Targeting interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) using a novel covalent inhibitor PRN694

Abstract

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.

Keywords: Enzyme Inhibitor; Immunology; Natural Killer Cells (NK cells); T-cell; T-cell Receptor (TCR).

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.

FIGURE 2.

PRN694 blocks cellular and molecular activation of T-lymphocytes and the Jurkat T-ALL cell line. A, gating strategy for flow cytometric analysis of CD69 surface expression in Jurkat cells. Singlet live lymphocytes were gated for CD69 surface expression in Jurkat cells. Jurkat cells (n = 4) (B), human primary CD4 T-cells (n = 6) (C), or human primary CD8 T-cells (n = 4) (D) were pretreated for 30 min with PRN694, BMS-509744, or PRN403 at the indicated doses or vehicle (DMSO), subjected to stimulation with anti-CD3/anti-CD28 for 6 h, and analyzed via flow cytometry for CD69 surface expression. Baseline (unstimulated) CD69 percentage was subtracted, and data were normalized to stimulated and vehicle-treated sample. Jurkat cells (E) or CD3 T-cells (F) isolated from healthy donors were pretreated with vehicle (DMSO), PRN694, BMS-509744, or PRN403 at the indicated doses and stimulated with anti-CD3/anti-CD28 for 6 h. Cell viability was evaluated by flow cytometry using LIVE/DEAD® stain (Invitrogen). Nuclear (G) or cytoplasmic (H) extracts from Jurkat cells pretreated for 30 min with 0.5 μm PRN694, 0.5 μm BMS-509744, 0.5 μm PRN403, 4 μm ibrutinib, or vehicle (DMSO) and stimulated with anti-CD3/anti-CD28 for 45 min were analyzed by immunoblot. Antibodies to actin, lamin B, and GAPDH were included as controls for nuclear versus cytoplasmic extractions. Data are representative of three experiments. For immunoblots G and H, densitometric ratios between phosphoprotein and total or loading control are provided. Error bars, S.E.

FIGURE 3.

TCR-proximal signaling is irreversibly blocked by PRN694, abrogating downstream pathway activation. A and B, freshly isolated primary CD4 T-cells were preincubated with vehicle (DMSO) or PRN694 (0.5 μm) and then stimulated for 45 min with anti-CD3/anti-CD28 antibodies. Nuclear extracts (A) or cytoplasmic extracts (B) were analyzed by immunoblot with the indicated antibodies. C and D, freshly isolated primary CD8 T-cells were treated as described for CD4 cells, and nuclear (C) and cytoplasmic (D) extracts were analyzed by immunoblot. E, Jurkat cells were pretreated with 25 nm PRN694 or vehicle, labeled with Fluo4AM, and then stimulated by the addition of anti-CD3 indicated by an arrow. Calcium flux was measured using the intensity of Fluo-AM measured over 5 min following the addition of anti-CD3. The graph shows cumulative data from 12 replicates conducted over two separate experiments. F, Jurkat cells were pretreated with PRN694 (1 nm to 1 μm), vehicle (DMSO), or BAPTA-AM (13 μm); labeled with Fluo4AM; and then stimulated by the addition of anti-CD3. Calcium flux was measured using the intensity of Fluo-AM measured over 5 min following the addition of anti-CD3. The graph shows the area under the fluorescent intensity/time curve. G and H, Jurkat cells were pretreated with 0.5 μm PRN694 or vehicle (DMSO) and then stimulated either with anti-CD3/anti-CD28 or 50 ng/ml phorbol 12-myristate 13-acetate plus 1 μg/ml ionomycin for 45 min. Nuclear (G) and cytoplasmic (H) extracts were analyzed by immunoblot. For immunoblots A, B, C, D, G, and H, densitometric ratios between phosphoprotein and total or loading control are provided. Error bars, S.E.

FIGURE 4.

