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Differences in methotrexate and 7-hydroxymethotrexate inhibition of folate-dependent enzymes of purine nucleotide biosynthesis.

ABSTRACT: 7-Hydroxymethotrexate (7-OH-MTX) is the major and, frequently the only, pteridine metabolite found in bone-marrow aspirates of patients chronically treated with low-dose oral methotrexate (MTX) [Sonneveld, Schultz, Nooter and Hahlen (1986) Cancer Chemother. Pharmacol. 18, 111-116]. The Ki values for MTX and 7-OH-MTX for avian liver 5-amino-imidazole-4-carboxamide ribonucleotide transformylase differ by 4.5-fold in favour of 7-OH-MTX as the better inhibitor, while Ki values for avian liver glycinamide ribonucleotide transformylase differ by 1.9-fold favouring MTX as the better inhibitor. Thus 7-OH-MTX possesses a different enzyme-inhibiting repertoire from its parent drug and this information may be useful in explaining the mechanism of action of low-dose MTX therapies used to treat autoimmune disease.

SUBMITTER: Baggott JE

PROVIDER: S-EPMC1138213 | biostudies-other | 1994 Jun

REPOSITORIES: biostudies-other

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Project description:We previously showed that classical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates bind to folate receptor (FR) ? and the target purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFTase) with different cis and trans conformations. In this study, we designed novel analogs of this series with an amide moiety in the bridge region that can adopt both the cis and trans lowest energy conformations. This provides entropic benefit, by restricting the number of side-chain conformations of the unbound ligand to those most likely to promote binding to FR? and the target enzyme required for antitumor activity. NMR of the most active compound 7 showed both cis and trans amide bridge conformations in ~1:1 ratio. The bridge amide group in the best docked poses of 7 in the crystal structures of FR? and GARFTase adopted both cis and trans conformations, with the lowest energy conformations predicted by Maestro and evidenced by NMR within 1?kcal/mol. Compound 7 showed ~3-fold increased inhibition of FR?-expressing cells over its non-restricted parent analog 1 and was selectively internalized by FR? over the reduced folate carrier (RFC), resulting in significant in vitro antitumor activity toward FR?-expressing KB human tumor cells. Antitumor activity of 7 was abolished by treating cells with adenosine but was incompletely protected by 5-aminoimidazole-4-carboxamide (AICA) at higher drug concentrations, suggesting GARFTase and AICA ribonucleotide formyltransferase (AICARFTase) in de novo purine biosynthesis as the likely intracellular targets. GARFTase inhibition by compound 7 was confirmed by an in situ cell-based activity assay. Our results identify a "first-in-class" classical antifolate with a novel amide linkage between the scaffold and the side chain aryl L-glutamate that affords exclusive selectivity for transport via FR? over RFC and antitumor activity resulting from inhibition of GARFTase and likely AICARFTase. Compound 7 offers significant advantages over clinically used inhibitors of this class that are transported by the ubiquitous RFC, resulting in dose-limiting toxicities.

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Differences in methotrexate and 7-hydroxymethotrexate inhibition of folate-dependent enzymes of purine nucleotide biosynthesis.

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