title: Quinazoline thymidylate synthase inhibitors: methods for assessing the contribution of polyglutamation to their in vitro activity.
abstract: Many quinazoline thymidylate synthase (TS) inhibitors undergo intracellular metabolism to polyglutamate forms which can significantly alter their activity and pharmacodynamics through improved TS inhibition and drug retention. When a series of quinazolines was tested for inhibitory activity towards TS (IC50 0.001-2 microM) and the growth of L1210 cells (IC50 0.005-10 microM), no direct correlation was observed. However, a very good correlation was apparent if a L1210 variant cell line (L1210: RD1694) was used. This line is deficient in its ability to form antifolate polyglutamates. A number of other intact cell methods have also been developed which estimate the contribution that intracellular polyglutamation makes to a compound's activity. These assays were validated using a series of quinazoline-based TS inhibitors with well-defined activity for TS, folypolyglutamate synthetase (FPGS) and the reduced-folate cell membrane carrier (RFC). Short-exposure growth-inhibition assays or the measurement of TS activity in situ after various incubation times, followed by different lengths of time in drug-free medium, can indicate both the speed and extent of appearance of retentive forms (usually polyglutamates). Continuous-exposure growth-inhibition assays, in the presence of leucovorin (LV), are also useful, since only the growth-inhibitory potency of polyglutamated analogues is significantly decreased by LV. Highly polyglutamated compounds, e.g. ZD1694, are virtually inactive in the presence of a high concentration of LV. It is proposed that these methods, when considered together, provide a greater degree of information concerning the rate and extent of polyglutamation of a particular compound than isolated FPGS assays alone.
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology; D002955:Leucovorin / Q000037:antagonists & inhibitors; D007939:Leukemia L1210 / Q000201:enzymology; D008727:Methotrexate / Q000378:metabolism; D010453:Peptide Synthases / Q000037:antagonists & inhibitors; D011099:Polyglutamic Acid / Q000378:metabolism; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D011799:Quinazolines / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*
chemical_list: D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D011624:Pteroylpolyglutamic Acids; D011799:Quinazolines; D011099:Polyglutamic Acid; C031662:CB 3717; D005492:Folic Acid; D013940:Thymidylate Synthase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D002955:Leucovorin; D008727:Methotrexate