Fragment-based Scaffold Hopping: Identification of Potent, Selective, and Highly Soluble Bromo and Extra Terminal Domain (BET) Second Bromodomain (BD2) Inhibitors.

The profound efficacy of pan-BET inhibitors is well documented, but these epigenetic agents have shown pharmacology-driven toxicity in oncology clinical trials. The opportunity to identify inhibitors with an improved safety profile by selective targeting of a subset of the eight bromodomains of the BET family has triggered extensive medicinal chemistry efforts. In this article, we disclose the identification of potent and selective drug-like pan-BD2 inhibitors such as pyrazole 23 (GSK809) and furan 24 (GSK743) that were derived from the pyrrole fragment 6. We transpose the key learnings from a previous pyridone series (GSK620 2 as a representative example) to this novel class of inhibitors, which are characterized by significantly improved solubility relative to our previous research.

MEK inhibitors overcome resistance to BET inhibition across a number of solid and hematologic cancers.

BET inhibitors exhibit broad activity in cancer models, making predictive biomarkers challenging to define. Here we investigate the biomarkers of activity of the clinical BET inhibitor GSK525762 (I-BET; I-BET762) across cancer cell lines and demonstrate that KRAS mutations are novel resistance biomarkers. This finding led us to combine BET with RAS pathway inhibition using MEK inhibitors to overcome resistance, which resulted in synergistic effects on growth and survival in RAS pathway mutant models as well as a subset of cell lines lacking RAS pathway mutations. GSK525762 treatment up-regulated p-ERK1/2 levels in both RAS pathway wild-type and mutant cell lines, suggesting that MEK/ERK pathway activation may also be a mechanism of adaptive BET inhibitor resistance. Importantly, gene expression studies demonstrated that the BET/MEK combination uniquely sustains down-regulation of genes associated with mitosis, leading to prolonged growth arrest that is not observed with either single agent therapy. These studies highlight a potential to enhance the clinical benefit of BET and MEK inhibitors and provide a strong rationale for clinical evaluation of BET/MEK combination therapies in cancer.

Osteogenic effects of a potent Src-over-Abl-selective kinase inhibitor in the mouse.

Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC(50) 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1-10 nM). Compound I (≥0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 µM compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, ß (ß-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by ß-CGRP.

(1-(4-(Naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine: a wingless beta-catenin agonist that increases bone formation rate.

A high-throughput screening campaign to discover small molecule leads for the treatment of bone disorders concluded with the discovery of a compound with a 2-aminopyrimidine template that targeted the Wnt beta-catenin cellular messaging system. Hit-to-lead in vitro optimization for target activity and molecular properties led to the discovery of (1-(4-(naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine (5, WAY-262611). Compound 5 has excellent pharmacokinetic properties and showed a dose dependent increase in the trabecular bone formation rate in ovariectomized rats following oral administration.

A cell-based Dkk1 binding assay reveals roles for extracellular domains of LRP5 in Dkk1 interaction and highlights differences between wild-type and the high bone mass mutant LRP5(G171V).

Dkk1 is a secreted antagonist of the LRP5-mediated Wnt signaling pathway that plays a pivotal role in bone biology. Because there are no well-documented LRP5-based assays of Dkk1 binding, we developed a cell-based assay of Dkk1/LRP5 binding using radioactive (125)I-Dkk1. In contrast to LRP6, transfection of LRP5 alone into 293A cells resulted in a low level of specific binding that was unsuitable for routine assay. However, co-transfection of LRP5 with the chaperone protein MesD (which itself does not bind Dkk1) or Kremen-2 (a known Dkk1 receptor), or both, resulted in a marked enhancement of specific binding that was sufficient for evaluation of Dkk1 antagonists. LRP5 fragments comprising the third and fourth beta-propellers plus the ligand binding domain, or the first beta-propeller, each inhibited Dkk1 binding, with mean IC(50)s of 10 and 196 nM, respectively. The extracellular domain of Kremen-2 ("soluble Kremen") was a weaker antagonist (mean IC(50) 806 nM). We also found that cells transfected with a high bone mass mutation LRP5(G171V) had a subtly reduced level of Dkk1 binding, compared to wild type LRP5-transfected cells, and no enhancement of binding by MesD. We conclude that (1) LRP5-transfected cells do not offer a suitable cell-based Dkk1 binding assay, unless co-transfected with either MesD, Kremen-2, or both; (2) soluble fragments of LRP5 containing either the third and fourth beta-propellers plus the ligand binding domain, or the first beta-propeller, antagonize Dkk1 binding; and (3) a high bone mass mutant LRP5(G171V), has subtly reduced Dkk1 binding, and, in contrast to LRP5, no enhancement of binding with MesD.

Isogenic human cell lines for drug discovery: regulation of target gene expression by engineered zinc-finger protein transcription factors.

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF-generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

In vitro and in vivo activities of C-terminally truncated PTH peptides reveal a disconnect between cAMP signaling and functional activity.

There is considerable evidence implicating the cAMP-signaling pathway in the anabolic action of PTH; and to date, all PTH and PTHrp peptides that stimulate cyclic AMP are active in animal models of osteogenesis. We have tested two C-terminally truncated peptides, PTH(1-29) and a modified PTH(1-21) (MPTH(1-21)), in in vitro and in vivo assays of PTH action. Each of the C-terminally truncated peptides was of low nanomolar potency in assays of receptor binding and cAMP stimulation. However, when we tested these peptides for functional response in Saos-2 cells stably transfected with a cyclic AMP response element (CRE) reporter, the C-terminally truncated peptides were two to four times less potent than would be expected from their binding and cAMP-stimulating properties. Furthermore, PTH(1-29), although active, was approximately 20-fold less potent than PTH(1-34) in a rat model of osteogenesis while MPTH(1-21) was inactive. The relative lack of activity of these peptides in vivo suggests that while activation of the cAMP pathway may be important for the anabolic effect of PTH fragments, it is not, of itself, predictive of their in vivo activity.