Integrated Biochemical and Cellular Validation of SIP0401 as an Isoform-preferential PDE4B Inhibitor

Phosphodiesterase-4B (PDE4B) regulates intracellular cAMP and drives pro-inflammatory cytokine production. Novel small-molecule PDE4B inhibitors with improved isoform selectivity are needed to broaden therapeutic options. We report the discovery and validation of SIP0401, a ChemBridge-derived small molecule prioritized via an integrated in silico pipeline combining pharmacophore modeling, molecular docking, and dynamics-based scoring.

SIP0401 was identified through virtual screening and ligand efficiency-guided filtering against the PDE4B catalytic domain. Experimental profiling included PDE-Glo™, HTRF cAMP, and differential scanning fluorimetry (DSF) with recombinant human PDE4B. Isoform preference (PDE4A/C/D) was assessed by PDE-Glo™. Aggregation, solubility, and luciferase interference were evaluated. Cellular assays included cAMP (HTRF) and TNF-α ELISA in THP-1 macrophages, and cytotoxicity (MTT) in BEAS-2B cells. Curve fitting used GraphPad Prism.

SIP0401 showed high predicted binding affinity (ΔG = - 8.6 kcal/mol) and favorable interaction stability in dynamics. It inhibited PDE4B with IC₅₀ = 282 nM (PDE-Glo) and 338.4 nM (HTRF); rolipram controls gave 91.5 nM and 106.6 nM. DSF showed Tₘ shift + 4.2 °C; luciferase interference was ≤ 6%. SIP0401 was selective for PDE4B over PDE4A/C/D (IC₅₀s 1.16-1.97 µM; 5.3-7.0×). In THP-1 cells, SIP0401 increased cAMP (EC₅₀ = 2.52 µM) and inhibited TNF-α (IC₅₀ = 3.24 µM). BEAS-2B viability > 90% up to 50 µM.

SIP0401, identified by structure-guided virtual screening, demonstrated moderate biochemical preference for PDE4B over other PDE4 isoforms under the tested conditions. Additionally, the observed cellular activity and favorable colloidal/solubility profiles supports its further optimization and in vivo assessment.