Summary /

ML384NCATS (patent compound 3) 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. ML384 inhibits NRF2 activity determined through ARE driven Luciferase response (gold circles) in A549 cell line, but does not affect cell viability in that same line (green triangles), and is not a broad transcriptional repressor as judged by activity in a HEK293-CMV-luciferase cell line (blue squares) nor a GR-agonist (black diamonds).

Summary /

ML384 (patent compound 3) showed a small reduction of the Nrf2 mRNA expression in the Real time RT-PCR assay after a 10 uM probe treatment for 72 hours. It also reduced the mRNA expression of downstream NRF2 target genes NQO1 but not GCLm.  Clonogenic assay showed inhibition of colony formation upon ML383 alone (at 5uM) and in combination with etoposide, carboplatin, and specially with cisplatin (Figure 2).

Figure 2. Clonogenic assay in A549 cells for ML384 at 2.5 uM and 5.0 uM, and in combination with etoposide, cisplatin and carboplatin at varying concentrations.

Summary /

ADME assessment of ML384 (patent compound 3) showed moderate profile with microsomal stability. Kinetic aqueous solubility was poor but due to robust on-target biological activity of the compounds, they were progressed to in vivo PK experiments. ML384 in vivo PK experiments showed a t_1/2 = 2.5 hours when dosed IP at 30 mg/kg (Figure 3).

Figure 3. In vivo PK profile of ML384.

Summary /

ML384 (patent compound 3) 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), Carboplatin dosed at 5 mg/kg 5d/wk for 2 wks then stopped (red), ML384 dosed at 60 mg/kg for 5d/wk for 2 wks, then stopped (green), and Carboplatin and ML384 co-dosed at 5 mg/kg and 60 mg/kg respectively, 5d/wk for 2 wks then stopped (purple).