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Supplementary Figure 1. Synthesis and pharmacokinetic characterization of AG-221. Supplementary Figure 2. Biochemical attributes of AG-221. Supplementary Figure 3. Biochemistry of AG-221 with respect to substrate and cofactor. Supplementary Figure 4. TF-1 IDH2R140Q cells treated with AG-221. Supplementary Figure 5. AG-221 can reverse the block in EPO-induced differentiation caused by the expression of IDH2R140Q in the TF-1 erythroleukemia cell line. Supplementary Figure 6. Pharmacokinetics/pharmacodynamics of AG-221 in IDH2R140Qmutant U87MG xenograft tumor-bearing mice. Supplementary Figure 7. AG-221 does not affect intrinsic hematological parameters or body weight. Supplementary Figure 8. AG-221 strongly reduces the number of human IDH2R140Q blasts in the liver and spleen in AML-1, AML-2, and AML-3. Supplementary Figure 9. Flow cytometry analyses of bone marrow-derived hCD45+ cells in primary human AML xenograft models. Supplementary Figure 10. Affinity of the IDH2R140Q homodimer for NADPH. Supplementary Figure 11. Electron density map diagrams for bound ligands for IDH2R140Q co-complex structures determined by X-ray crystallography.

Supplementary Table 1. Drug metabolism and pharmacokinetic attributes of AG-221. Supplementary Table 2. Selectivity of AG-221 confirmed by testing against a panel of kinases. Supplementary Table 3. Clinical characteristics of patients with IDH2R140Q-mutated AML. Supplementary Table 4. Treated NSG mice (AML-1, AML-2, AML-3) engrafted with human IDH2R140Q mononuclear cells display stable levels of AG-221 in serum. Supplementary Table 5. AG-221 inhibits 2HG production in models AML-1, AML-2, and AML-3. Supplementary Table 6. Summary of pharmacokinetics/pharmacodynamics in primary human acute myeloid leukemia xenograft model (AML-4). Supplementary Table 7. Summary of data collection and refinement statistics. Supplementary Table 8. Percentage of human chimerism in peripheral blood in models AML-1 and AML-2.

Supplementary Methods

AG-221 or enasidenib is a first-in-class selective inhibitor of mutated isocitrate dehydrogenase 2 (IDH2) with early demonstrated clinical efficacy in acute myeloid leukemia as a single agent, yet with persistence of mutant IDH2 clones. Two papers in this issue of Cancer Discovery provide further insight into the biological activity of AG-221 in promoting differentiation of IDH2 mutant cells and reversing aberrant DNA methylation over time, and demonstrating pre-clinical activity in combination with a targeted FLT3 kinase inhibitor to eliminate IDH2 mutant clones.

Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR. We first identified compound 1 ( N -3-[2-(4-biphenyl)-6-chloro-5-methyl]indolyl-acetyl- l -lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core. Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR. Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells. Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO, such as ( i ) the activation of a STAT-dependent luciferase reporter gene in BAF3 cells expressing human EPOR, ( ii ) supporting the proliferation of several tumor cell lines expressing the human or mouse EPOR, and ( iii ) the in vitro differentiation of human progenitor cells into colonies of erythrocytic lineage. These data demonstrate that a nonpeptide molecule is capable of inducing EPOR dimerization and mimicking the biological activities of EPO.

C5a is a 74-amino-acid glycoprotein whose receptor is a member of the rhodopsin superfamily. While antagonists have been generated to many of these receptors, similar efforts directed at family members whose natural ligands are proteins have met with little success. The recent development of hexapeptide analogs of C5a has allowed us to begin elucidation of the molecular events that lead to activation by combining a structure/activity study of the ligand with receptor mutagenesis. Removal of the hexapeptide's C-terminal arginine reduces affinity by 100-fold and eliminates the ability of the ligand to activate the receptor. Both the guanidino side chain and the free carboxyl of the arginine participate in the interaction. The guanidino group makes the energy-yielding contact with the receptor, while the free carboxylate negates “electrostatic” interference with Arg-206 of the receptor. It is the apparent movement Arg-206 induced by this set of interactions that is responsible for activation, since conversion of Arg-206 to alanine eliminates the agonist activity of the hexapeptides. Surprisingly, activation is a nearly energy-neutral event and may reflect the binding process rather than the final resting site of the ligand.

The guanine nucleotide-binding protein-coupled receptor superfamily binds a vast array of biological messengers including lipids, odorants, catecholamines, peptides, and proteins. While some small molecules bind to these receptors at a single interhelical site, we find that the binding domain on the receptor for the inflammatory protein C5a is more complex and consists of two distinct subsites. This more elaborate motif appears to be an evolutionary adaptation of the simpler paradigm to which a second interaction site has been added in the receptor N terminus. Surprisingly, occupation of only one of the subsites is required for receptor activation. The two-site motif is not unique to the C5a receptor but appears to be widely used by the superfamily to accommodate macromolecular ligands.