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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

Inhibition of the

The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (

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.

Fragment-based drug design (FBDD) has become an important and successful approach to drug discovery. In this review, we discuss two classes of simulation technologies that we routinely employ as part our of computational FBDD efforts. The first class centers on simulation methods in torsion space to develop high-quality protein models suitable for FBDD. These algorithms allow for fast molecular dynamics and modal Monte Carlo simulations. The torsion space dynamics techniques have been applied to develop models for the bound conformations of a variety of proteins including the HIV-1 protease, p38 MAP kinase, and the 5′-AMP-activated protein kinase. The second class of simulations is comprised of the Grand Canonical Monte Carlo and systematic sampling methods, which are used to explore the interactions of individual fragments with the protein target. Previously published validation studies for the binding of molecules to T4 lysozyme and the p38 MAP kinase are discussed. We review previous work to computationally assemble whole molecules from fragment binding data, a potential bottleneck in the FBDD approach. One effect of the fragment simulations is that an approximate value for the free energy of binding of a given molecule with the protein may be computed from the fragment simulations, with an estimated standard error approaching 1 kcal/mol, which is comparable to the performance of a variety of other methods reported in the literature. Drug Dev Res 72:130–137, 2011. © 2010 Wiley-Liss, Inc.

We introduce TICRA (transplant-insert-constrain-relax-assemble), a method for modeling the structure of unknown protein-ligand complexes using the X-ray crystal structures of homologous proteins and ligands with known activity. We present results from modeling the structures of protein kinase-inhibitor complexes using p38 and Lck as examples. These examples show that the TICRA method may be used prospectively to create and refine models for protein kinase-inhibitor complexes with an overall backbone rmsd of less than 0.75 Å for the kinase domain, when compared to published X-ray crystal structures. Further refinement of the models of the kinase domains of p38 and Lck in complex with their cognate ligands from the published crystal structures was able to improve the rmsd's of the model complexes to below 0.5 Å. Our results show that TICRA is a useful approach to the problem of structure-based drug design in cases where little structural information is available for the target proteins and the binding mode of active compounds is unknown.

The development of small-molecule drugs to counter the threat of bioterrorism will differ from classical drug discovery because it will be impossible to evaluate efficacy in clinical trials for many agents. This difference focuses biodefense on the identification of multiple drug candidates for each threat organism so that multiple treatments can be mounted simultaneously when needed to maximize the probability of success. Accordingly, drug discovery will become the rate- and cost-limiting phase of the overall drug development process. We address the potential of computational chemistry to optimize efficiency and efficacy in the discovery phase. The major elements required for a successful computational approach are the calculation of binding free energy, accounting for changes in solvation on ligand binding, and compensating for protein flexibility. Drug Dev Res 70:279–287, 2009. © 2009 Wiley-Liss, Inc.

A novel class of antagonists of the human glucagon receptor (hGCGR) has been discovered. Systematic modification of the lead compound identified substituents that were essential for activity and those that were amenable to further optimization. This SAR exploration resulted in the synthesis of 13, which exhibited good potency as an hGCGR functional antagonist (IC50 = 34 nM) and moderate bioavailability (36% in mice).

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.

A library of 48 di- and trisubstituted guanidines was synthesized on a soluble, tetravalent support. Support-bound intermediates and cleaved products were isolated in parallel by size exclusion chromatography using a semiautomated system. Products were generally obtained in good yield and purity.

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.

Discovery of a new class of DFG-out p38α kinase inhibitors with no hinge interaction is described. A computationally assisted, virtual fragment-based drug design (vFBDD) platform was utilized to identify novel non-aromatic fragments which make productive hydrogen bond interactions with Arg 70 on the αC-helix. Molecules incorporating these fragments were found to be potent inhibitors of p38 kinase. X-ray co-crystal structures confirmed the predicted binding modes. A lead compound was identified as a potent (p38α IC(50)=22 nM) and highly selective (≥ 150-fold against 150 kinase panel) DFG-out p38 kinase inhibitor.

In silico fragment-based drug discovery has become an integral component of the new fragment-based approach that has evolved over the past decade. Protein structure of high quality is essential in carrying out computational designs, and protein flexibility has been shown to impact prospective designs or docking experiments. Here we introduce methodology to calculate protein normal modes and protein molecular dynamics in torsion space which enable the development of multiple protein states to address the natural flexibility of proteins. We also present two fragment-based sampling methods, grand canonical Monte Carlo and systematic sampling, which are used to study protein-fragment interactions by generating fragment ensembles and we discuss the process by which these ensembles are linked to design ligands.

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.

A variety of spectroscopic and potentiometric studies have been carried out detailing the nature of the complex formation between lanthanide ions and malic acid. Formation constants calculated below pH 7 (where the complexes are mononuclear) demonstrate that both Gd3+ and Dy3+ complexes of DL-malic are more stable than corresponding complexes of either D- or l-malic acid. Studies of intermolecular energy transfer, on the other hand, indicate that above pH 7 (where the complexes are found to self-associate) complexes of resolved ligand associate to a greater extent than complexes prepared from racemic ligand. Supporting evidence is obtained from measurements of differential absorption. It appears that the weaker D- or L-MAL complexes are more prone to hydrolysis than the DL-MAL complexes, and evidence is presented from measurements of luminescent titrations that dimeric complex units are apparently bridged by either one or two -OH bridges. No evidence was found at 1:5 metal-to-ligand ratios for the existence of 1:3 lanthanide.malic acid complexes and no experimental effects could be detected which pointed toward participation of the malic acid hydroxyl group in the complex bonding.

The lanthanide complexes of glycine, diglycine and triglycine have been studied by a variety of physical means. Measurements of differential absorption of Ho(III) complexes, luminescence intensities of Tb(III) complexes, and intermolecular energy transfer from Tb(III) to Eu(III) complexes were all used to characterize the nature of the solution chemistry. The complexes tend to remain mononuclear at low pH, but associate through hydroxide linkages above neutral pH. The degree of association is much less for complexes of diglycine relative to those of glycine, suggesting that the steric nature of the amino acid ligand plays a dominant role in determining the degree of association.

The photodecomposition of the two title compounds has been carried out at 254 nm, and a single decarboxylation of the ligand was observed in each case. The decarboxylated complexes that resulted were characterized by visible and infrared spectroscopy, and it was concluded that uranium(IV) products were produced as a result of the reaction. The quantum yield of the ethylenediaminetetraacetic acid (EDTA) complex was found to be 0.84 ± 0.02, and the quatum yield of the diethylenetriaminepentaacetic acid (DTPA) was 0.50 + 0.01. The photoproducts could be isolated free from starting material or secondary photoproducts, since these other materials were not water-soluble. When the photoproduct was obtained in solid form, however, it formed as a plastic-like material, and this observation suggested polymerization of the uranium(IV) complexes due to radical reactions that occurred during the photolysis process.