In vitro and in vivo induction of fetal hemoglobin with a reversible and selective DNMT1 inhibitor.

Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2'-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.

Exploiting binding-site arginines in drug design: Recent examples.

Active or allosteric site arginines can form diverse interactions with ligands including different types of cation-π interactions, H-bond interactions and non-bond, non-canonical interactions. This provides many opportunities for creative structure-based drug design to improve potency, introduce novelty, and modulate MoA (mode of action), and even to achieve selectivity. This digest will use some recent drug targets of interest as examples to illustrate different types of interactions and how these interactions impact on potency, MoA, and selectivity.

Endemic plant species are more palatable to introduced herbivores than non-endemics.

Islands harbour a spectacular diversity and unique species composition. This uniqueness is mainly a result of endemic species that have evolved in situ in the absence of mammal herbivores. However, island endemism is under severe threat by introduced herbivores. We test the assumption that endemic species are particularly vulnerable to generalist introduced herbivores (European rabbit) using an unprecedented dataset covering an entire island with enormous topographic, climatic and biological diversity (Tenerife, Canary Islands). With increasing endemism, plant species are more heavily browsed by rabbits than non-endemic species with up to 67% of endemics being negatively impacted by browsing, indicating a dramatic lack of adaptation to mammal herbivory in endemics. Ecosystems with high per cent endemism are most heavily browsed, suggesting ecosystem-specific vulnerability to introduced herbivores, even within islands. Protection of global biodiversity caused by disproportionally high endemism on oceanic islands via ecosystem-specific herbivore control and eradication measures is of utmost importance.

Nucleoside protein arginine methyltransferase 5 (PRMT5) inhibitors.

Protein Arginine Methyltransferase 5 (PRMT5) is known to symmetrically dimethylate numerous cytosolic and nuclear proteins that are involved in a variety of cellular processes. Recent findings have revealed its potential as a cancer therapeutic target. PRMT5 selective inhibitors, GSK3326595, a substrate competitive inhibitor, and JNJ64619178, a SAM (S-adenosyl-l-methionine) mimetic/competitive inhibitor, have entered clinic trials for multiple cancer types. This review focuses on the recent developments in SAM mimetic nucleoside PRMT5 inhibitors, their SAR and structural insight based on published co-crystal structures.

Synthesis and structure-activity relationships of imidazo[1,2-a]pyrimidin-5(1H)-ones as a novel series of beta isoform selective phosphatidylinositol 3-kinase inhibitors.

A series of PI3K-beta selective inhibitors, imidazo[1,2-a]-pyrimidin-5(1H)-ones, has been rationally designed based on the docking model of the more potent R enantiomer of TGX-221, identified by a chiral separation, in a PI3K-beta homology model. Synthesis and SAR of this novel chemotype are described. Several compounds in the series demonstrated potent growth inhibition in a PTEN-deficient breast cancer cell line MDA-MB-468 under anchorage independent conditions.

Synthesis and structure-activity relationships of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones as novel series of potent ß isoform selective phosphatidylinositol 3-kinase inhibitors.

A series of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones with excellent enzyme inhibition, improved isoform selectivity, and excellent inhibition of downstream phosphorylation of AKT has been identified. Several compounds in the series demonstrated potent (∼ 0.100 µM IC(50)) growth inhibition in a PTEN deficient cancer cell line.

Preclinical activity of eltrombopag (SB-497115), an oral, nonpeptide thrombopoietin receptor agonist.

Eltrombopag is a first-in-class, orally bioavailable, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), which is being developed as a treatment for thrombocytopenia of various etiologies. In vitro studies have demonstrated that the activity of eltrombopag is dependent on expression of TpoR, which activates the signaling transducers and activators of transcription (STAT) and mitogen-activated protein kinase signal transduction pathways. The objective of this preclinical study is to determine if eltrombopag interacts selectively with the TpoR to facilitate megakaryocyte differentiation in platelets. Functional thrombopoietic activity was demonstrated by the proliferation and differentiation of primary human CD34(+) bone marrow cells into CD41(+) megakaryocytes. Measurements in platelets in several species indicated that eltrombopag specifically activates only the human and chimpanzee STAT pathways. The in vivo activity of eltrombopag was demonstrated by an increase of up to 100% in platelet numbers when administered orally (10 mg/kg per day for 5 days) to chimpanzees. In conclusion, eltrombopag interacts selectively with the TpoR without competing with Tpo, leading to the increased proliferation and differentiation of human bone marrow progenitor cells into megakaryocytes and increased platelet production. These results suggest that eltrombopag and Tpo may be able to act additively to increase platelet production.

