Macrocyclic Modalities Combining Peptide Epitopes and Natural Product Fragments.

Stéphanie M Guéret,Herbert Waldmann,Alleyn T Plowright,Sasikala Thavam,Göran Dahl,Anita Dellsén,Niklas Larsson,Malin Lemurell,Rodrigo J Carbajo,Eric Valeur,Marco Potowski,Tom N Grossmann

doi:10.1021/jacs.0c00269

Abstract

“Hot loop” protein segments have variable structure and conformation and contribute crucially to protein–protein interactions. We describe a new hot loop mimicking modality, termed PepNats, in which natural product (NP)-inspired structures are incorporated as conformation-determining and -restricting structural elements into macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were synthesized on solid support employing macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors. NP-inspired fragment absolute configuration determines the conformation of the peptide part responsible for binding. These results demonstrate that combination of NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop mimics with conformationally constrained and biologically relevant structure.

Highlights

Loop segments composed of peptide sequences displaying diverse structures and with their termini positioned in spatial proximity (“Ω loops”) were identified as frequently occurring protein structural motifs, mediating numerous PPIs (“hot loops”).[4,5]
In individual cases hybrid macrocycles which incorporate sp3-configured stereocenters inspired by natural product (NP) structure have recently been reported with the cyclization in general performed in solution after solid-phase peptide synthesis (SPPS) of precursors.[21,22]
We describe a new principle for the design and synthesis of a hot loop mimicking modality, termed Peptide-Natural product-inspired hybrids (PepNats)

Summary

■ INTRODUCTION

For small-molecule modulation of protein−protein interactions (PPIs) mediated by extended binding surfaces,[1] new approaches and chemical modalities are in high demand.[2,3] Recently, loop segments composed of peptide sequences displaying diverse structures and with their termini positioned in spatial proximity (“Ω loops”) were identified as frequently occurring protein structural motifs, mediating numerous PPIs (“hot loops”).[4,5]. It should be noted that linkers not incorporating stereogenic centers such as disulfide-bridged compound 24, ortho ether aromatic unit VIII in peptidomimetic 26 and phenylmethanamine IX in cyclic peptidomimetic 27, require separate and lengthy syntheses (for synthesis details, see Supporting Information, section 5) These syntheses only afforded a single DINNN analogue that did not have improved affinity for the protein of interest, hSPSB2. To investigate whether combination of adjustable NPinspired scaffolds with the AGRP109−118 hot loop would yield selective ligands with distinct peptide conformations, binding and functional activity of 33 cyclic PepNats were determined (Supplementary Table S5). Analysis of the NMR spectra of PepNat K1major and K1-minor in deuterated methanol at room temperature revealed that these differences in affinity and selectivity for the diastereomers correlate with two distinct preferred major conformations in solution (Supplementary Figure S3b and Supporting Information section 6). This further corresponds to the PepNat conformation in solution described for the model PepNat H1 as described above (Figure 5, Supporting Information)

■ CONCLUSION

■ ACKNOWLEDGMENTS

■ REFERENCES