FIGURE 1.

Structure-based design and selectivity of the novel ITK/RLK inhibitor PRN694. A, chemical structure of PRN694. B, model of PRN694 covalently bound to ITK. Molecular modeling predicts that the aminobenzimidazole scaffold makes two hydrogen bonds to the backbone amides of the hinge residues of the ATP binding pocket. The CF2-thiophene was found to provide high potency for either a phenylalanine or threonine gatekeeper residue. A methylpyrolidine linker presents the acrylamide in a position easily accessible for covalent bond formation with Cys-442. C, the dissociation of reversible versus irreversible inhibitors. Ligand binding to ITK as a function of time (hours) in a biochemical off-rate assay utilizing time-resolved FRET was determined as described under “Experimental Procedures.” The completely reversible inhibitor BMS-509744 demonstrated extremely rapid dissociation from ITK (residence time (τ) = 32 ± 17 min), whereas PRN694 remained bound to ITK for the entire assay period, consistent with its irreversible covalent binding. D, the in vitro selectivity and potency of PRN694 against a panel of 250 kinases. Screening of PRN694 was performed at 0.1 and 1.0 μm. The results are displayed on a human kinase dendrogram (reproduced courtesy of Cell Signaling Technology, Inc.). E, PRN694 was tested in a microfluidics format kinase assay that separated phosphorylated from unphosphorylated peptide substrate based on capillary electrophoresis. PRN694 was tested against each target that contains a Cys in a homologous position to Cys-442 in ITK. IC₅₀ values were calculated from plots of percentage inhibition of activity as a function of inhibitor concentration. All assays were performed at Nanosyn Inc. F, the BioMap cell screening panel (performed by BioSeek, South San Francisco, CA) is a set of cell-based assays used to understand the cellular potency and selectivity of inhibitory compounds. A variety of cell culture and co-culture systems are stimulated with a range of molecules. Biomarker readouts are measured as an indicator of various pathway activities in the culture systems. The biomarker readouts measured in each system are indicated along the x axis. The y axis shows the log10 expression ratios of the readout level measurements relative to solvent (DMSO buffer) controls. Three concentrations of PRN694 were tested for the ability to modulate the various readouts. The three culture systems that showed dose-dependent modulation of biomarkers in response to PRN694 were the SAg system, the BT system, and the TH2 system. The SAg system consists of primary human umbilical vein endothelial cells cultured with peripheral blood mononuclear cells stimulated with superantigens. The BT system consists of B cells co-cultured with peripheral blood mononuclear cells stimulated with anti-IgM and mild T-cell receptor stimulation. The TH2 system consists of primary human umbilical vein endothelial cells co-cultured with 14-day TH2 polarized CD4 T-cell blasts stimulated with TCR and IL-2. PRN694 had essentially no inhibitory activity in cell types that do not express ITK and RLK, including 3C (venular endothelial cells (HuVEC)/IL-1β, TNF, and IFNγ), 4H (HuVEC/IL-4 and histamine), LPS (PBMC and HuVEC/LPS), BF4T (bronchial epithelial cells and human dermal fibroblasts/TNF and IL-4), BE3C (bronchial epithelial cells/IL-1β, TNF, and IFNγ), CASM3C (coronary artery smooth muscle cells/IL-1β, TNF, and IFNγ), HDF3CGF (human dermal fibroblasts/IL-1β, TNF, IFNγ, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor-BB), KF3CT (keratinocytes and dermal fibroblasts/IL-1β, TNF, and IFNγ), MyoF (lung fibroblasts/TNF and TGF-β), and Mphg (HuVEC and macrophages/TLR2). Weak inhibitory signals were only detected at the highest PRN694 concentration in the venular endothelia system (3C), the PBMC/HUVEC system (LPS), and the human dermal fibroblast system (HDF3CGF), potentially due to low level off-target effects.