Discovery of structural diverse reversible BTK inhibitors utilized to develop a novel in vivo CD69 and CD86 PK/PD mouse model.

For the past two decades, BTK a tyrosine kinase and member of the Tec family has been a drug target of significant interest due to its potential to selectively treat various B cell-mediated diseases such as CLL, MCL, RA, and MS. Owning to the challenges encountered in identifying drug candidates exhibiting the potency block B cell activation via BTK inhibition, the pharmaceutical industry has relied on the use of covalent/irreversible inhibitors to address this unmet medical need. Herein, we describe a medicinal chemistry campaign to identify structurally diverse reversible BTK inhibitors originating from HITS identified using a fragment base screen. The leads were optimized to improve the potency and in vivo ADME properties resulting in a structurally distinct chemical series used to develop and validate a novel in vivo CD69 and CD86 PD assay in rodents.

Utilizing structure based drug design and metabolic soft spot identification to optimize the in vitro potency and in vivo pharmacokinetic properties leading to the discovery of novel reversible Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine kinase (BTK) is an essential node on the BCR signaling in B cells, which are clinically validated to play a critical role in B-cell lymphomas and various auto-immune diseases such as Multiple Sclerosis (MS), Pemphigus, and rheumatoid arthritis (RA). Although non-selective irreversible BTK inhibitors have been approved for oncology, due to the emergence of drug resistance in B-cell lymphoma associated with covalent inhibitor, there an unmet medical need to identify reversible, selective, potent BTK inhibitor as viable therapeutics for patients. Herein, we describe the identification of Hits and subsequence optimization to improve the physicochemical properties, potency and kinome selectivity leading to the discovery of a novel class of BTK inhibitors. Utilizing Met ID and structure base design inhibitors were synthesized with increased in vivo metabolic stability and oral exposure in rodents suitable for advancing to lead optimization.

Optimization of novel reversible Bruton's tyrosine kinase inhibitors identified using Tethering-fragment-based screens.

Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton's tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for the pharmaceutical industry has been the identification of reversible, selective, potent molecules for inhibition of BTK. Herein, we report application of Tethering-fragment-based screens to identify low molecular weight fragments which were further optimized to improve on-target potency and ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors suitable for pre-clinical proof-of-concept studies.

A mouse Fcgamma-Fcepsilon protein that inhibits mast cells through activation of FcgammaRIIB, SH2 domain-containing inositol phosphatase 1, and SH2 domain-containing protein tyrosine phosphatases.

BACKGROUND: A human Fcgamma-Fcepsilon fusion protein (GE2) designed to inhibit FcepsilonRI signaling by coaggregating FcepsilonRI with the inhibitory receptor FcgammaRIIB has been shown to inhibit mast cell activation and block cutaneous anaphylaxis. A critical issue remained as to whether the mechanism of GE2 inhibition is competition for IgE binding or inhibitory signaling through FcgammaRIIB. OBJECTIVE: Our aim was to define the in vitro and in vivo mechanism of action of a mouse homolog of GE2 (mGE) and to assess the potential of human GE2 (hGE2) for therapeutic administration. METHODS: The in vitro activity of mGE on mediator release and signaling pathways was characterized in IgE-sensitized bone marrow-derived mast cells (BMMCs). The in vivo activity of mGE was examined in mouse passive cutaneous and passive systemic anaphylaxis models, and the therapeutic activity of hGE2 was evaluated in Ascaris suum-sensitized cynomolgus monkeys. RESULTS: mGE inhibited release of histamine and cytokines by BMMCs from wild-type mice but not by BMMCs from FcgammaRIIB-deficient mice. In mice mGE blocked IgE-dependent anaphylaxis mediated by mast cells with sustained efficacy. In BMMCs mGE decreased spleen tyrosine kinase and extracellular signal-regulated kinases 1/2 phosphorylation and induced FcgammaRIIB phosphorylation and the subsequent recruitment of SH2 domain-containing inositol polyphosphate 5' phosphatase (SHIP) 1 and SH2 domain-containing protein tyrosine phosphatase (SHP) 1/2 phosphatases. When administered therapeutically, hGE2 protected sensitized monkeys from local anaphylaxis for 3 weeks. CONCLUSION: mGE-mediated inhibition of mast cell activation is associated with inhibitory signaling through FcgammaRIIB that results from activation of SHIP-1 and SHP-1/2 phosphatases.

Systemic administration of an Fcgamma-Fc(epsilon)-fusion protein in house dust mite sensitive nonhuman primates.

Crosslinking Fc(epsilon)RI and FcgammaRIIB receptors inhibits mast cell and basophil activation, decreasing mediator release. In this study, a fusion protein incorporating human Fcgamma and Fc(epsilon) domains, hGE2, was shown to inhibit degranulation of human mast cells and basophils, and to exhibit efficacy in a nonhuman primate model of allergic asthma. hGE2 increased the provocative concentration of dust mite aeroallergen that induced an early phase asthmatic response. The treatment effect lasted up to 4 weeks and was associated with reduction in the number of circulating basophils and decreased expression of Fc(epsilon)RI on repopulating basophils. Repeat hGE2 dosing induced production of serum antibodies against human Fcgamma and Fc(epsilon) domains and acute anaphylaxis-like reactions. Immune serum induced histamine release from human IgE or hGE2-treated cord blood-derived mast cells and basophils in vitro. These results indicate that repeat administration with hGE2 induced an antibody response to the human molecule that resulted in activation rather than inhibition of allergic responses.

Homeostasis and anergy of CD4(+)CD25(+) suppressor T cells in vivo.

CD4(+)CD25(+) suppressor T (TS) cells play a critical role in the maintenance of peripheral tolerance. We examined here proliferative and functional responses as well as differential gene expression in T(S) cells. We found that T(S) cells were hyporesponsive to antigenic stimuli in vivo and unable to flux Ca(2+) upon T cell receptor (TCR) engagement. However, T(S) cells were not impaired in their proliferative response to lymphopenia, which was dependent on major histocompatibility complex class II expression. Homeostatic proliferation did not abolish T(S) cell anergy; rather, it substantially augmented T(S) cell function. DNA array analyses identified genes that may inhibit responsiveness at a number of levels in multiple signaling cascades in T(S) cells, as well as several anti-apoptotic genes that may mediate their survival.