ART5537 is a selective EXO1 inhibitor with an EC50 of 55 nM, an IC50 of 3.4 nM, and a Kd of 6.8 nM. It inhibits the homologous recombination (HR) process in HAP1 parental cells with an EC50 of 7.2 nM. The selectivity of ART5537 for the broad-spectrum nuclease superfamily members is over 200-fold higher. Its biological effects are entirely driven by EXO1 inhibition. ART5537 enhances sensitivity to ionizing radiation and exhibits synergism with Olaparib. It is applicable in research on breast and colon cancers.

ART5537 demonstrates inhibition of homologous recombination in HAP1 parental cells at concentrations ranging from 0.001 to 10 μM, with an EC50 of 7.2 nM. It exhibits dose-dependent target binding with an EC50 of 55 nM. In HAP1 EXO1 knockout cells, ART5537 (0.001-10 μM) is inactive, failing to exceed the approximately 50% inhibition seen with EXO1 knockout alone, and induces dose-dependent retention of GFP2-EXO1 on chromatin in U-2 OS Flp-In cells. ART5537, over 7 days, shows limited broad-spectrum cytotoxicity and mild EXO1-dependent sensitivity in cell lines such as A549 and the BRCA1 knockout MDA-MB-436 (EC50 = 4.0 nM), HCC1937 (EC50 = 5.3 nM), SUM149PT (EC50 = 5.0 nM), and HCC38 (EC50 = 8.3 nM). During a 5-day treatment, ART5537 in combination with Olaparib does not exhibit general cytotoxicity in HCT116 cells. When combined with Olaparib over 7 days, ART5537 shows strong synergy in irradiated wild-type HCT116 cells but not in irradiated EXO1 knockout clones. ART5537 (0.01-100 μM, 6-day treatment) significantly enhances cancer cell eradication and efficacy in irradiated HCT116 cells in combination with Olaparib through potent EXO1 inhibition. In HCT116 cells, ART5537 inhibits EXO1 and induces a sensitization effect exceeding 40-fold, possesses favorable physicochemical properties for use as a cellular probe (including high thermodynamic solubility and good passive permeability), but is unstable in human and mouse liver microsomes. Cell Proliferation AssayCell Line: HCT116 cells Concentration: 0.01, 0.1, 10, and 100 μM Incubation Time: 6 days Resulted in a >40-fold sensitization, potently sensitizing cells irradiated with 3 × 1 Gy (EC50 = 144 nM).

ART5537 demonstrates unfavorable pharmacokinetic properties in mice, specifically exhibiting high systemic clearance and very low oral bioavailability (less than 5%).