D19-07 Target-selective Modulators of Erk1/2 Signaling Attenuate Airway Smooth Muscle Cell Proliferation

Rationale Airway smooth muscle (ASM) hyperplasia contributes to fixed airflow limitation in chronic asthma and correlates with disease severity. ERK1/2 signaling via mitogen-activated protein kinases regulates ASM proliferation. The use of ATP-competitive inhibitors of ERK results in unintended off-target side effects. To overcome this, we developed target-selective modulators of ERK1/2 that bind the FRS, the substrate-binding domain of ERK2, which is known to interact with and activate transcription factor Activator Protein-1 (AP-1), using computational approaches. We hypothesize that these novel modulators inhibit ASM proliferation without exerting untoward cytotoxicity. Methods Human ASM cells were treated with different concentrations of target-selective ERK1/2 modulators (referred to as SP compounds), followed by 10 ng/ml PDGF. Cell proliferation (CyQUANT), AP-1 activity (luciferase assay), expression of AP-1 components (FRA-1, c-Jun, FosB, c-Fos), Cyclin D1, and α-SMA (western blot), and ASM cells 3D spheroid model (fluorescence/immunoblotting) were analyzed. VX-11e, an ATP-competitive inhibitor of ERK1/2 was used as a positive control. Results SP25, SP38, SP90, SP96, and SP98 significantly reduced HASM cell proliferation, as assessed by CyQUANT assay without exerting cytotoxicity in ASM cells. This anti-proliferative effect was associated with a selective downregulation of PDGF-induced FRA-1 and FosB protein expression, key components of the AP-1 transcription factor complex, in a time-dependent manner. In contrast, c-Fos and c-Jun expression remained unchanged, highlighting the selective modulation of AP-1 by SP compounds. Notably, the ERK inhibitor VX-11e broadly suppressed all AP-1 proteins, indicating global ERK pathway inhibition that may disrupt cellular homeostasis. SP compound treatment also led to a marked decrease in Cyclin D1, a downstream target of AP-1 and key regulator of cell cycle progression, confirming the anti-proliferative effect of SP compounds. In AP-1 luciferase reporter assays, PDGF-induced AP-1 promoter activity was suppressed by SP compounds. Finally, in a 3D spheroid culture model, SP compounds attenuated PDGF-induced α-SMA expression, as assessed using a confocal microscope and western blotting. Conclusion Our study demonstrates that a novel class of target-selective ERK1/2 modulators effectively suppresses ASM proliferation by targeting AP-1 components. These findings highlight the therapeutic potential of substrate-selective inhibitors for asthma-related airway remodeling with minimal off-target effects or toxicity.