Pemetrexed (LY-231514 Disodium Hydrate), a guanine-derived antineoplastic agent, binds to and inhibits the enzyme thymidylate synthase (TS).

GARFT:65 nM(ki), DHFR:7.2 nM(ki), TS:1.3 nM(ki)

Pemetrexed (LY231514) disodium is a novel classical antifolate whose antitumor activity likely results from the simultaneous inhibition of several key folate-requiring enzymes via its polyglutamated metabolites. It is one of the best substrates for the enzyme FPGS (Km=1.6 μM, Vmax/Km=621). Polyglutamation and the polyglutamated metabolites of LY231514 play significant roles in determining its selectivity and antitumor activity. LY231514 only moderately inhibits TS (Ki=340 nM, recombinant mouse), but its pentaglutamate form is 100-fold more potent (Ki=3.4 nM), making it one of the most potent folate-based TS inhibitors[1].

The group of mice treated with PC61 combined with Pemetrexed exhibited significantly longer survival compared to those treated with PC61 alone, rat IgG combined with Pemetrexed, or left untreated. Survival analysis further confirmed that the PC61 and Pemetrexed combination resulted in significantly improved survival rates [2].

AICARFT inhibition assays are carried out at room temperature by monitoring the formation of [6S]-5,6,7,8-tetrahydrofolate from 10-formyl-[6R,S]-5,6,7,8-tetrahydrofolate at A298. All solutions are purged with N2 gas prior to use. The reaction solution contains 33 mM Tris-Cl, pH 7.4, 25 mM KCl, 5 mM 2-Mercaptoethanol, 0.05 mM AICA ribonucleotide, and 16 nM (2 milliunits/mL) of AICARFT. 10-Formyl-[6R,S]-5,6,7,8-tetrahydrofolate concentrations of 0.037, 0.074, and 0.145 mM are used (0.61, 1.23, and 2.45 times its Km value, respectively). LY231514 is tested as an inhibitor at 0.08-0.8 mM (four concentrations). When the tri- and pentaglutamates of LY231514 are used as inhibitors, the concentrations are 0.0005-0.009 mM (eight concentrations). Enzyme assays are initiated by the addition of enzyme. Data is analyzed using the ENZFITTER program for competitive inhibition.

Pemetrexed is dissolved in DMSO and stored, and then diluted with cell culture medium before use[1]. Dose-response curves are generated to determine the concentration required for 50% inhibition of growth (IC50). Pemetrexed is dissolved initially in DMSO at a concentration of 4 mg/mL and further diluted with cell culture medium to the desired concentration. CCRF-CEM leukemia cells in complete medium are added to 24-well Cluster plates at a final concentration of 4.8×l04 cells/well in a total volume of 2 mL. Test compounds at various concentrations are added to duplicate wells so that the final volume of DMSO is 0.5%. The plates are incubated for 72 h at 37°C in an atmosphere of 5% CO2 in air. At the end of the incubation, cell numbers are determined on a ZBI Coulter counter. Control wells usually contain 4×105 to 6×105 cells at the end of the incubation. For several studies, IC50s are determined for each compound in the presence of either 300 μM AICA, 5 μM thymidine, 100 μM hypoxanthine, or combination of 5 μM hymidine plus 100 μM hypoxanthine[1].