Cellular proliferation, driven by cyclin-dependent kinases (CDKs) and their cyclin partners, is deregulated in cancer. Anti-estrogens, such as tamoxifen, antagonise estrogen-induced ERalpha transactivation of cyclin D1, resulting in reduced CDK4/6 activity, p27(Kip1)-mediated inhibition of CDK2 and growth arrest. We hypothesised that direct inhibition of CDK2 and CDK1 may overcome the major clinical problem of anti-estrogen resistance.
Poly(ADP-ribose) polymerase-1 (PARP) inhibitors (PARPi) exploit tumour-specific defects in homologous recombination DNA repair and continuous dosing is most efficacious. Early clinical trial data with rucaparib suggested that it caused sustained PARP inhibition. Here we investigate the mechanism of this durable inhibition and potential exploitation. Uptake and retention of rucaparib and persistence of PARP inhibition were determined by radiochemical and immunological assays in human cancer cell lines. The pharmacokinetics and pharmacodynamics of rucaparib were determined in tumour-bearing mice and the efficacy of different schedules of rucaparib was determined in mice bearing homologous recombination DNA repair-defective tumours. Rucaparib accumulation is carrier mediated (Km=8.4±1.2 μ M, Vmax=469±22 pmol per 106 cells per 10 min), reaching steady-state levels >10 times higher than the extracellular concentration within 30 min. Rucaparib is retained in cells and inhibits PARP ⩾50% for ⩾72 h days after a 30-min pulse of 400 nM. In Capan-1 tumour-bearing mice rucaparib accumulated and was retained in the tumours, and PARP was inhibited for 7 days following a single dose of 10 mg kg−1 i.p or 150 mg kg−1 p.o. by 70% and 90%, respectively. Weekly dosing of 150 mg kg−1 p.o once a week was as effective as 10 mg kg−1 i.p daily for five days every week for 6 weeks in delaying Capan-1 tumour growth. Rucaparib accumulates and is retained in tumour cells and inhibits PARP for long periods such that weekly schedules have equivalent anticancer activity to daily dosing in a pre-clinical model, suggesting that clinical evaluation of alternative schedules of rucaparib should be considered.
Supplementary Fig. S2 from Preclinical evaluation of a novel pyrimidopyrimidine for the prevention of nucleoside and nucleobase reversal of antifolate cytotoxicity
<div>Abstract<p>Antifolates have been used to treat cancer for the last 50 years and remain the mainstay of many therapeutic regimes. Nucleoside salvage, which depends on plasma membrane transport, can compromise the activity of antifolates. The cardiovascular drug dipyridamole inhibits nucleoside transport and enhances antifolate cytotoxicity <i>in vitro</i>, but its clinical activity is compromised by binding to the plasma protein α<sub>1</sub>-acid glycoprotein (AGP). We report the development of a novel pyrimidopyrimidine analogue of dipyridamole, NU3153, which has equivalent potency to dipyridamole, remains active in the presence of physiologic levels of AGP, inhibits thymidine incorporation into DNA, and prevents thymidine and hypoxanthine rescue from the multitargeted antifolate, pemetrexed. Pharmacokinetic evaluation of NU3153 suggested that a soluble prodrug would improve the <i>in vivo</i> activity. The valine prodrug of NU3153, NU3166, rapidly broke down to NU3153 <i>in vitro</i> and <i>in vivo</i>. Plasma NU3153 concentrations commensurate with rescue inhibition <i>in vitro</i> were maintained for at least 16 hours following administration of NU3166 to mice at 120 mg/kg. However, maximum inhibition of thymidine incorporation into tumors was only 50%, which was insufficient to enhance pemetrexed antitumor activity <i>in vivo</i>. Comparison with the cell-based studies revealed that pemetrexed enhancement requires substantial (≥90%) and durable inhibition of nucleoside transport. In conclusion, we have developed non-AGP binding nucleoside transport inhibitors. Pharmacologically active concentrations of the inhibitors can be achieved <i>in vivo</i> using prodrug approaches, but greater potency is required to evaluate inhibition of nucleoside rescue as a therapeutic maneuver. [Mol Cancer Ther 2009;8(7):1828–37]</p></div>
4156 The clinical response to DNA-damaging anticancer therapies may be compromised by cellular DNA repair processes, and agents that impede DNA repair are thus of potential therapeutic interest as chemo- and radio-sensitising agents in the treatment of cancer. The phosphatidylinositol-3-kinase related kinase (PIKK) family member DNA-dependent protein kinase (DNA-PK) is a key player in the non-homologous end-joining pathway of DNA double-strand break (DSB) repair. Identification of the lead benzo[h]chromen-4-one DNA-PK inhibitor NU7026 (IC50 = 0.23 μM), guided the subsequent development of the potent and selective ATP-competitive chromenone NU7441 (DNA-PK IC50 = 13 nM). Although proof-of-principle studies with NU7441 confirmed promising activity in vitro as a chemo- and radio-potentiator in a range of human tumour cell lines, further biological studies were hampered by sub-optimal pharmaceutical properties. Structure-activity relationship studies for DNA-PK inhibition by chromenone-derivatives were conducted in conjunction with homology modelling. This approach predicted the 1-, 8-, and 9-positions on the pendant dibenzothiophen-4-yl substituent of NU7441 as tolerant to substitution without detriment to DNA-PK inhibitory activity. The introduction of suitable functionality (e.g. OH, NH2 CO2H) at these positions provided a platform for the synthesis of focussed libraries of compounds bearing water-solubilising amine substituents. This could be achieved directly at the dibenzothiophene 1-position through nitration and subsequent functional group modification. By contrast, functionalisation of the dibenzothiophene 8- and 9-positions of NU7441 entailed incorporation of the required substituent into a synthetic precursor, prior to construction of the dibenzothiophene ring and coupling to the chromenone scaffold. Interestingly, substitution with a methyl or allyl group at the 3-position of the dibenzothiophen-4-yl ring enabled the separation by chiral hplc of atropisomers, as a consequence of restricted rotation about the dibenzothiophene-chromenone bond, albeit with a marked loss of potency (R = 3-Me, IC50 = 2.5 μM). Library synthesis was undertaken employing a solution multiple-parallel approach, by O-alkylation (dibromoethane or methyl bromoacetate) or N-acylation (chloroacetyl chloride or 2-bromopropionyl chloride) of the appropriately substituted NU7441 derivatives, respectively, followed by reaction with a range of amines to afford the target compounds. These studies resulted in the identification of compounds that combined potent DNA-PK inhibition with excellent aqueous solubility (20-40 mg/mL as acid salts), without compromising cellular activity. Prominent amongst these derivatives is KU-0060648 (DNA-PK IC50 = 8.6 nM), which exhibits 20-1000 fold selectivity for DNA-PK over related PIKK enzymes and PI3K family members.
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