Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases

Abstract

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.

Max‐Bergmann award lecture:A RaPID way to discover bioactive nonstandard peptides assisted by the flexizyme and FIT systems

“…We also solved the tertiary structures of some of the above thioether macrocyclic peptides by X‐ray crystallographic analyses (Figure A‐F). It is quite remarkable that we have witnessed a wide range of tertiary structures, eg, a ®‐sheet structure (PlexB1, Figure A; and KDM4A, Fig B), a macrocyclic structure containing an α‐helix motif (CmABCB1, Figure C), a macrocyclic structure containing a water molecule in the ring (SIRT2, Figure D), and a compact macrocyclic structure with a tail linear peptide (human α‐amylase; MATE, Figure E; iPGM, Figure F). The above results indicate how the RaPID system effectively samples a wide range of tertiary structures of macrocyclic peptides from the libraries.…”

“…We also found antagonists against human intracellular enzymes, a ubiquitin ligase (E6AP), a kinase (AKT2), and histone deacetylase and demethylase (SIRT2 and KDM4A). We recently reported antagonists against a pathogenic microorganism enzyme, a cofactor‐independent phosphoglycerate mutase (iPGM), and an inhibitor against Zaire Ebola virus protein 24 (VP24) disrupting the protein‐protein interaction with and human karyopherin alpha 5‐KPNA5 . Moreover, thioether‐macrocyclic peptides against a cancer stem cell marker, EpCAM were reported to devise a potential imaging probe .…”

“…The above thioether‐macrocyclic peptides discovered by the RaPID system displayed remarkable potencies, for which K D values and IC 50 values were in the range of low to sub‐nM in most cases. Their mode of actions turned out to be quite diverse, eg, competing with the substrates in the binding site (human α‐amylase, AKT2, SIRT2, and KDM4A), clogging the transporter site (MATE), targeting allosteric or allosteric‐like site (CmABCB1, PlexB1, and VP24), binding a unique remote‐inhibitory site (iPGM), and dimerizing the intercellular kinase domain via dimerization of the extracellular domain (cMET).…”

Cyclic peptides can engage a single binding pocket through multiple, entirely divergent modes

“…Indeed, such display platforms have been used to isolate macrocyclic peptide ligands against diverse protein targets, including enzymes, proteases, cellular receptors, and growth factors [3][4][5][6][7][8] .…”

Recent Advances in In Vitro Translation and Selection of Bioactive Nonstandard Peptides

“…7 When an mRNA template is tethered with a puromycin at the 3 0 end, puromycin leads to the covalent ligation of mRNA and its encoded peptide so that the mRNA-puromycin-peptide complex is released as a translation product. 9 Crystallographic analysis of the selected macrocyclic peptide-iPGM complex revealed that the peptide ligand was cradled in a pocket at the bi-domain interface, slightly shifted from the substrate binding site (Figure 2(b)). After a number of cycles, enriched peptide "hits" with high target affinity are sorted out and characterized by DNA sequencing (Figure 2(a)).…”