Application of cultured human mast cells (CHMC) for the design and structure-activity relationship of IgE-mediated mast cell activation inhibitors.

Here we report the optimization of small molecule inhibitors of human mast cell degranulation via anti-IgE-mediated tryptase release following cross-linking and activation of IgE-loaded FcεR1 receptors. The compounds are selective upstream inhibitors of FcεR1-dependent human mast cell degranulation and proved to be devoid of activity in downstream ionomycin mediated degranulation. Structure-activity relationship (SAR) leading to compound 26 is outlined.

Design, synthesis of diaminopyrimidine inhibitors targeting IgE- and IgG-mediated activation of Fc receptor signaling.

Using cultured human mast cells (CHMC) the optimization of 2,4-diaminopyrimidine compounds leading to 22, R406 is described. Compound 22 is a potent upstream inhibitor of mast cell degranulation and its mechanism of action is via inhibition of Syk kinase. Compound 22 has significant activity in inhibiting both IgE- and IgG-mediated activation of Fc receptor (FcR) in mast cells and basophils, and in addition inhibits Syk kinase-dependent activity of FcR-mediated activation of monocytes, macrophages, neutrophils, and B cell receptor (BCR)-mediated activation of B lymphocytes. Overall, the biological activity of 22 suggests that it has potential for application as a novel therapeutic for the treatment of an array of autoimmune maladies and hematological malignancies.

R723, a selective JAK2 inhibitor, effectively treats JAK2V617F-induced murine myeloproliferative neoplasm.

The activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs.

Tuning the Reactivity of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Activators for Optimal in Vivo Efficacy.

Dimethyl fumarate 1 is approved for the treatment of multiple sclerosis but is also associated with off-target activation of the niacin receptor. By using a tetrazolone or triazolone bioisostere approach to the fumarate and vinyl sulfone series of Nrf2 activators, we have optimized the electrophilicity of the double bond to tune the on-target Nrf2 activation with PK properties to achieve efficacy in animal models of multiple sclerosis. The study linked highly potent, highly electrophilic molecules to low plasma stability and, subsequently, limited efficacy. By contrast, a sulfonylvinyltriazolone 17 retains on-target potency but shows much weaker electrophilic potential. As a consequence, in vivo high exposures of 17 are obtained, resulting in efficacy in the EAE model similar to that observed for DMF. 17 (R079) is Ames negative, is not cytotoxic to cells, and shows little inhibition of either the niacin receptor or a panel of off-target receptors.

Regulation of bone homeostasis by MERTK and TYRO3.

The fine equilibrium of bone homeostasis is maintained by bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we show that TAM receptors MERTK and TYRO3 exert reciprocal effects in osteoblast biology: Osteoblast-targeted deletion of MERTK promotes increased bone mass in healthy mice and mice with cancer-induced bone loss, whereas knockout of TYRO3 in osteoblasts shows the opposite phenotype. Functionally, the interaction of MERTK with its ligand PROS1 negatively regulates osteoblast differentiation via inducing the VAV2-RHOA-ROCK axis leading to increased cell contractility and motility while TYRO3 antagonizes this effect. Consequently, pharmacologic MERTK blockade by the small molecule inhibitor R992 increases osteoblast numbers and bone formation in mice. Furthermore, R992 counteracts cancer-induced bone loss, reduces bone metastasis and prolongs survival in preclinical models of multiple myeloma, breast- and lung cancer. In summary, MERTK and TYRO3 represent potent regulators of bone homeostasis with cell-type specific functions and MERTK blockade represents an osteoanabolic therapy with implications in cancer and beyond.

Metabolism of fostamatinib, the oral methylene phosphate prodrug of the spleen tyrosine kinase inhibitor R406 in humans: contribution of hepatic and gut bacterial processes to the overall biotransformation.

The metabolism of the spleen tyrosine kinase inhibitor N4-(2,2-dimethyl-3-oxo-4-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine (R406) and its oral prodrug N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine disodium hexahydrate (R788, fostamatinib) was determined in vitro and in humans. R788 was rapidly converted to R406 by human intestinal microsomes, and only low levels of R788 were observed in plasma of human subjects after oral administration of (14)C-R788. R406 was the major drug-related compound in plasma from human subjects, and only low levels of metabolites were observed in plasma. The plasma metabolites of R406 were identified as a sulfate conjugate and glucuronide conjugate of the para-O-demethylated metabolite of R406 (R529) and a direct N-glucuronide conjugate of R406. Elimination of drug-related material into the urine accounted for 19% of the administered dose, and the major metabolite in urine from all the human subjects was the lactam N-glucuronide of R406. On average, 80% of the total drug was recovered in feces. Two drug-related peaks were observed; one peak was identified as R406, and the other peak was identified as a unique 3,5-benzene diol metabolite of R406. The 3,5-benzene diol metabolite appeared to result from the subsequent O-demethylations and dehydroxylation of R529 by anaerobic gut bacteria because only R529 was converted to this metabolite after the in vitro incubation with human fecal samples. These data indicate that the major fecal metabolite of R406 observed in humans is a product of a hepatic cytochrome P450-mediated O-demethylation and subsequent O-demethylations and dehydroxylation by gut bacteria.