Lead Optimization of Small Molecule ENL YEATS Inhibitors to Enable In Vivo Studies: Discovery of TDI-11055.

Eleven-nineteen leukemia (ENL) is an epigenetic reader protein that drives oncogenic transcriptional programs in acute myeloid leukemia (AML). AML is one of the deadliest hematopoietic malignancies, with an overall 5-year survival rate of 27%. The epigenetic reader activity of ENL is mediated by its YEATS domain that binds to acetyl and crotonyl marks on histone tails and colocalizes with promoters of actively transcribed genes that are essential for leukemia. Prior to the discovery of TDI-11055, existing inhibitors of ENL YEATS showed in vitro potency, but had not shown efficacy in in vivo animal models. During the course of the medicinal chemistry campaign described here, we identified ENL YEATS inhibitor TDI-11055 that has an improved pharmacokinetic profile and is appropriate for in vivo evaluation of the ENL YEATS inhibition mechanism in AML.

Small-Molecule Inhibition of the Acyl-Lysine Reader ENL as a Strategy against Acute Myeloid Leukemia.

The chromatin reader eleven-nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9-mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line- and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach. SIGNIFICANCE: AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483.

A Chemical Strategy toward Novel Brain-Penetrant EZH2 Inhibitors.

Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received U.S. Food and Drug Administration approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain penetrance, limiting their use in patients with central nervous system malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity, and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound, we discovered TDI-11904 (compound 21), a novel, highly potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.

Structure-dependent impairment of intracellular apolipoprotein E4 trafficking and its detrimental effects are rescued by small-molecule structure correctors.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

Sustained viral response in a hepatitis C virus-infected chimpanzee via a combination of direct-acting antiviral agents.

Efforts to develop novel, interferon-sparing therapies for treatment of chronic hepatitis C (HCV) infection are contingent on the ability of combination therapies consisting of direct antiviral inhibitors to achieve a sustained virologic response. This work demonstrates a proof of concept that coadministration of the nucleoside analogue MK-0608 with the protease inhibitor MK-7009, both of which produced robust viral load declines as monotherapy, to an HCV-infected chimpanzee can achieve a cure of infection.

Synthesis and evaluation of novel tricyclic benzo[4.5]cyclohepta[1.2]pyridine derivatives as NMDA/NR2B antagonists.

A novel series of annulated tricyclic compounds was synthesized and evaluated as NMDA/NR2B antagonists. Structure-activity development was directed towards in vitro optimization of NR2B activity and selectivity over the hERG K(+) channel. Preferred compounds were subsequently evaluated for selectivity in an alpha(1)-adrenergic receptor binding counter-screen and a cell-based assay of NR2B activity.

Synthesis of the first sulfur-35-labeled hERG radioligand.

The synthesis of the first high specific activity S-35-labeled hERG radioligand, [(35)S]MK-0499, for use in HTS assays of drug candidates for hERG interaction is described. The radioligand is prepared by [(35)S]sulfonylation of a high diastereomeric excess (de) aniline precursor prepared from unlabeled MK-0499.

Antiarrhythmic efficacy of combined I(Ks) and beta-adrenergic receptor blockade.

Suppression of malignant ventricular arrhythmias by selective blockade of the cardiac slowly activating delayed rectifier current (I(Ks)) has been demonstrated with the benzodiazepine L-768673 [(R)-2-(2,4-trifluoromethyl-phenyl)-N-[2-oxo-5-phenyl-1-(2,2,2-trifluoro-ethyl)-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl]acetamide] in canine models of recent and healed myocardial infarction. The present study extends the initial antiarrhythmic assessment of I(Ks) blockade by demonstrating prevention of ischemic malignant arrhythmias in dogs with recent (8.0 +/- 0.4 days) anterior myocardial infarction with the coadministration of a subeffective dose of L-768673 and a subeffective, minimally beta-adrenergic blocking dose of timolol. Administered individually, neither 0.3 microg/kg i.v. L-768673 nor 1.0 microg/kg i.v. timolol prevented the induction of ventricular tachyarrhythmia (VT) by programmed ventricular stimulation (PVS) or the development of malignant ventricular arrhythmia in response to acute coronary artery thrombosis. In contrast, coadministration of 0.3 microg/kg i.v. L-768673 + 1.0 microg/kg i.v. timolol suppressed the induction of VT by PVS (8/10, 80% rendered noninducible versus 1/10, 10% noninducible in vehicle group; p < 0.01) and prevented the development of acute ischemic lethal arrhythmias (3/10, 30% incidence versus 8/10, 80% incidence in vehicle group; p < 0.05). Concomitant administration of low-dose L-768673 + timolol produced modest increases in QTc and paced QT intervals (4.5 +/- 1.2 and 5.5 +/- 1.4%; both p < 0.01), increases in noninfarct zone relative and effective refractory periods (7.0 +/- 1.7 and 12.3 +/- 3.0%; both p < 0.01), and lesser increases in infarct zone relative and effective refractory periods (5.3 +/- 1.6 and 5.8 +/- 1.4%; both p < 0.01). These findings suggest that concomitant low-dose I(Ks) and beta-adrenergic blockade may constitute a potential pharmacologic strategy for prevention of malignant ischemic ventricular arrhythmias.