Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.

Design, synthesis and biological evaluation of 6-pyridylmethylaminopurines as CDK inhibitors.

The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSßR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 µM, and CDK9-cyclinT with IC(50)=0.11 µM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 µM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts.

Targeting the alpha-folate receptor with cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase.

The alpha-FR has been reported to be overexpressed in many carcinomas, in particular those of the ovary and uterus. The high expression of alpha-FR in some tumours compared with normal tissues has been exploited over the last decade for folate-mediated targeting of macromolecules, anticancer drugs, imaging agents and nucleic acids to cancer cells. CB300638, a cyclopenta[g]quinazoline-based inhibitor of thymidylate synthase (TS), has been reported to have high affinity for the receptor and selectivity for alpha-FR overexpressing tumour cell lines. In this study, the structural features of the molecule, in particular modifications at the 2-position, have been investigated with respect to TS inhibition, affinity for the alpha-FR and reduced folate carrier (RFC) and activity in A431-FBP cells (transfected with human alpha-FR) compared with neo-transfected A431 cells. Compounds 1a,b, 2a,b and 3a,b were synthesised utilising multistep sequences. It was found that the 2-substituent does not affect the affinity for the alpha-FR; however, it greatly affects selectivity for A431-FBP cells, and suggests that there are factors other than TS inhibition and alpha-FR affinity that are important for the activity of these compounds. Compound 2b (2-CH2OH derivative) displayed the highest selectivity for the A431-FBP cells compared with A431 cells.

An evaluation of the ability of pifithrin-alpha and -beta to inhibit p53 function in two wild-type p53 human tumor cell lines.

The small-molecule compound pifithrin-alpha (PFT-alpha) has been reported to inhibit p53 function and protect against a variety of genotoxic agents. We show here that PFT-alpha is unstable in tissue culture medium and is rapidly converted to its condensation product PFT-beta. Both compounds showed limited solubility with PFT-alpha precipitating out of tissue culture medium at concentrations >30 micromol/L. PFT-alpha and -beta exhibited cytotoxic effects in vitro towards two human wild-type p53-expressing tumor cell lines, A2780 ovarian and HCT116 colon (IC(50) values for both cell lines were 21.3 +/- 8.1 micromol/L for PFT-alpha and 90.3 +/- 15.5 micromol/L for PFT-beta, mean +/- SD, n = 4). There was no evidence of protection by clonogenic assay with either compound in combination with ionizing radiation. Indeed, there was some evidence that PFT-alpha enhanced cytotoxicity, particularly at higher concentrations of PFT-alpha. Neither compound had any effect on p53, p21, or MDM-2 protein expression following ionizing radiation exposure and there was no evidence of any abrogation of p53-dependent, ionizing radiation-induced cell cycle arrest. Similarly, there was no evidence of cellular protection, or of effects on p53-dependent gene transcription, or on translation of MDM-2 or p21 following UV treatment of these human tumor cell lines. In addition, there was no effect on p53 or p21 gene transactivation or p38 phosphorylation after UV irradiation of NIH-3T3 mouse fibroblasts. In conclusion, neither PFT-alpha nor -beta can be regarded as a ubiquitous inhibitor of p53 function, and caution should be exercised in the use of these agents as specific p53 inhibitors.

Structural and mechanistic studies on anthocyanidin synthase catalysed oxidation of flavanone substrates: the effect of C-2 stereochemistry on product selectivity and mechanism.

