RP-3500: A Novel, Potent, and Selective ATR Inhibitor that is Effective in Preclinical Models as a Monotherapy and in Combination with PARP Inhibitors

Abstract

Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC₅₀ values of 1.0 and 0.33 nmol/L in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mammalian target of rapamycin (mTOR) and more than 2,000-fold selectivity over ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and phosphatidylinositol 3-kinase alpha (PI3Kα) kinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5 to 7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor inhibition of phosphorylated checkpoint kinase 1 (pCHK1) (IC₈₀ = 18.6 nmol/L) and induction of phosphorylated H2A.X variant histone (γH2AX), phosphorylated DNA-PK catalytic subunit (pDNA-PKcs), and phosphorylated KRAB-associated protein 1 (pKAP1). RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC₈₀ for 10 to 12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.

Graphical abstract

Figure 1.

RP-3500 is a potent ATR kinase inhibitor. A, Chemical structure of RP-3500. B, Cell growth inhibition in a panel of cell lines with defective ATM or BRCA1/2 pathways measured in a 5-day CellTiter-Glo assay. IC₅₀ values were calculated using a dose-response curve normalized to DMSO-treated cells (Prism v7.02). Mean ± SD; n ≥ 3 independent experiments. C, ATR kinase targets or DNA damage markers detected by immunoblotting lysates from ATM pathway-defective human cancer cell lines treated with 1 μmol/L RP-3500 for the indicated times. h, hours.

Figure 2.

RP-3500 is efficacious as a single agent in ATM-deficient tumor models. A, LoVo tumor xenograft volume in mice treated with RP-3500 orally once daily × 17 days. Results are expressed as mean ± SEM, N = 10/group. ^aOne mouse sacrificed due to body weight loss on day 12. B, Tumor volume of a human gastric cancer PDX A with a biallelic loss of ATM in mice treated with RP-3500 at 5 and 10 mg/kg once daily x 28 days. Results expressed as mean ± SEM, N = 10/group. Statistical significance relative to vehicle control for A and B established by one-way ANOVA with Fisher LSD test; **P < 0.01; ****P < 0.0001. C, Proportion of pCHK1 (Ser345) inhibition relative to circulating free RP-3500 plasma levels. Tumors and plasma sampled at 1 and 3 hours after RP-3500 administration. The pCHK1/total CHK1 signal is represented relative to vehicle-treated tumors. D, Free circulating plasma concentrations of RP-3500 over time in SCID mice. Green, measured concentrations; red, simulated PK at 5 mg/kg. Horizontal line indicates in vivo LoVo tumor pCHK1 IC₈₀ determined by nonlinear curve fit of C. Vertical grey bar indicates window of duration over IC₈₀ at the MED (5–7 mg/kg). h, hours. E, pKAP1 (Ser824) in LoVo tumors from mice treated with RP-3500 once daily x 7 days and harvested 24 hours after the last dose. pKAP1/total KAP1 quantified by immunoblot are represented as fold increase over vehicle control signal. F, Quantitative γH2AX and pKAP1 staining of tissue sections from the gastric PDX-bearing mice treated with RP-3500 once daily for 3 days harvested 24 hours after the last dose. PD, pharmacodynamics.

Figure 3.

Intermittent dose scheduling for RP-3500 improves efficacy while minimizing toxicity. A and B,In vitro treatment of RPE1-hTERT Cas9 TP53-KO ATM WT and ATM KO cells with RP-3500 (30 nmol/L; in green) followed by washout periods and stained with nuclear γH2AX by immunofluorescence (IF) after 120 hours. Mean ± SD; N = 3 independent experiments. C, Tumor xenograft volume. D, Red blood cell; and E, Reticulocyte counts from mice bearing Granta-519 tumors treated orally with RP-3500 at MTD on different weekly dosing schedules. RP-3500 was administered once daily according to the indicated schedule. Blood parameters were measured on day 14. Mean ± SEM, N = 9/group. Dashed lines indicate reference RBC range from female, age- and strain-matched mice (Charles River). Statistical significance relative to vehicle control was established by one-way ANOVA with Fisher LSD test (GraphPad Prism v9). *P < 0.05; **P < 0.01; ****P < 0.0001, NS, not significant; h, hours.

Figure 4.

Concomitant treatment with RP-3500 and olaparib is more synergistic and effective than sequential treatment in BRCA1 mutated SUM149PT cells. A,In vitro RP-3500 and olaparib combination treatment schedule in SUM149PT cells with endpoint viability measured at day 8 by CellTiter-Glo. B, Maximum ZIP synergy scores for each schedule (SynergyFinder; ref. 32). C, The synergy matrix ZIP score for concomitant and sequential compound treatment. Score ≥10, (red) synergy; ≤–10 (green) antagonism; dashed lines, concentrations of each agent generating maximum synergy. Max., maximum. D, Viability of cells treated with either RP-3500, olaparib, or the combination at the indicated concentrations and schedules as in A. Values represent the percent viability relative to untreated controls. Circles are values from three independent biological replicates; bars indicate mean ± SD. P values calculated with a two-tailed unpaired Student t test; **P < 0.01.

Figure 5.

Low-dose PARPi administered concomitantly on an intermittent schedule with RP-3500 is more effective without additional toxicity in vivo. A, Combinations of RP-3500 with olaparib or niraparib were administered on the schedule shown with concomitant or sequential (PARPi first) weekly schedule. Tumor xenograft volume from SUM149PT (B) or Granta-519 (C) tumor-bearing mice. Mean ± SEM; N = 9 mice/group. Mice were treated orally with RP-3500 once daily for 3 days and B, Olaparib twice daily for 3 days; or C, Niraparib once daily for 3 days or 5 days at the doses indicated. D and E, Blood parameters measured on day 21 from Granta-519 tumor-bearing mice; mean ± SEM; N = 9 mice/group. Dashed lines indicate reference RBC range from female age- and strain-matched mice (Charles River). Statistical significance relative to vehicle control was established by one-way ANOVA with Fisher LSD test (GraphPad Prism v9). NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.