title: LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes.
abstract: N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]-benzoyl]-L-glutamic acid (LY231514) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate currently undergoing extensive Phase II clinical trials. Previous studies have established that LY231514 and its synthetic gamma-polyglutamates (glu3 and glu5) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit other key folate-requiring enzymes, including dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). For example, the Ki values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In contrast, although a similar high level of inhibitory potency was observed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (Ki) for the parent monoglutamate are significantly weaker for TS (109 nM) and GARFT (9,300 nM). The effects of LY231514 and its polyglutamates on aminoimidazole carboxamide ribonucleotide formyltransferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formyltetrahydrofolate synthetase were also evaluated. The end product reversal studies conducted in human cell lines further support the concept that multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a major site of action for LY231514 at concentrations near the IC50, higher concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demonstrated that LY231514 requires polyglutamation and transport via the reduced folate carrier for cytotoxic potency. Therefore, our data suggest that LY231514 is a novel classical antifolate, the antitumor activity of which may result from simultaneous and multiple inhibition of several key folate-requiring enzymes via its polyglutamated metabolites.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
authors.forename: C
authors.initials: C
authors.lastname: Shih
authors.forename: V J
authors.initials: VJ
authors.lastname: Chen
authors.forename: L S
authors.initials: LS
authors.lastname: Gossett
authors.forename: S B
authors.initials: SB
authors.lastname: Gates
authors.forename: W C
authors.initials: WC
authors.lastname: MacKellar
authors.forename: L L
authors.initials: LL
authors.lastname: Habeck
authors.forename: K A
authors.initials: KA
authors.lastname: Shackelford
authors.forename: L G
authors.initials: LG
authors.lastname: Mendelsohn
authors.forename: D J
authors.initials: DJ
authors.lastname: Soose
authors.forename: V F
authors.initials: VF
authors.lastname: Patel
authors.forename: S L
authors.initials: SL
authors.lastname: Andis
authors.forename: J R
authors.initials: JR
authors.lastname: Bewley
authors.forename: E A
authors.initials: EA
authors.lastname: Rayl
authors.forename: B A
authors.initials: BA
authors.lastname: Moroson
authors.forename: G P
authors.initials: GP
authors.lastname: Beardsley
authors.forename: W
authors.initials: W
authors.lastname: Kohler
authors.forename: M
authors.initials: M
authors.lastname: Ratnam
authors.forename: R M
authors.initials: RM
authors.lastname: Schultz
mesh_terms: D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D000217:Acyltransferases / Q000037:antagonists & inhibitors; D000619:Aminohydrolases / Q000037:antagonists & inhibitors; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D005574:Formate-Tetrahydrofolate Ligase / Q000037:antagonists & inhibitors; D005971:Glutamates / Q000737:chemistry / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D008727:Methotrexate / Q000494:pharmacology; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000037:antagonists & inhibitors; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D010088:Oxidoreductases / Q000037:antagonists & inhibitors; D000068437:Pemetrexed; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D051026:Phosphoribosylglycinamide Formyltransferase; D011099:Polyglutamic Acid / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects*; D013763:Tetrahydrofolates / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000964:Antimetabolites, Antineoplastic; D005493:Folic Acid Antagonists; D005971:Glutamates; D009097:Multienzyme Complexes; D011799:Quinazolines; D013763:Tetrahydrofolates; D013876:Thiophenes; D000068437:Pemetrexed; D011099:Polyglutamic Acid; D006147:Guanine; C045894:lometrexol; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D010088:Oxidoreductases; D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D042965:Methylenetetrahydrofolate Reductase (NADPH2); D013762:Tetrahydrofolate Dehydrogenase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D013940:Thymidylate Synthase; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000217:Acyltransferases; D000619:Aminohydrolases; D005574:Formate-Tetrahydrofolate Ligase; C068874:raltitrexed; D008727:Methotrexate