LLK203 is a potent dual-target inhibitor of USP2/USP8 with IC50 values of 0.89 μM and 0.52 μM, respectively. It promotes ERα degradation and induces apoptosis in breast cancer MCF-7 cells, demonstrating antitumor activity in the 4T1 tumor mice model [1].

USP2:0.89 μM, USP8:0.52 μM

LLK203 exhibits high inhibitory activity on MCF-7 cells (IC50 = 3.4 μM) within the 0-100 μM range over 36 hours, with greater specificity compared to ML364 (IC50 = 9.3 μM). It enhances activity towards USP2 by fourfold and USP8 by ninefold relative to ML364 [1]. At concentrations of 10-50 μM for 24 hours, LLK203 increases the proportion of apoptotic MCF-7 cells, maintaining most cells in the G1 phase [1]. LLK203, at 2-50 μM over 24 hours, facilitates dose-dependent degradation of proteins such as MDM2, Cyclin D1, Her2, and ERα [1]. At a concentration of 10 μM sustained over 7 days, it exhibits strong inhibition of colony formation [1]. In cell cytotoxicity assays, LLK203 demonstrates lower cytotoxicity towards MCF10A cells (IC50 = 20.4 μM) while showing higher inhibitory activity against BC cells (MCF-7; IC50 = 3.4 μM), increases the ratio of apoptotic cells, largely maintains MCF-7 cells in the G1 phase, and degrades various proteins in a dose-dependent manner through Western blot analysis [1].

LLK203 administered intraperitoneally at a dose of 20 mg/kg daily for 23 consecutive days, significantly inhibited tumor growth in a 4T1 cell-bearing BALB/c mouse model [1]. Additionally, the pharmacokinetic parameters of LLK203 were assessed in male Sprague-Dawley rats, revealing the following: when administered intravenously (5 mg/kg), the Tmax (h) was 6, Cmax (ng/mL) was 36630, AUC0-t (h*ng/mL) was 60824, T1/2 (h) was 20, and CL (mL/h/kg) was 58, with an F (%) of 2.2%. When administered orally (50 mg/kg), the values were Tmax (h) 6, Cmax (ng/mL) 1572, AUC0-t (h*ng/mL) 19144, and T1/2 (h) 6.14 [1].