of the B-chain is important for RXFP2 binding and the A-chain is necessary for activation,98, 99, 101, 102 the minimised INSL3 peptidomimetic INSL3:A5–26/B7–27 was developed as an RXFP2 agonist [Figs. 7B, 5].101 This peptide contains truncated A- and B-chains and is almost one-quarter smaller than the native peptide and thus easier and cheaper to make and represents a potential lead peptide for further development to probe biological function of RXFP2. A high-affinity RXFP2 antagonist
Insulin-like peptide 5 (INSL5) and relaxin family peptide receptor 4 (RXFP4)
Human insulin-like peptide 5 (INSL5) has a predicted structure with 2 chains (A-chain 21 amino acids and B chain 24 amino acids; Molecular weight 5048) connected by three disulfide bridges [Figs. 1A, 8A]. INSL5 was originally identified as a novel peptide hormone with high expression in the gut.14 The solution NMR structure of human INSL5108 determined by our laboratory (2009) indicates that the B-chain has an extended C-terminal α-helix compared with H3 relaxin Fig. 8A].109 Subsequent studies
Binding and activation mechanism
Human INSL5 is one of the most difficult peptides to produce chemically as both A and B chains are unusually resistant to standard synthesis protocols and require highly optimised conditions for their acquisition.110 Therefore, preliminary SAR studies were carried out using mouse INSL5 whose sequences were predicted to be easier to assemble. Interestingly, mouse INSL5 was found to be almost 10 times more potent than hINSL5 on human RXFP4.111 We identified a residue, KA15, in the A-chain of
INSL5 peptidomimetics
These SAR studies led to the design and synthesis of the simplified peptidomimetics, mINSL5:A8–21118 and hINSL5:A8–21K15 [Figs. 8B, 5] 112. The latter analogue contains one mutation where human residue TA15 was replaced with equivalent mouse residue KA15.112 These analogues have 2 disulfide bridges and a truncated A-chain structure. They exhibited native hINSL5-like RXFP4 affinity and potency and were potent full agonists of both cAMP inhibition and pERK activation. They are good template for
Conclusion
This review has highlighted the distinct mechanisms utilized by insulin and relaxin-insulin-like peptides for binding and activation of their native receptors. Knowledge of these mechanisms has led the development of peptidomimetics which act as agonists or antagonists. However, the distinct differences in peptide structure and receptor interaction means that each ligand-receptor pairing has required a different approach for mimetic design. Studies on this unique peptide family therefore
Acknowledgements
This research was partly funded by NHMRC (Australia) project grants (1122170, 1023321, 1065481, and 1100676) and ARC linkage grant (LP120100654) to M.A.H. and R.A.D.B. Research at The Florey Institute of Neuroscience and Mental Health is supported by the Victorian Government Operational Infrastructure Support Program. Some of the figures were prepared by Praveen, a PhD student at the Florey.
Conflict of interest
The authors declare no conflict of interest.
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