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, 288 (39), 28195-206

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

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Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

John D Harling et al. J Biol Chem.

Abstract

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.

Keywords: Asthma; Drug Discovery; IL-2-inducible Tyrosine Kinase; Kinase Inhibitor; Medicinal Chemistry; Nonreceptor Tyrosine Kinase; T Cell.

Figures

SCHEME 1.
SCHEME 1.
Time-dependent irreversible inhibition. a, in addition to competitive inhibition, irreversible inhibition also involves a reaction that irreversibly transforms the inhibitor-enzyme complex into an “inactive“ form (denoted by E*I, where kinact describes the rate at which inhibitor-enzyme complex is irreversibly transformed into E*I). The parameter KI is the inhibitor concentration that results in half the maximal rate of inactivation (23). b, the velocity of product formation approximated for irreversible inhibition is denoted, where S represents substrate concentration, and I is inhibitor concentration. Due to the irreversible formation of E*I, the functional catalytic enzyme concentration E(cat)(t) decreases in time, resulting in a time-dependent maximal velocity Vmax(t) = kcat·E(cat)(t).
FIGURE 1.
FIGURE 1.
a, inhibition of Itk by compound 12 is time-dependent. The rate of binding of compound 12 to Itk was assessed in the LanthaScreen binding assay. Concentrations of compound 12 were as follows: 0 nm (■), 0.3 nm (□), 0.8 nm (●) 1.3 nm (○), 1.9 nm (▴), 3.7 nm (▵), 8 nm (♦), and 17 nm (♢), and 50 nm (▾). A rapid initial binding event is followed by a slower onset of increased inhibition. b, Kitz-Wilson plot of compound 12. Shown is Kitz-Wilson analysis for compound 12 binding to Itk (kobs data derived from Fig. 1a). The kinact and KI parameters can be derived from the reciprocals of the y and x intercepts, respectively. The non-zero y intercept is consistent with a two-step inactivation event. c, inhibition of Itk by compound 5 is not time-dependent. The rate of binding of compound 5 to Itk was assessed in the LanthaScreen binding assay. Concentrations of compound 5 were as follows: 0 nm (●), 15 nm (▿), 46 nm (▴), 137 nm (▵), 412 nm (■), 1,235 nm (□), and 3,704 nm (▾). A single, rapid binding event is consistent with simple reversible binding.
FIGURE 2.
FIGURE 2.
a, x-ray crystallography of drug-kinase complex. Shown is the x-ray crystal structure of compound 7 covalently complexed with the Itk kinase domain (Y512E) at 2.18 Å resolution obtained by replacement soaking a co-crystal of the kinase domain complexed with a non-covalently bound tool compound. b, an FoFc difference “omit” map contoured at 3σ (gray mesh) highlighting the active site of complex C (of the four complexes (A–D) in the asymmetric unit) and calculated after omitting compound 7 and Cys-442 from the model. It reveals that compound 7 has displaced the tool compound (conclusively in this site) and is consistent with Cys-442 adopting two rotamer conformations, one of which is covalently bound to compound 7.
FIGURE 3.
FIGURE 3.
T cell versus B cell selectivity. Compound 12 shows inhibition T cell response (CytoStim (●) or anti-CD3/CD28 (■)) with ∼20–100-fold greater potency than B cell response (anti-IgM (▴)). Inhibition of CD3/CD28- or CytoStim-induced IFNγ production was used as a marker of T cell inhibition. Inhibition of anti-IgM-induced CD69 was used as a marker of B cell inhibition. Results are shown as a mean ± S.E. (error bars) of seven donors for CytoStim, four donors for anti-CD3/CD28, and three donors for anti-IgM. Note that different donors were used for each of the three assay types.
FIGURE 4.
FIGURE 4.
Compound 12 does not selectively inhibit TH2 cytokine release. Compound 12 produced a concentration-dependent inhibition of IL-2 (■), IFNγ (●), IL-17 (▴), and IL-13 (▾) release following activation of human PBMCs with either CytStim (a) or CD2/CD3/CD28 (b) for 24 h. Results are shown as mean ± S.E. (error bars) of seven donors.
FIGURE 5.
FIGURE 5.
Compound 12 shows additive inhibition with a glucocorticoid. Human PBMCs were stimulated with either CytoStim- or anti-CD2/CD3/CD28-coated beads in the presence of increasing concentrations of glucocorticoid (FP) alone (●) or FP combined with increasing concentrations of compound 12 (0.2 nm (■), 1 nm (▴), or 5 nm (▾)). Co-administration of FP with compound 12 produced an upward shift of the FP concentration response curve, indicative of an additive effect. Inhibition by vehicle or Compound 12 alone is shown in open symbols. Results are shown as percentage inhibition of cytokine release following 24-h incubation, expressed as mean ± S.E. (error bars) of four donors.
FIGURE 6.
FIGURE 6.
Compound 12 inhibits IL-2, IFNγ, and IL-17 release from fragments of human lung parenchyma. Compound 12 (●) and glucocorticoid (FP) (▴) produced a concentration-dependent inhibition of IL-2 (a), IFNγ (b), and IL-17 (c) release from fragments of human lung parenchyma following a 72-h incubation with PHA. Results are expressed as mean ± S.E. of 5–6 donors.
FIGURE 7.
FIGURE 7.
Compound 12 retains long duration of action activity following washout. The washout profile was determined for both reversible (compound 5) and irreversible (compound 12) Itk inhibitors. Control curves show the concentration-dependent inhibition of IL-2 release from PBMC in response to activation by anti-CD3/CD28 in the presence of compound. Cells were washed to remove free compound and incubated for a further 2 or 19 h before activation with anti-CD3/CD28. Results are shown as mean ± S.E. (error bars) of four donors.
FIGURE 8.
FIGURE 8.
Itk pulse-chase. Jurkat cells, in the presence of absence of compound 12, were biosynthetically pulse-labeled with [35S]methionine for 30 min, followed by a cold methionine chase for 0–24 h. Cell extracts were prepared, and Itk was immunoprecipitated and analyzed by SDS-PAGE and fluorography (Itk IP). Identical cell extracts were prepared in parallel from non-radiolabeled Jurkat cells and analyzed by Western blotting with antibodies to Itk (Itk WB) and actin (Actin WB) as a loading control. In lane 11 (downward arrow) was loaded 20 ng of purified Itk protein as a control.
FIGURE 9.
FIGURE 9.
Rat PD model following inhaled administration of compound 12. Lung cell suspensions were prepared from rats who had inhaled air alone (a) or compound 12 (b). Cells were unstimulated (control) or activated ex vivo with anti-CD3, followed by measurement of CD25 expression by flow cytometry on CD4+ cells. Specificity of the anti-CD25 staining is demonstrated with anti-CD3 activated cells by the isotype control (isotype). The CD4+/CD25+ cells are boxed, and the percentage of CD4+ cells with increased CD25 staining is shown.

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