During the biosynthesis of the tricyclic flavonoid natural products in plants, oxidative modifications to the central C-ring are catalysed by Fe(ii) and 2-oxoglutarate dependent oxygenases. The reactions catalysed by three of these enzymes; flavone synthase I, flavonol synthase and anthocyanidin synthase (ANS), are formally desaturations. In comparison, flavanone 3beta-hydroxylase catalyses hydroxylation at the C-3 pro-R position of 2S-naringenin. Incubation of ANS with the unnatural substrate (+/-)-naringenin results in predominantly C-3 hydroxylation to give cis-dihydrokaempferol as the major product; trans-dihydrokaempferol and the desaturation product, apigenin are also observed. Labelling studies have demonstrated that some of the formal desaturation reactions catalysed by ANS proceed via initial C-3 hydroxylation followed by dehydration at the active site. We describe analyses of the reaction of ANS with 2S- and 2R-naringenin substrates, including the anaerobic crystal structure of an ANS-Fe-2-oxoglutarate-naringenin complex. Together the results reveal that for the 'natural' C-2 stereochemistry of 2S-naringenin, C-3 hydroxylation predominates (>9 : 1) over desaturation, probably due to the inaccessibility of the C-2 hydrogen to the iron centre. For the 2R-naringenin substrate, desaturation is significantly increased relative to C-3 hydroxylation (ca. 1 : 1); this is probably a result of both the C-3 pro-S and C-2 hydrogen atoms being accessible to the reactive oxidising intermediate in this substrate. In contrast to the hydroxylation-elimination desaturation mechanism for some ANS substrates, the results imply that the ANS catalysed desaturation of 2R-naringenin to form apigenin proceeds with a syn-arrangement of eliminated hydrogen atoms and not via an oxygenated (gem-diol) flavonoid intermediate. Thus, by utilising flavonoid substrates with different C-2 stereochemistries, the balance between C-3 hydroxylation or C-2, C-3 desaturation mechanisms can be altered.

Metabolism and pharmacokinetics of the cyclin-dependent kinase inhibitor R-roscovitine in the mouse.

R-roscovitine (seliciclib, CYC202) is a cyclin-dependent kinase inhibitor currently in phase II clinical trials in patients with cancer. Here, we describe its mouse metabolism and pharmacokinetics as well as the identification of the principal metabolites in hepatic microsomes, plasma, and urine. Following microsomal incubation of R-roscovitine at 10 microg/mL (28 micromol/L) for 60 minutes, 86.7% of the parent drug was metabolized and 60% of this loss was due to formation of one particular metabolite. This was identified as the carboxylic acid resulting from oxidation of the hydroxymethyl group of the amino alcohol substituent at C2 of the purine core present in R-roscovitine. Identification was confirmed by chemical synthesis and comparison of an authentic sample of the R-roscovitine-derived carboxylate metabolite (COOH-R-roscovitine). Other minor metabolites were identified as C8-oxo-R-roscovitine and N9-desisopropyl-R-roscovitine; these accounted for 4.9% and 2.6% of the parent, respectively. The same metabolic pattern was observed in vivo, with a 4.5-fold lower AUC(infinity) for R-roscovitine (38 micromol/L/h) than for COOH-R-roscovitine (174 micromol/L/h). Excretion of R-roscovitine in the urine up to 24 hours post-dosing accounted for an average of only 0.02% of the administered dose of 50 mg/kg, whereas COOH-R-roscovitine represented 65% to 68% of the dose irrespective of the route of administration (i.v., i.p., or p.o.). A partially deuterated derivative (R-roscovitine-d9) was synthesized to investigate if formation of COOH-R-roscovitine could be inhibited by replacement of metabolically labile protons with deuterium. After 60 minutes of incubation of R-roscovitine-d9 or R-roscovitine with mouse liver microsomes, formation of COOH-R-roscovitine-d9 was decreased by approximately 24% compared with the production of COOH-R-roscovitine. In addition, the levels of R-roscovitine-d9 remaining were 33% higher than those of R-roscovitine. However, formation of several minor R-roscovitine metabolites was enhanced with R-roscovitine-d9, suggesting that metabolic switching from the major carbinol oxidation pathway had occurred. Synthetic COOH-R-roscovitine and C8-oxo-R-roscovitine were tested in functional cyclin-dependent kinase assays and shown to be less active than R-roscovitine, confirming that these metabolic reactions are deactivation pathways.