PRN694 blocks Fc receptor-induced cellular and molecular activation in primary NK cells. A, immunoblot analysis of nuclear and cytoplasmic extracts from freshly isolated healthy primary NK cells pretreated with 0.5 μm PRN694 or vehicle (DMSO) and stimulated with plate-coated alemtuzumab (10 μg/ml) for 45 min. B, healthy primary NK cells were pretreated with PRN694 at several doses or with vehicle (DMSO) and then stimulated with plate-coated alemtuzumab (10 μg/ml) for 6 h. Surface expression of CD107a/b was analyzed via flow cytometry. Data show the percentage of positive cells of the purified NK cell population. C, health primary NK effector cells were isolated from PBMCs and pretreated with PRN694 or PRN403 at the indicated concentrations. Effector cells were cultured at a 25:1 ratio with 1 μg/ml rituximab biosimilar-coated Jeko-1 target cells for 16 h. Specific lysis of target cells was quantified by measuring lactate dehydrogenase release into the supernatant. Isotype-coated target cells were used as a non-killing control. Error bars, S.E. For all immunoblots, densitometric ratios between phosphoprotein and total or loading control are provided.

FIGURE 5.

PRN694 does not inhibit FcR-induced macrophage, dendritic cell, or mast cell activation. Monocyte-derived macrophages (A) or dendritic cells (B) were stimulated with immobilized human IgG for 24 h. Supernatant levels of TNF were measured. C, monocyte-derived dendritic cells were stimulated with immobilized human IgG for 24 h. Supernatant levels of IL-12 were measured. A–C, data are derived from three individual experimental donors. E, RBL-2H3 mast cells were coated with anti-DNP IgE and subsequently stimulated with DNP for 30 min. Supernatant levels of histamine were measured via ELISA. F, RBL-2H3 mast cells were coated with anti-DNP IgE and subsequently stimulated with DNP for 30 min. Molecular activation was characterized by immunoblot analysis of pERK1/2. In addition, BTK and ITK expression were tested. Jurkat and Mec-1 cells were included as ITK- and BTK-expressing controls. NS, not significant. Error bars, S.E.

FIGURE 6.

PRN694 selectively inhibits ITK and RLK kinase activity. Shown is immunoblot analysis of Jurkat ITK-WT (A) and Jurkat ITK-C442A (B) nuclear and cytoplasmic lysates after pretreatment with 0.5 μm PRN694, 0.5 μm BMS-509744, 0.5 μm PRN403, 1 μm ibrutinib, or vehicle (DMSO) followed by stimulation with anti-CD3/anti-CD28 for 45 min. C, immunoblot analysis of cytoplasmic lysates from parental Jurkat, Jurkat ITK-WT, and Jurkat ITK-C442A cell lines with or without anti-CD3/CD28 stimulation for 45 min. Blots were probed with ITK and GAPDH. Immunoblot analysis of Jurkat-EV (empty vector) (D) and Jurkat-RLK (E) nuclear and cytoplasmic lysates after pretreatment with 0.5 μm PRN694, 0.5 μm BMS-509744, 0.5 μm PRN403, 1 μm ibrutinib, or vehicle (DMSO) followed by stimulation with anti-CD3/anti-CD28 for 45 min. For all immunoblots, densitometric ratios between phosphoprotein and total or loading control are provided.

FIGURE 7.

