The target of FM-381 is Janus Kinase 3 (JAK3). It exhibits picomolar affinity and unprecedented kinome-wide selectivity for JAK3, achieved by covalent reversible targeting of a JAK3-specific cysteine residue and a ligand-induced binding pocket. [1]

FM-381 is tested at 100 nM and 500 nM concentrations against a panel of 410 kinases. At 100 nM, FM-381 has no discernible impact on the activity of any of the examined kinases other than JAK3. Eleven additional kinases, including JAK3, are moderately inhibited by FM-381 at 500 nM, with residual activity less than 50%. A selectivity panel of frequently hit BRDs (BRD4, BRPF, CECR, FALZ, TAF1, BRD9) reveals that FM-381 is inactive. Human CD4+ T cells with specific JAK3 signaling inhibition from FM-381. In the dose-dependent BRET experiment, FM-381 exhibits an apparent EC50 of 100 nM. It also inhibits phosphorylation of STAT5 induced by IL2 (JAK3/JAK1 dependent) at 100 nM, but not JAK3 independent IL6 (JAK1/2/TYK dependent) stimulated STAT3 signaling in human CD4+ T cells up to 1 µM[1].

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1. Kinase selectivity: FM-381 shows unprecedented kinome-wide selectivity for JAK3, with picomolar inhibitory activity against JAK3. It does not exhibit significant inhibitory effects on other JAK isoforms (JAK1, JAK2, TYK2) or other kinases in the kinome [1]
2. Inhibition of JAK3-mediated cytokine signaling: Human CD4⁺ T cells were pre-incubated with FM-381 for 1 hour at different concentrations, then stimulated with cytokines for 30 minutes. FM-381 selectively inhibited JAK3-mediated cytokine signaling pathways: it suppressed the phosphorylation of STAT5 induced by IL-2 (which activates JAK3/JAK1) and the phosphorylation of STAT6 induced by IL-4 (which activates JAK3/JAK1). In contrast, it had no significant effect on the phosphorylation of STAT3 induced by IL-6 (which activates JAK2/JAK1/TYK2) or the phosphorylation of STAT1 induced by IFN-α (which activates JAK1/TYK2), demonstrating its high selectivity for JAK3 [1]
3. Binding kinetics and residence time: In BRET experiments using HeLa cells expressing NanoLuc-tagged JAK3, FM-381 displaced the fluorescent tracer in a dose-dependent manner. Residence time experiments showed that after equilibrating HeLa cells expressing NanoLuc-JAK3 with 1 μM FM-381, washing out the inhibitor and treating with high concentrations of tracer, the displacement of FM-381 was slow, with BRET levels of full occupancy control reached after approximately 1 hour, indicating a long residence time of FM-381 on JAK3. Mutant studies (JAK3 R953A and R911A/R953A) showed that both mutants retained the slow binding kinetics of FM-381 [1]

1. Co-crystal structure analysis: Co-crystal structures of JAK3 with FM-381 (compound 5 in the literature) were resolved, including both non-covalently and covalently bound forms (PDB: 5LWN). The 2Fₒ − Fc omitted electron density map confirmed the binding mode of FM-381 to JAK3. The analysis revealed that FM-381 binds to a novel induced fit binding pocket of JAK3, with rearrangement of amino acid side chains (R911, D912, R953) forming a unique arginine pocket that is not observed in other JAK3 crystal structures (e.g., PDB: 3LXK, 4QT1, 4QPS, etc.). This induced pocket is critical for the high selectivity of FM-381 for JAK3 [1]
2. BRET-based binding assay: HeLa cells were transfected to express NanoLuc-tagged JAK3. Cells were equilibrated with different concentrations of FM-381, then a fluorescent tracer was added. The displacement of the tracer by FM-381 was monitored by measuring BRET signals, which reflected the binding affinity and dose-dependent interaction between FM-381 and JAK3. For residence time measurement, cells were first equilibrated with 1 μM FM-381, washed to remove unbound inhibitor, then treated with high concentrations of tracer. The change in BRET signals over time was recorded to determine the residence time of FM-381 on JAK3 [1]

1. Human CD4⁺ T cell cytokine signaling assay: Human CD4⁺ T cells were isolated and cultured in vitro. The cells were pre-incubated with different concentrations of FM-381 (or control compounds like Tofacitinib) for 1 hour, then stimulated with IL-2 (1 hr pre-incubation + 30 min stimulation), IL-4 (1 hr pre-incubation + 30 min stimulation), IL-6 (1 hr pre-incubation + 30 min stimulation), or IFN-α (1 hr pre-incubation + 30 min stimulation). After stimulation, cell lysates were prepared, and western blotting was performed using phospho-specific antibodies to detect the phosphorylation levels of STAT5 (IL-2-induced), STAT6 (IL-4-induced), STAT3 (IL-6-induced), and STAT1 (IFN-α-induced). Actin was used as a loading control to ensure equal protein loading. The results demonstrated the selective inhibition of JAK3-mediated STAT phosphorylation by FM-381 [1]
2. Mutant JAK3 binding assay in HeLa cells: HeLa cells were transfected to express NanoLuc-tagged JAK3 mutants (R953A and R911A/R953A). The cells were treated with FM-381 (1 μM) for equilibration, then subjected to washout and tracer displacement steps as in the wild-type JAK3 BRET assay. BRET signals were measured over time to evaluate the binding kinetics of FM-381 to the mutant JAK3 proteins, confirming that the induced fit binding pocket (involving R911, R953) is critical for the slow binding kinetics but not the core binding of FM-381 to JAK3 [1]

[1]. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket. Cell Chem Biol. 2016 Nov 17;23(11):1335-1340.

1. FM-381 is a novel JAK3 inhibitor with a covalently reversible binding mode that targets a newly discovered inducible-fit binding pocket on JAK3. Due to the high structural conservatism among JAK subtypes, JAK subtype-selective targeted therapy faces challenges, hence FM-381 was developed to address this problem [1]. 2. The unique mechanism of FM-381 involves the simultaneous covalently and reversibly targeting of JAK3-specific cysteine residues and ligand-induced binding pockets (formed by the rearrangement of R911, D912, and R953), thereby endowing it with unprecedented kinase selectivity and picomolar-level JAK3 affinity [1]. 3. JAK3 functions only on immune cells, therefore subtype-selective JAK3 inhibitors like FM-381 are of great value for achieving precise anti-inflammatory effects and reducing off-target toxicity in clinical practice. FM-381 is a powerful chemical probe that can be used to elucidate the specific biological functions of JAK3 [1].

C24H24N6O2

428.49

428.196

2226521-65-7

122197584

Light yellow to yellow solid powder

1.4±0.1 g/cm3

749.8±70.0 °C at 760 mmHg

407.3±35.7 °C

0.0±2.5 mmHg at 25°C

1.709

3.77

1

5

4

32

785

0

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)