In vitro activity: NU2058 is an O(6)-Cyclohexylmethylguanine-based CDK2 (cyclin-dependent kinases 2) inhibitor with IC50 of 17 μM in an enzymatic assay. It potentiates cisplatin cytotoxicity in vitro. it can also potentiate melphalan (DMF 2.3), and monohydroxymelphalan (1.7), but not temozolomide or ionising radiation. NU2058 increased cisplatin cytotoxicity, by clonogenic assay, with a dose modification factor (DMF) of 3.1. NU2058 increased total intracellular platinum levels 1.5-fold, and platinum-DNA adduct levels twofold. Furthermore, the cisplatin-DNA adducts formed were more toxic in the presence of NU2058.

Kinase Assay: NU2058 is an O(6)-Cyclohexylmethylguanine-based CDK2 (cyclin-dependent kinases 2) inhibitor with IC50 of 17 μM in an enzymatic assay. Exponentially growing parental and derivative LNCaP cells (8–10 × 105) were exposed to Casodex or NU2058 for 1 h. Cells were washed in PBS, harvested and lysed in reaction lysis buffer (50 mM Tris, pH 7.5, 150 mMNaCl, 0.2 mM Na3VO4, 0.5% NP40, 1 mM PMSF, 1 mM dithiothreitol, 25 μg/ml leupeptin, 25 μg/ml aprotinin and 25 μg/ml pepstatin) on ice for 30 min. Lysates were centrifuged at 14 000 g for 3 min. Supernatants were precleared with 20 μl protein G sepharose (PGS) after three washes in reaction lysis buffer and rotated at 4°C for 4 h. PGS and nonspecific-bound protein were removed by centrifugation at 14 000 gfor 5 min. Immunoprecipitation was performed with 2 μg of polyclonal anti-CDK2 antibody (Santa Cruz Biotechnology) and samples were incubated overnight at 4°C with rotation. After incubation, a further 20 μl of PGS were added to immunoprecipitated samples and returned to 4°C for 1 h with rotation. PGS with bound protein complexes was recovered by centrifugation at 14 000 g for 5 min and beads were washed once in wash buffer (PBS, 0.2% Triton X-100) and twice in PBS. Phosphorylation of histone H1 was measured by incubating the beads for 10 min at 30°C with γ[32P]ATP, 1 mM ATP, CDK buffer (50 mM Tris, pH 7.5, 5 mM MgCl2) and histone H1 (5 mg/ml) as substrate. The reaction was stopped by the addition of 50 μl Laemmli buffer. Samples were analysed by 12% SDS–PAGE and the gel was dried and subjected to autoradiography.

Cell Assay: Cells (350,000 cells/well) are seeded into six-well plates and allowed to attach overnight. On the day of the experiment, growth media are replaced with media containing 100 μM of either NU2058, NU6102, NU6230 or DMSO (0.1% (v/v) final concentration) for 2 h followed by a further 2 h in the additional presence of cytotoxic drugs, unless otherwise indicated. For the radiation experiments, cells are treated for a total of 4 h with NU2058, and irradiated after the first 2 h. After treatment, the cells are washed twice with PBS, trypsinised, and replated into 100 mm Petri dishes at various cell densities (300-50,000 cells per plate). After approximately 12 days, media is removed, and cells are fixed with Carnoy's reagent (75% (v/v) methanol, 25% (v/v) acetic acid), stained with crystal violet (0.4% (w/v) in water) and colonies are counted.