A miniaturized mode-of-action profiling platform enables high throughput characterization of the molecular and cellular dynamics of EZH2 inhibition.

The market approval of Tazemetostat (TAZVERIK) for the treatment of follicular lymphoma and epithelioid sarcoma has established "enhancer of zeste homolog 2" (EZH2) as therapeutic target in oncology. Despite their structural similarities and common mode of inhibition, Tazemetostat and other EZH2 inhibitors display differentiated pharmacological profiles based on their target residence time. Here we established high throughput screening methods based on time-resolved fluorescence energy transfer, scintillation proximity and high content analysis microscopy to quantify the biochemical and cellular binding of a chemically diverse collection of EZH2 inhibitors. These assays allowed to further characterize the interplay between EZH2 allosteric modulation by methylated histone tails (H3K27me3) and inhibitor binding, and to evaluate the impact of EZH2's clinically relevant mutant Y641N on drug target residence times. While all compounds in this study exhibited slower off-rates, those with clinical candidate status display significantly slower target residence times in wild type EZH2 and disease-related mutants. These inhibitors interact in a more entropy-driven fashion and show the most persistent effects in cellular washout and antiproliferative efficacy experiments. Our work provides mechanistic insights for the largest cohort of EZH2 inhibitors reported to date, demonstrating that-among several other binding parameters-target residence time is the best predictor of cellular efficacy.

Discovery and characterization of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists.

PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders.

Systematic identification of biomarker-driven drug combinations to overcome resistance.

The ability to understand and predict variable responses to therapeutic agents may improve outcomes in patients with cancer. We hypothesized that the basal gene-transcription state of cancer cell lines, coupled with cell viability profiles of small molecules, might be leveraged to nominate specific mechanisms of intrinsic resistance and to predict drug combinations that overcome resistance. We analyzed 564,424 sensitivity profiles to identify candidate gene-compound pairs, and validated nine such relationships. We determined the mechanism of a novel relationship, in which expression of the serine hydrolase enzymes monoacylglycerol lipase (MGLL) or carboxylesterase 1 (CES1) confers resistance to the histone lysine demethylase inhibitor GSK-J4 by direct enzymatic modification. Insensitive cell lines could be sensitized to GSK-J4 by inhibition or gene knockout. These analytical and mechanistic studies highlight the potential of integrating gene-expression features with small-molecule response to identify patient populations that are likely to benefit from treatment, to nominate rational candidates for combinations and to provide insights into mechanisms of action.

The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies.

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.

Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia.

While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.

Efficient Routes to a Diverse Array of Amino Alcohol-Derived Chiral Fragments.

Efficient syntheses of chiral fragments derived from chiral amino alcohols are described. Several unique scaffolds were readily accessed in 1-5 synthetic steps leading to 45 chiral fragments, including oxazolidinones, morpholinones, lactams, and sultams. These fragments have molecular weights ranging from 100 to 255 Da and are soluble in water (0.085 to >15 mM).

A Short Synthesis of Aphanamol I in Both Racemic and Enantiopure Forms.

A short synthesis of the biologically active sesquiterpene natural product (+)-aphanamol I in both racemic and enantiopure forms is reported. Key steps include: a catalytic enantioselective conjugate addition, an oxidative radical cyclization, and a ring-expanding Claisen rearrangement.

Discovery of 6-substituted indole-3-glyoxylamides as lead antiprion agents with enhanced cell line activity, improved microsomal stability and low toxicity.

A series of highly potent indole-3-glyoxylamide based antiprion agents was previously characterized, focusing on optimization of structure-activity relationship (SAR) at positions 1-3 of the indole system. New libraries interrogating the SAR at indole C-4 to C-7 now demonstrate that introducing electron-withdrawing substituents at C-6 may improve biological activity by up to an order of magnitude, and additionally confer higher metabolic stability. For the present screening libraries, both the degree of potency and trends in SAR were consistent across two cell line models of prion disease, and the large majority of compounds showed no evidence of toxic effects in zebrafish. The foregoing observations thus make the indole-3-glyoxylamides an attractive lead series for continuing development as potential therapeutic agents against prion disease.

Structure-activity relationship refinement and further assessment of indole-3-glyoxylamides as a lead series against prion disease.

Structure-activity relationships within the indole-3-glyoxylamide series of antiprion agents have been explored further, resulting in discovery of several new compounds demonstrating excellent activity in a cell line model of prion disease (EC50 <10 nM). After examining a range of substituents at the para-position of the N-phenylglyoxylamide moiety, five-membered heterocycles containing at least two heteroatoms were found to be optimal for the antiprion effect. A number of modifications were made to probe the importance of the glyoxylamide substructure, although none were well tolerated. The most potent compounds did, however, prove largely stable towards microsomal metabolism, and the most active library member cured scrapie-infected cells indefinitely on administration of a single treatment. The present results thereby confirm the indole-3-glyoxylamides as a promising lead series for continuing in vitro and in vivo evaluation against prion disease.

Intentional retrieval of viabil stent-grafts from the biliary system.

The present report describes the intentional retrieval of six Viabil stent-grafts from the biliary system. The six stent-grafts were successfully removed from five patients with a mean time from placement to retrieval of 38 days. Successful retrieval was independent of underlying disease etiology and included strictures associated with benign disease, malignant disease, disease of unknown etiology, and stent-graft misplacement. There were no complications resulting from stent-graft retrieval. Intentional removal of Viabil stent-grafts is technically feasible. Further study is necessary to establish its potential role in the management of biliary disease.