2,3,5-Trisubstituted pyridines as selective AKT inhibitors. Part II: Improved drug-like properties and kinase selectivity from azaindazoles.

A novel series of AKT inhibitors containing 2,3,5-trisubstituted pyridines with novel azaindazoles as hinge binding elements are described. Among these, the 4,7-diazaindazole compound 2c has improved drug-like properties and kinase selectivity than those of indazole 1, and displays greater than 80% inhibition of GSK3beta phosphorylation in a BT474 tumor xenograft model in mice.

2,3,5-Trisubstituted pyridines as selective AKT inhibitors-Part I: Substitution at 2-position of the core pyridine for ROCK1 selectivity.

2,3,5-Trisubstituted pyridines have been designed as potent AKT inhibitors that are selective against ROCK1 based on the comparison between AKT and ROCK1 structures. Substitution at the 2-position of the core pyridine is the key element to provide selectivity against ROCK1. An X-ray co-crystal structure of 9p in PKA supports the proposed rationale of ROCK1 selectivity.

Tetrasubstituted pyridines as potent and selective AKT inhibitors: Reduced CYP450 and hERG inhibition of aminopyridines.

The synthesis and evaluation of tetrasubstituted aminopyridines, bearing novel azaindazole hinge binders, as potent AKT inhibitors are described. Compound 14c was identified as a potent AKT inhibitor that demonstrated reduced CYP450 inhibition and an improved developability profile compared to those of previously described trisubstituted pyridines. It also displayed dose-dependent inhibition of both phosphorylation of GSK3beta and tumor growth in a BT474 tumor xenograft model in mice.

Discovery of Isoxazole Amides as Potent and Selective SMYD3 Inhibitors.

We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure-activity relationship of the high-throughput screening (HTS) lead compound 1 provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.

Discovery of 5-pyrrolopyridinyl-2-thiophenecarboxamides as potent AKT kinase inhibitors.

A pyrrolopyridinyl thiophene carboxamide 7 was discovered as a tractable starting point for a lead optimization effort in an AKT kinase inhibition program. SAR studies aided by a co-crystal structure of 7 in AKT2 led to the identification of AKT inhibitors with subnanomolar potency. Representative compounds showed antiproliferative activity as well as inhibition of phosphorylation of the downstream target GSK3beta.

Discovery of Potent and Selective Covalent Protein Arginine Methyltransferase 5 (PRMT5) Inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is known to symmetrically dimethylate numerous cytosolic and nuclear proteins that are involved in a variety of cellular processes. Recent findings have revealed its potential as a cancer therapeutic target. PRMT5 possesses a cysteine (C449) in the active site, unique to PRMT5. Therefore, covalent PRMT5 inhibition is an attractive chemical approach. Herein, we report an exciting discovery of a series of novel hemiaminals that under physiological conditions can be converted to aldehydes and react with C449 to form covalent adducts, which presumably undergo an unprecedented elimination to form the thiol-vinyl ethers, as indicated by electron density in the co-crystal structure of the PRMT5/MEP50 complex.

Nuisance Compounds, PAINS Filters, and Dark Chemical Matter in the GSK HTS Collection.

High-throughput screening (HTS) hits include compounds with undesirable properties. Many filters have been described to identify such hits. Notably, pan-assay interference compounds (PAINS) has been adopted by the community as the standard term to refer to such filters, and very useful guidelines have been adopted by the American Chemical Society (ACS) and subsequently triggered a healthy scientific debate about the pitfalls of draconian use of filters. Using an inhibitory frequency index, we have analyzed in detail the promiscuity profile of the whole GlaxoSmithKline (GSK) HTS collection comprising more than 2 million unique compounds that have been tested in hundreds of screening assays. We provide a comprehensive analysis of many previously published filters and newly described classes of nuisance structures that may serve as a useful source of empirical information to guide the design or growth of HTS collections and hit triaging strategies.

Discovery and characterization of a selective, nonpeptidyl thrombopoietin receptor agonist.