PRN694 inhibits TCR-induced primary T-cell proliferation and proinflammatory cytokine production without direct cytotoxicity. CD3 T-cells isolated from healthy donors (n = 3) were stained with 1 μm CFSE; pretreated with 0.1 μm PRN694, 0.1 μm PRN869, 0.1 μm PRN403, or 0.1 μm ibrutinib for 30 min; and then incubated without or with anti-CD3/CD28 for 6 days. A, representative flow cytometry analysis of CFSE staining. Shown is a graphical representation of results for CD8 T-cells (B) and CD4 T-cells (C). The division index is the average number of cell divisions that a cell in the original population has undergone. D, cytotoxicity of PRN694 in primary healthy T-cells. CD3 T-cells were isolated from healthy donors (n = 3), pretreated with PRN694 as indicated, and analyzed by annexin/propidium iodide flow cytometry at 24, 48, and 72 h. Data show absolute percentages of CD3-positive cells negative for both annexin and propidium iodide. E–G, CD3 T-cells from healthy donors (n = 4) were pretreated with or without 0.1 μm PRN694, 0.1 μm BMS-509744, 0.1 μm PRN403, or vehicle (DMSO) for 30 min and then stimulated with anti-CD3/CD28 for 48 h. Cytokines, including IL-2 (E), TNF (F), and IFNγ (G), present in the supernatant were assayed by a cytometric bead assay. H, effect of PRN694 on TCR-induced Th17 activation. PBMCs from healthy donors (n = 6) were pre-enriched for CD4⁺/CXCR3⁻/CCR6⁺ cells, which include a high fraction of Th17 cells. These cells were pretreated with vehicle (DMSO), 0.5 μm PRN694, 0.5 μm BMS-509744, or 0.5 μm PRN403 and then stimulated with anti-CD3/anti-CD28 for 12 h. TCR-induced intracellular IL-17a was detected by flow cytometry. Data are shown as the percentage of IL-17a-positive CD4 T-cells normalized to DMSO. I, healthy primary CD8 T-cells were pretreated with 1 μm PRN694 or DMSO control and subsequently cultured at a 25:1 ratio with ⁵¹Cr-labeled allogeneic PBMCs for 4 h. Afterward, specific lysis of allogeneic PBMCs was quantified by measurement of released ⁵¹Cr as compared with maximum and minimum lysis controls. For all figures: *, p < 0.05, **, p < 0.01, ***, p < 0.001. Error bars, S.E.

FIGURE 8.

PRN694 attenuates TCR-induced signaling in primary human T-PLL. A, total RNA from 13 cryopreserved primary T-PLL samples and 4 healthy donors was interrogated for ITK and GAPDH mRNA by quantitative RT-PCR. -Fold change relative to GAPDH is represented along with the mean and S.E. B, HH cells were pretreated with DMSO or 0.1 μm PRN694 for 30 min and then stimulated with anti-CD3/anti-CD28 for 45 min. Nuclear and cytoplasmic cellular extracts were interrogated for activation of NFAT1, JunB, PLCγ1-Y783, and IκBα. C, primary cryopreserved T-PLL samples (n = 8) were stimulated via anti-CD3/anti-CD28 for 45 min, and pPLCγ1-Y783 was examined by phosphoflow cytometry. The mean fluorescence intensity for each sample was normalized to unstimulated. D, immunoblot analysis of TCR-induced (anti-CD3/anti-CD28 for 45 min) primary T-PLL cells freshly obtained from peripheral blood. For immunoblots B and D, densitometric ratios between phosphoprotein and total or loading control are provided. Error bars, S.E.

FIGURE 9.

In vivo pharmacokinetic and pharmacodynamic profile for PRN694 and in vivo inhibition of delayed type hypersensitivity in mice. A, thymocytes were harvested from mice at the indicated time points following intraperitoneal administration of 20 mg/kg PRN694. The thymocytes were treated with an irreversible fluorescent probe for ITK to measure the target occupancy. After separation by SDS-PAGE, the amount of probe binding was measured by fluorescence scanning of the gel. Plasma was also harvested, and the concentration of PRN694 in the plasma was determined by mass spectrometry. B, mice were sensitized with oxazolone on the shaved abdomen and then 7 days later were challenged with oxazolone on both sides of the right ear or vehicle on both sides of the left ear. One hour prior to the challenges, mice were dosed intraperitoneally with vehicle, 0.5 mg/kg dexamethasone (positive control), or PRN694 (20 mg/kg). Twenty-four hours after the challenge, ear edema was measured by weighing 7-mm ear cores. *, p < 0.05. Error bars, S.E.