Discovery of Allosteric, Potent, Subtype Selective, and Peripherally Restricted TrkA Kinase Inhibitors.

Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are activated by hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). Moreover, the NGF antibody tanezumab has provided clinical proof of concept for inhibition of the TrkA kinase pathway in pain leading to significant interest in the development of small molecule inhibitors of TrkA. However, achieving TrkA subtype selectivity over TrkB and TrkC via a Type I and Type II inhibitor binding mode has proven challenging and Type III or Type IV allosteric inhibitors may present a more promising selectivity design approach. Furthermore, TrkA inhibitors with minimal brain availability are required to deliver an appropriate safety profile. Herein, we describe the discovery of a highly potent, subtype selective, peripherally restricted, efficacious, and well-tolerated series of allosteric TrkA inhibitors that culminated in the delivery of candidate quality compound 23.

Discovery of novel irreversible inhibitors of interleukin (IL)-2-inducible tyrosine kinase (Itk) by targeting cysteine 442 in the ATP pocket.

IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both TH1 and TH2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.

Experiences in implementing uHTS--cutting edge technology meets the real world.

Driven by growing corporate compound files, the demands of target biology, and attempts to cut cost, the number of solutions to HTS has spiralled. In quick succession new assay technologies and screening platforms are appearing on the market, with the promise of screening faster than ever in low volume high density formats whilst providing high quality data. Within this world of rapid change, Pfizer has applied cutting edge technology to HTS by introducing screening in 1 microl formats utilising single molecule detection technology. Instead of resource intensive in-house development, Pfizer entered into a collaboration with Evotec OAI / Evotec Technologies and introduced their Mark-II EVOscreen platform. In this article we will outline the benefits of the approach taken at Pfizer, Sandwich, and introduce the Mark-II EVOscreen platform, illustrating the potential but also possible pitfalls of HTS miniaturisation.