Summary /

ML383 (patent compound 1) inhibited NRF2 activity in A549 ARE-luciferase assay in a dose response manner (gold circles) but did not affect A549 cell viability (green triangles). Moreover, this probe did not show any broad transcriptional repressor activity as it did not inhibit the HEK293 CMV-Luciferase (blue squares) and the GR-agonist (black squares) counterscreens (Figure 1).

Figure 1. Dose Response Curve for ML383. Result showed selective inhibition for the NRF2 ARE-Luc.

Summary /

ML383 (patent compound 1) showed a significant reduction of the Nrf2 mRNA expression in the Real time RT-PCR assay after a 10 uM probe treatment for 72 hours. In addition, it also reduced the mRNA expression of downstream NRF2 target genes GCLm and NQO1 (Figure 2B).  Clonogenic assay also showed inhibition of colony formation upon ML383 alone and in combination with etoposide, cisplatin, and carboplatin (Figure 2A).

Figure 2. Cellular Activity for ML383. A) Clonogenic assay in A549 cells for ML383 alone at 10 uM, and in combination with etoposide, cisplatin and carboplatin. B) RT-PCR analysis for A549 cells treated with 10 uM ML383. β-actin was used for normalization.

Summary /

ADME assessment of ML383 (patent compound 1) showed moderate profile with microsomal stability >30 minutes. Kinetic aqueous solubility was poor but due to robust on-target biological activity of the compounds, they were progressed to in vivo PK experiments. The in vitro microsomal stability of all three probes tracked well into their in vivo PK experiments with ML383 having a t_1/2 = 9.7 hours when dosed IP at 30 mg/kg (Figure 3).

Figure 3. In vivo PK profile of ML383.

Summary /

ML383 (patent compound 1) showed inhibition in tumor size after 4 weeks of treatment alone. The the decrease in tumor size was more significant with the combination treatment of carboplatin and ML383 (Figure 4). In brief, A549 cells (5 × 10⁶) were injected subcutaneously into the flank of athymic nude mice and tumor dimensions were measured by caliper at an interval of 3-5 days. The tumor volumes were calculated using the following formula: [length (mm) × width (mm) × width (mm) × 0.52]. Once the tumor volumes were approximately 50-100 mm³, mice were randomly allocated into 4 groups: vehicle, small molecule (ML383, ML384, or ML385), carboplatin, small molecule+carboplatin. Vehicle, carboplatin (5 mg/kg daily Monday to Friday), small molecule (30 mg/kg daily Monday to Friday), and a combination of small molecule and carboplatin were administered intraperitoneally for three weeks. At the end of treatment period, mice were sacrificed and tumor, blood, lung, and liver samples were collected. Tumor volume was measured and plotted against sacrificed days (Figure 4).

Figure 4. In vivo xenograft experiment with a treatment schedule of vehicle (blue), ML383 dosed at 30 mg/kg, 5d/wk for 4 wks (red), carboplatin dosed at 5 mg/kg 5d/wk (green) for 4 weeks, and carboplatin and ML383 co-dosed at 5 mg/kg and 30mg/kg respectively, 5d/wk for 4 weeks (purple).