Discovery of Glu2Py: a water-soluble glycosylated MAP4K4 inhibitor for targeted ischemia-reperfusion therapy.

Abstract

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4/HGK) is a validated upstream activator of the JNK/p38 stress-signaling axis implicated in cardiac ischemia-reperfusion injury (IRI), inflammatory disease, and cancer. Despite its therapeutic relevance, no MAP4K4 inhibitor has advanced to clinical use, partly due to the poor aqueous solubility of existing lead scaffolds. Here we report the design, synthesis, and structure-activity relationships of a pyrimidine derived from the potent but insoluble parent compound DMX-5804. Systematic exploration of rings A, B, and C established three cardinal SAR rules, (i) electron-donating substituents at ring A are required for electrostatic complementarity with the hinge region; (ii) phenyl aromaticity and planarity at ring B are indispensable for π-π stacking with Tyr36; and (iii) the endocyclic -CONH- linker at ring C is the non-negotiable hinge hydrogen-bond donor to Cys108 and Glu106. Saturation of ring B or replacement of the -CONH- linker with exocyclic amines abolished the activity in both cases, consistent with molecular docking against the MAP4K4 crystal structure. To address the solubility liability of the parent scaffold, glycosylation at the ring A phenolic position was pursued as an orthogonal strategy. The glucose conjugate Glu2Py (IC50 = 11.4 nM; K i = 5.7 nM) and the ribofuranose analog Ribo-7A (IC50 = 9.68 nM; K i = 4.8 nM) retained sub-12 nM potency while achieving complete aqueous solubility up to 1250 μg mL-1, as confirmed by kinetic turbidity assay. Molecular docking revealed that Glu2Py uniquely forms a supplementary hydrogen bond between the glucose C2-OH and Asp115, with MM-GBSA binding free energies equal to or exceeding the co-crystallized reference ligand. Glu2Py is nominated as the optimal lead compound, combining sub-12 nM MAP4K4 potency, complete aqueous solubility, and a well-defined binding mode that provides a rational foundation for further optimization.

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