OBJECTIVE: Peptide and other small molecule agonists have been described for several cytokines and growth factors. Hydrazone compounds described here as thrombopoietin receptor agonists were identified as activating STAT proteins in a Tpo responsive cell line. METHODS: STAT activation and analysis of signal transduction pathways in cell lines and normal human platelets was elucidated by Western blot and electrophoretic mobility shift assays. Proliferation assays in cell types responsive to other cytokines determined specificity for Tpo receptor. Flow cytometry quantified differentiation of CD34(+) cells into CD41(+) megakaryocytes and platelet production in vitro. RESULTS: Activation of STAT5, mitogen-activated protein kinase, p38, and early response genes by SB 394725 was similar to that induced by Tpo. SB 394725 induced a reporter gene response under a STAT activation promoter as well as the megakaryocyte-specific gpIIb promoter. The compound induced proliferation of Tpo responsive lines but demonstrated no activity in cell lines responding to other cytokines, i.e., erythropoietin, granulocyte-colony stimulating factor, interleukin-3, interferon-gamma. The response of normal human Tpo receptors was elucidated by measuring growth and differentiation of human bone marrow in vitro. Activation of endogenous Tpo receptors by SB 394725 was demonstrated in human and chimp platelets, but not in platelets of other species including mouse, dog, rabbit, or cynomolgus monkey. CONCLUSIONS: SB 394725, a small molecule with a molecular weight of 452 Da, is capable of activating Tpo-specific signal transduction, proliferation, and differentiation responses similar to the responses and functions of the protein growth factor, Tpo.

Discovery of a Novel 2,6-Disubstituted Glucosamine Series of Potent and Selective Hexokinase 2 Inhibitors.

A novel series of potent and selective hexokinase 2 (HK2) inhibitors, 2,6-disubstituted glucosamines, has been identified based on HTS hits, exemplified by compound 1. Inhibitor-bound crystal structures revealed that the HK2 enzyme could adopt an "induced-fit" conformation. The SAR study led to the identification of potent HK2 inhibitors, such as compound 34 with greater than 100-fold selectivity over HK1. Compound 25 inhibits in situ glycolysis in a UM-UC-3 bladder tumor cell line via (13)CNMR measurement of [3-(13)C]lactate produced from [1,6-(13)C2]glucose added to the cell culture.

Identification of a pharmacophore for thrombopoietic activity of small, non-peptidyl molecules. 1. Discovery and optimization of salicylaldehyde thiosemicarbazone thrombopoietin mimics.

High-throughput screening has resulted in the discovery of thiosemicarbazone thrombopoietin mimics. A shared pharmacophore hypothesis between this series and a previously identified class, the pyrazol-4-ylidenehydrazines, led to the rapid optimization of both potency and efficacy of the thiosemicarbazones. The application of high-throughput chemistry and purification techniques allowed for the rapid elucidation of structure-activity relationships.

Identification of a pharmacophore for thrombopoietic activity of small, non-peptidyl molecules. 2. Rational design of naphtho[1,2-d]imidazole thrombopoietin mimics.

The invention of a new class of naphtho[1,2-d]imidazole thrombopoietin mimics based on a pharmacophore hypothesis for small-molecule thrombopoietic agonists is discussed. Parallel array synthesis and purification techniques allowed for the rapid exploration of structure-activity relationships within this class and for the improvement in TPO mimetic potencies and efficacies.

An efficient synthesis of 3-substituted 3H-pyrimidin-4-ones.

[reaction: see text] A novel and practical synthesis of 3-substituted 3H-pyrimidin-4-ones is described. The key step involves the cyclization of enamide esters, derived from readily available beta-keto esters, with trimethylaluminum and various primary amines.

[3a,4]-Dihydropyrazolo[1,5a]pyrimidines: Novel, Potent, and Selective Phosphatidylinositol-3-kinase ß Inhibitors.

A series of novel [3a,4]dihydropyrazolo[1,5a]pyrimidines were identified, which were highly potent and selective inhibitors of PI3Kß. The template afforded the opportunity to develop novel SAR for both the hinge-binding (R3) and back-pocket (R4) substitutents. While cellular potency was relatively modest due to high protein binding, the series displayed low clearance in rat, mouse, and monkey.