The EED protein-protein interaction inhibitor A-395 inactivates the PRC2 complex.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

SAR of amino pyrrolidines as potent and novel protein-protein interaction inhibitors of the PRC2 complex through EED binding.

Herein we disclose SAR studies of a series of dimethylamino pyrrolidines which we recently reported as novel inhibitors of the PRC2 complex through disruption of EED/H3K27me3 binding. Modification of the indole and benzyl moieties of screening hit 1 provided analogs with substantially improved binding and cellular activities. This work culminated in the identification of compound 2, our nanomolar proof-of-concept (PoC) inhibitor which provided on-target tumor growth inhibition in a mouse xenograft model. X-ray crystal structures of several inhibitors bound in the EED active-site are also discussed.

SAR and characterization of non-substrate isoindoline urea inhibitors of nicotinamide phosphoribosyltransferase (NAMPT).

Herein we disclose SAR studies that led to a series of isoindoline ureas which we recently reported were first-in-class, non-substrate nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. Modification of the isoindoline and/or the terminal functionality of screening hit 5 provided inhibitors such as 52 and 58 with nanomolar antiproliferative activity and preclinical pharmacokinetics properties which enabled potent antitumor activity when dosed orally in mouse xenograft models. X-ray crystal structures of two inhibitors bound in the NAMPT active-site are discussed.

Superoxide produced by mitochondrial site IQ inactivates cardiac succinate dehydrogenase and induces hepatic steatosis in Sod2 knockout mice.

Superoxide produced by mitochondria has been implicated in numerous physiologies and pathologies. Eleven different mitochondrial sites that can produce superoxide and/or hydrogen peroxide (O2.-/H2O2) have been identified in vitro, but little is known about their contributions in vivo. We introduce novel variants of S1QELs and S3QELs (small molecules that suppress O2.-/H2O2 production specifically from mitochondrial sites IQ and IIIQo, respectively, without compromising bioenergetics), that are suitable for use in vivo. When administered by intraperitoneal injection, they achieve total tissue concentrations exceeding those that are effective in vitro. We use them to study the engagement of sites IQ and IIIQo in mice lacking functional manganese-superoxide dismutase (SOD2). Lack of SOD2 is expected to elevate superoxide levels in the mitochondrial matrix, and leads to severe pathologies and death about 8 days after birth. Compared to littermate wild-type mice, 6-day-old Sod2-/- mice had significantly lower body weight, lower heart succinate dehydrogenase activity, and greater hepatic lipid accumulation. These pathologies were ameliorated by treatment with a SOD/catalase mimetic, EUK189, confirming previous observations. A 3-day treatment with S1QEL352 decreased the inactivation of cardiac succinate dehydrogenase and hepatic steatosis in Sod2-/- mice. S1QEL712, which has a distinct chemical structure, also decreased hepatic steatosis, confirming that O2.- derived specifically from mitochondrial site IQ is a significant driver of hepatic steatosis in Sod2-/- mice. These findings also demonstrate the ability of these new S1QELs to suppress O2.- production in the mitochondrial matrix in vivo. In contrast, suppressing site IIIQo using S3QEL941 did not protect, suggesting that site IIIQo does not contribute significantly to mitochondrial O2.- production in the hearts or livers of Sod2-/- mice. We conclude that the novel S1QELs are effective in vivo, and that site IQ runs in vivo and is a significant driver of pathology in Sod2-/- mice.

Discovery and metabolic stabilization of potent and selective 2-amino-N-(adamant-2-yl) acetamide 11beta-hydroxysteroid dehydrogenase type 1 inhibitors.

Starting from a rapidly metabolized adamantane 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) inhibitor 22a, a series of E-5-hydroxy-2-adamantamine inhibitors, exemplified by 22d and (+/-)-22f, was discovered. Many of these compounds are potent inhibitors of 11beta-HSD1 and are selective over 11beta-HSD2 for multiple species (human, mouse, and rat), unlike other reported species-selective series. These compounds have good cellular potency and improved microsomal stability. Pharmacokinetic profiling in rodents indicated moderate to large volumes of distribution, short half-lives, and a pharmacokinetic species difference with the greatest exposure measured in rat with 22d. One hour postdose liver, adipose, and brain tissue 11beta-HSD1 inhibition was confirmed with (+/-)-22f in a murine ex vivo assay. Although 5,7-disubstitued-2-adamantamines provided greater stability, a single, E-5-position, polar functional group afforded inhibitors with the best combination of stability, potency, and selectivity. These results indicate that adamantane metabolic stabilization sufficient to obtain short-acting, potent, and selective 11beta-HSD1 inhibitors has been discovered.

The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia.

Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2) is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1), but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

Discovery and development of potent and selective inhibitors of histone methyltransferase g9a.

G9a is a histone lysine methyltransferase responsible for the methylation of histone H3 lysine 9. The discovery of A-366 arose from a unique diversity screening hit, which was optimized by incorporation of a propyl-pyrrolidine subunit to occupy the enzyme lysine channel. A-366 is a potent inhibitor of G9a (IC50: 3.3 nM) with greater than 1000-fold selectivity over 21 other methyltransferases.

Discovery of adamantane ethers as inhibitors of 11beta-HSD-1: Synthesis and biological evaluation.

A novel class of adamantane ethers 11beta-hydroxysteroid hydrogenase type I inhibitors has been discovered. These compounds have excellent HSD-1 potency and selectivity against HSD-2. The structure-activity relationships, selectivity, metabolism, PK, ex vivo pharmacodynamic data, and an X-ray crystal structure of one of these inhibitors bound to h-HSD-1 are discussed.

The synthesis and biological evaluation of a novel series of antimicrobials of the oxazolidinone class.

A novel series of antimicrobials of the oxazolidinone class is disclosed. These compounds are characterized relative to previously described analogues by a 'halostilbene-derived' pharmacophore and demonstrate enhanced antimicrobial activity against key Gram-positive pathogens when compared to Linezolid.