Covalent Inhibitors of Interleukin‑2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood Assay

We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concentrations. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogues are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approximately 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk

Discovery and Preclinical Profiling of 3‑[4-(Morpholin-4-yl)‑7<i>H</i>‑pyrrolo[2,3‑<i>d</i>]pyrimidin-5-yl]benzonitrile (PF-06447475), a Highly Potent, Selective, Brain Penetrant, and in Vivo Active LRRK2 Kinase Inhibitor

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson’s disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of <b>14</b> (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies

Acyl Glucuronide Metabolites of 6‑Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]‑1<i>H</i>‑indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-Activated Protein Kinase (AMPK)

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1β1γ1 isoform have culminated in the identification of PF-06409577 (<b>1</b>), PF-06885249 (<b>2</b>), and PF-06679142 (<b>3</b>) as potential clinical candidates. Compounds <b>1</b>–<b>3</b> are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of <b>1</b>–<b>3</b>. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of β1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1β1γ1 isoform with <b>1</b>–<b>3</b> and M1–M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates