A highly selective JAK3 inhibitor is developed for treating rheumatoid arthritis by suppressing γc cytokine-related JAK-STAT signal

Abstract

Janus kinases (JAKs) play a critical role in immune responses by relaying signals from more than 50 cytokines, making them attractive therapeutic targets for autoimmune diseases. Although approved JAK inhibitors have demonstrated clinical efficacy, they target a broad spectrum of cytokines, which results in side effects. Therefore, next-generation inhibitors maintain efficacy, while sparing adverse events need to be developed. Among members of the JAK family, JAK3 only regulates a narrow spectrum of γc cytokines and becomes a potentially ideal target. Here, a highly JAK3-selective inhibitor Z583 is developed, which showed a potent inhibition of JAK3 with an IC₅₀ of 0.1 nM and exhibited a 4500-fold selectivity for JAK3 than other JAK subtypes. Furthermore, Z583 completely inhibited the γc cytokine signaling and sufficiently blocked the development of inflammatory response in RA model, while sparing hematopoiesis. Collectively, the highly selective JAK3 inhibitor Z583 is a promising candidate with significant therapeutic potential for autoimmune diseases.

Fig. 1.. The biological characteristics of selective JAK3 inhibitor Z583.

(A) The structure of compound Z583. (B) Inhibitory activity and selectivity of Z583 for JAK family kinases at K_m ATP and 1 mM ATP concentration. (C) The IC₅₀ of Z583 and ritlecitinib inhibiting JAK3 at 1 mM ATP concentration. (D) KINOMEscan kinase selectivity profile for Z583 at a concentration of 0.1 μM. (E) The selective inhibition of Z583 against a panel of 48 kinases. (F) The inhibitory potent and selectivity of Z583 for JAK family kinases and kinases with cysteine residue.

Fig. 2.. The binding property of Z583 with JAK3 subtype.

(A) Z583 can bind to Cys⁹⁰⁹ of the x-ray crystal of JAK3 (PDB ID: 4Z16). (B) Z583 irreversibly inhibits the expression of p-STAT5 after stimulation by γc cytokines of IL-2 or IL-15 in CD4⁺ T cells from C57BL/6, even after being washed extensively with phosphate-buffered saline. (C) LC-MS/MS experiments identified a single modified peptide LVMEYLPSGC*LR (C*, cysteine residue) by Z583 at Cys⁹⁰⁹ position. m/z, mass/charge ratio.

Fig. 3.. Z583 selectively inhibited the JAK3-dependent signaling pathway in hPBMCs.

(A) Four JAK subtypes (JAK1, JAK2, JAK3, and Tyk2) relay intracellular signal transduction with combinations of heterodimers or homodimers. (B) JAKs and STATs engage associated cytokines to mediate biological function. hPBMCs or CD34⁺ cells (for EPO) with or without various concentrations of Z583 were stimulated with JAK3-particular γc cytokines (C and E) or other cytokines (D and F). Western blots were carried out to evaluate the expression of cytokine-associated STAT phosphorylation. All data are from three independent experiments. Data are presented as means ± SEM. ##P < 0.01, ###P < 0.001 versus control group and *P < 0.05, **P < 0.01, and ***P < 0.001 versus alone cytokine-stimulating group. ns, no statistical significance.

Fig. 4.. Z583 inhibited the phenotypic maturation of DCs in vitro.

(A) Representative morphology of DC population after different treatment as shown by phase-contrast microscopy. iDCs from monocytes of C57BL/6 mice were pretreated with Z583 (10, 1, and 0.1 μM) for 1 hour and then followed by LPS stimulation for another 24 hours; percentage of CD80⁺CD11c⁺ (B) and CD86⁺CD11c⁺ (C) cells were analyzed by flow cytometry. The production of proinflammatory cytokines TNF-α (D), IL-6 (E), and IL-12 (F) in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). All data are from three independent experiments. Data are presented as means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 versus LPS-treated control group.

Fig. 5.. Selective JAK3 inhibition with Z583 suppressed the naïve T cell proliferation and differentiation.

Naïve CD4⁺ T cells from lymph nodes of C57BC/6 mice were isolated to perform the proliferation and differentiation assays. (A) Naïve CD4⁺ T cells were stimulated with anti-CD3/anti-CD28; the effect of Z583 on T cell proliferation was analyzed by flow cytometry. CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 in the presence or absence of Z583 (10, 1, and 0.1 μM) for 72 hours; the expression of IFN-γ (B) and T_H17A (D) was analyzed by flow cytometry. The production of IFN-γ (C) and T_H17A (E) by CD4⁺ T cells in the supernatant was measured by ELISA; the IC₅₀ values of Z583 (0.01, 0.3, 1, 3, and 10 μM) for inhibiting T cell differentiation were calculated. All data are from three independent experiments. Data are presented as means ± SEM; **P < 0.01 and ***P < 0.001 versus anti-CD3/anti-CD28–treated control group.

Fig. 6.. Selective JAK3 inhibitor Z583 treatment alleviated CIA in mice.

(A) Experimental scheme of the prophylactic effect of Z583 for the analysis of CIA; DBA/1 mice were immunized on days 0 and 21 and were treated with different doses of Z583 (3, 1, and 0.3 mg/kg) from day 27 for 3 weeks. (B) Representative hind paws from each treatment group. Clinical score (C) and CIA incidence (D) were assessed every 3 days in control, CIA mice, and Z583-treated CIA mice. (E) Ankle joint sections were stained by hematoxylin and eosin (H&E) or saffron O/Fast green staining. Quantification of synovitis (F), cartilage degradation (G), and bone erosion (H) in ankle joint was assessed using clinical scores on day 48. (I) Representative micro-CT images of hind paws and knees. (J) Bone volume of the right hind paw second to fourth metatarsophalangeal joint was assessed using Mimics software. (K) Experimental scheme of the therapeutic effect of Z583 for the analysis of CIA. DBA/1 mice were immunized on days 0 and 21 and were treated with different doses of Z583 (10, 3, and 1 mg/kg) from day 43 for 4 weeks. (L) Clinical score was observed every 3 days in control, CIA mice, and Z583-treated CIA mice. Data are presented as means ± SEM, n = 8. ##P < 0.01 and ###P < 0.001 versus control group; *P < 0.05, **P < 0.01, and ***P < 0.001 versus CIA group.

Fig. 7.. Safety and pharmacokinetic property of Z583.

(A) Signal dose acute oral toxicity was performed by up-and-down procedure according to the Organization for Economic Cooperation and Development for chemical testing guidelines no. 425 and limited test with single dose at 2 g/kg. (B) Chinese hamster ovary cells were used to measure the possible inhibition of Z583 for hERG potassium current by patch-clamp recording. (C) S. typhimurium strains TA100 and TA102 were used to evaluate the mutagenic potentials of Z583 by bacterial reverse mutation assay; data are presented as means ± SEM, n = 3; ***P < 0.001 versus positive control; ns, no statistical significance of positive group versus blank or vehicle group. White blood cells (D), monocytes (E), lymphocytes (F), neutrophils (G), red blood cells (H), hemoglobin (I), hematocrit (J), and platelets (K) were measured in the blood of rats dosed for 4 weeks with Z583 at 20, 60, and 180 mg/kg (n = 10); data are presented as means ± SEM. ns, no statistical significance of control group versus three treatment group. (L) Preliminary pharmacokinetic property of Z583 by oral (p.o.) and intravenous (i.v.) route in mice (n = 8) was analyzed by LC-MS/MS.