Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways

Abstract

A member of the Janus kinase (JAK) family, Tyrosine Kinase 2 (TYK2), is crucial in mediating various cytokine-signaling pathways such as interleukin-23 (IL23), interleukin-12 (IL12) and type I Interferons (IFN) which contribute to autoimmune disorders (e.g., psoriasis, lupus, and inflammatory bowel disease). Thus, TYK2 represents an attractive target to develop small-molecule therapeutics for the treatment of cytokine-driven inflammatory diseases. Selective inhibition of TYK2 over other JAK isoforms is critical to achieve a favorable therapeutic index in the development of TYK2 inhibitors. However, designing small molecule inhibitors to target the adenosine triphosphate (ATP) binding site of TYK2 kinase has been challenging due to the substantial structural homology of the JAK family catalytic domains. Here, we employed an approach to target the JAK homology 2 (JH2) pseudokinase regulatory domain of the TYK2 protein. We developed a series of small-molecule TYK2 pseudokinase ligands, which suppress the TYK2 catalytic activity through allosteric regulation. The TYK2 pseudokinase-binding small molecules in this study simultaneously achieve high affinity-binding for the TYK2 JH2 domain while also affording significantly reduced affinity for the TYK2 JAK homology 1 (JH1) kinase domain. These TYK2 JH2 selective molecules, although possessing little effect on suppressing the catalytic activity of the isolated TYK2 JH1 catalytic domain in the kinase assays, can still significantly block the TYK2-mediated receptor-stimulated pathways by binding to the TYK2 JH2 domain and allosterically regulating the TYK2 JH1 kinase. These compounds are potent towards human T-cell lines and primary immune cells as well as in human whole-blood specimens. Moreover, TYK2 JH2-binding ligands exhibit remarkable selectivity of TYK2 over JAK isoforms not only biochemically but also in a panel of receptor-stimulated JAK1/JAK2/JAK3-driven cellular functional assays. In addition, the TYK2 JH2-targeting ligands also demonstrate high selectivity in a multi-kinase screening panel. The data in the current study underscores that the TYK2 JH2 pseudokinase is a promising therapeutic target for achieving a high degree of biological selectivity. Meanwhile, targeting the JH2 domain represents an appealing strategy for the development of clinically well-tolerated TYK2 inhibitors that would have superior efficacy and a favorable safety profile compared to the existing Janus kinase inhibitors against autoimmune diseases.

Deucravacitinib, a tyrosine kinase 2 pseudokinase inhibitor, protects human beta cells against proinflammatory insults

“…Based on our findings, it will be interesting to test whether novel small molecule TYK2 pseudokinase ligands [49] could also protect beta cells from IFNα deleterious effects. Nevertheless, we must bear in mind that completely inhibiting TYK2 may be counterproductive, as it might lead to susceptibility to microorganisms (e.g.…”

Development and Therapeutic Implications of Tyrosine Kinase 2 Inhibitors

“…112 Brepocitinib and ropsacitinib were found to inhibit functional responses of other JAK family members at varying levels, while TYK2 JH2 ligands displayed robust inhibition on TYK2-dependent signaling but no obvious influence on other JAK-related pathways. 112 This further confirmed the advantages of TYK2 JH2 ligands in both potency and selectivity. It was noted that 82-1 showed much lower activity against JAK1-dependent IL-6 induced phosphorylation of STAT3 in TF-1 cells, when compared to BMS986165 (IC 50 : 5900 nM for 82-1 vs 100 nM for BMS986165), suggesting a higher selectivity against JAK1 of 82-1.…”

“…It was noted that 82-1 showed much lower activity against JAK1-dependent IL-6 induced phosphorylation of STAT3 in TF-1 cells, when compared to BMS986165 (IC 50 : 5900 nM for 82-1 vs 100 nM for BMS986165), suggesting a higher selectivity against JAK1 of 82-1. 112,152 In addition, this company claimed another series of BMS-986165 analogues by replacing the cyclopropyl amide at C6 with substituted aminopyridine, exemplified by compound 82-2 (US20220002267). 153 82-2 showed potent binding affinity with TYK2 JH2 with K d of 0.0052 nM in a KdELECT competition binding assay, and functional potency against IL-23 and IFNα signaling pathways with IC 50 values of 5.9 and 83 nM, respectively, in Kit225 T cell assays.…”

“…According to the binding site to TYK2, the reported TYK2 inhibitors are generally divided into two types: JH1-targeting inhibitors (or orthosteric inhibitors) and JH2 ligands (or allosteric inhibitors) that bind to the JH2 domain. Accumulating evidence reveals that allosteric inhibitors possess much higher selectivity for TYK2 over other JAK family members, when compared with JH1-targeting inhibitors. , Some of TYK2 inhibitors have progressed into clinical trials (Table ), and most strikingly, a selective TYK2 allosteric inhibitor, deucravacitinib (BMS-986165, 11 ), has been approved by the FDA for the treatment of moderate-to-severe plaque psoriasis . Besides, a dual TYK2 and JAK1 inhibitor, brepocitinib (PF06700841, 12 ), , as well as a selective TYK2 inhibitor, ropsacitinib (PF06826647, 13 ), are currently in phase II clinical trials for multiple indications.…”

“…This further confirmed the advantages of TYK2 JH2 ligands in both potency and selectivity. It was noted that 82 - 1 showed much lower activity against JAK1-dependent IL-6 induced phosphorylation of STAT3 in TF-1 cells, when compared to BMS986165 (IC 50 : 5900 nM for 82 - 1 vs 100 nM for BMS986165), suggesting a higher selectivity against JAK1 of 82 - 1 . , In addition, this company claimed another series of BMS-986165 analogues by replacing the cyclopropyl amide at C6 with substituted aminopyridine, exemplified by compound 82 - 2 (US20220002267)…”