Doramapimod (BIRB 796) is a highly potent inhibitor of p38 MAPK (Kd: 0.1 nM), but weakly inhibits c-RAF, Fyn, Lck, ERK-1, SYK, IKK2, and ZAP-70.

p38 MAPK:0.1 nM (Kd, cell free)

Doramapimod (BIRB796) 是一种高效的 p38 MAPK 抑制剂（Kd：0.1 nM），能够阻断 LPS 刺激的 THP-1 细胞中 TNFα 的释放（IC50：18 nM）[1]。BIRB796 同时抑制 SAPK3/p38gamma 的活性及其激活，并阻断应激诱导的脚手架蛋白 SAP97 的磷酸化[2]。此外，BIRB 796 抑制了由 17-AAG 加 bortezomib 引起的 Hsp27 磷酸化，从而增强了细胞毒性。在骨髓基质细胞（BMSC）中，BIRB 796 抑制了由肿瘤坏死因子-α 或肿瘤生长因子-β1 触发的 p38 MAPK 的磷酸化和 IL-6 及血管内皮生长因子的分泌。BIRB 796 还抑制了 BMSCs 因黏附到 MM 细胞而诱导的 IL-6 分泌，从而抑制了肿瘤细胞增殖[3]。

未经治疗的dTGRs的收缩压为204 mm Hg，但经过Doramapimod (30 mg/kg 每日) 处理后有所降低（166 mm Hg），而Sprague-Dawley大鼠保持正常血压。与dTGRs相比，Doramapimod处理后的心脏中β-肌球蛋白重链表达显著降低。Doramapimod的治疗显著减少了心脏纤维化、结缔组织生长因子、肿瘤坏死因子-α、白细胞介素-6和巨噬细胞浸润[4]。

Binding studies are conducted in a buffer containing 20 mM Bis-Tris Propane, pH 7.0, 2 mM EDTA, 0.01% (w/v) NaN3 and 0.15% (w/v) n-octylglucoside. Kinetic data for the association of SK&F 86002 to p38 MAP kinase is collected on a Kintech fluorescence detector system equipped with a stopped-flow controller. The data are fit simultaneously to an appropriate equation describing kinetic binding for a simple one-step binding mechanism. The data for the binding of the fluorescent analog of BIRB 796 is corrected for background fluorescence of unbound ligand. The exchange curve assays are run as two half-reactions using an SLM Aminco Bowman Series 2 Model SQ-340 fluorescence detector. In the first half-reaction, p38 MAP kinase and SK&F 86002 are preincubated for 3 min. In the second half-reaction, p38 MAP kinase is preincubated with Doramapimod for 60 min. A net dissociation of the fluoroprobe is observed for the first half-reaction, and a net association is observed for the second half-reaction. The raw data from both half reactions are fit simultaneously to an equation describing simple competitive inhibition. p38 is preactivated by treatment with constitutively active recombinant MKK6 (prepared by mutagenizing the two activation residues, Ser189 and Thr193, to Glu residues). Activated p38 is purified and used as a source of enzyme in a standard kinase activity assay monitoring the incorporation of radioactive phosphate into recombinant human MAPKAP k2. Cellular assays follow published procedures. Briefly, human THP.1 cells are stimulated with 1 μg/mL LPS, in the presence or absence of compound, followed by the determination of released TNF using a commercial ELISA kit [1].

Human embryonic kidney (HEK) 293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium at 37 °C, supplemented with 10% fetal calf serum, 50 units of penicillin/ml, 50 μg/ml streptomycin, and 2 mM glutamine. Mouse embryonic fibroblasts were cultured as described previously, and C2C12 myoblasts were cultured. Cells were exposed to 0.5 M sorbitol for 30 min or 100 ng/ml EGF for 10 min and then lysed in buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 50 mM sodium β-glycerophosphate, 5 mM pyrophosphate, 0.27 M sucrose, 0.1 mM phenylmethylsulfonyl fluoride, 1% (v/v) Triton X-100) plus 0.1% (v/v) 2-mercaptoethanol and Complete proteinase inhibitor mixture from Roche Applied Science. Lysates were centrifuged at 18,000 × g for 5 min at 4 °C, and the supernatants were removed, quick-frozen in liquid nitrogen, and stored at –20 °C until use. When required, cells were preincubated for 1 h without or with 10 μM SB 203580 or 10 μM PD 184352 or with different concentrations of BIRB796 for the times indicated in the figures [2].

We studied male transgenic dTGRs and age-matched nontransgenic Sprague–Dawley (SD) rats (MDC). Local authorities approved the studies, and American Physiological Society guidelines for animal care were followed. We performed 2 different protocols. In protocol 2, untreated dTGR (n=15), dTGR+BIRB796 (30 mg/kg per day in the diet for 3 weeks; n=11), and SD (n=8 each group) rats were analyzed. Systolic blood pressure was measured weekly by tail cuff. Twenty-four– hour urine samples were collected in metabolic cages from weeks 5 to 7. Serum was collected at week 7. Serum creatinine and cystatin C were measured by clinical routine assays. Urinary rat albumin was determined by enzyme-linked immunosorbent assay. The aim of protocol 2 was to focus on electrophysiological alterations and mortality. Untreated dTGR (n=10), dTGR+BIRB796 (n=10), and SD (n=10) rats were studied up to week 8. Cardiac magnetic field mapping (CMFM) was performed at week 7 under isoflurane anesthesia. Echocardiography was performed as described earlier [4].