Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Mareike Wiedmann,Patricia K Dranchak,Mahesh Aitha,Bryan Queme,Christopher D Collmus,Maithri M Kashipathy,Liza Kanter,Laurence Lamy,Joseph M Rogers,Dingyin Tao,Kevin P Battaile,Ganesha Rai,Scott Lovell,Hiroaki Suga,James Inglese

doi:10.1016/j.jbc.2021.100628

Abstract

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.

Highlights

Cofactor-dependent phosphoglycerate mutase is a 2, 3-bisphophoglycerate cofactor–utilizing homodimer consisting of 23-kDa subunits, while the parasitic enzyme is a 56-kDa monomer cofactorindependent enzyme consisting of a phosphatase domain connected by a flexible hinge to the phosphotransferase domain
The low specific catalytic activity of Escherichia coli independent phosphoglycerate mutase (iPGM) having 45% identity with C. elegans iPGM limits the ability to measure IC50 values below 50 nM for this enzyme, with Ce-2d showing no significant inhibition up to 30 μM.§ E. coli iPGM appears to demarcate the point in sequence similarity sensitive to the Ce-2d ipglycermide, whereas the enzyme activity of nematode orthologs tested (% sequence identity is between 71 and 75% of C. elegans) was stoichiometrically inhibited by Ce-2 and potently inhibited by Ce-2d (Fig. 1C)
From the cocrystal structure of the ipglycermide Ce-2d analog bound to C. elegans iPGM, we observe that the ipglycermide core is a compact structure with an internal H-bond network strongly mediated by Asp6 (Fig. 1D and Table S1)

Summary

Introduction

To calculate enrichment scores for each mutant (Fig. 4A, black circles), the fraction of DNA reads relative to the WT Ce-Table 1 Rate and dissociation constants of ipglycermide analogs binding to iPGM orthologs RaPID scanning mutagenesis provided a broad and revealing structure–activity relationship analysis for ipglycermide and was used to guide subsequent SPPS of Ce-2 and Ce-2d analogs for further ortholog selectivity analysis in functional enzyme inhibition assays, binding kinetics, and cocrystal structural determinations.

Results

Conclusion