Inhibition of folylpolyglutamate synthetase by substrate analogues with an ornithine side chain

Article abstract of DOI:10.1002/jhet.5570330457

N(α)-[4-[[(4-Aminopteridin-6-yl)methyl]amino]benzoyl]-L-ornithine (dAPA-Orn) was synthesized, and its ability to inhibit folylpolyglutamate synthetase from mouse liver was compared with that of the corresponding 2,4-diamino analogue APA-Orn. Also compared were the inhibitory activities of the deaza analogues 5-deazaAPA-Orn, 8-deazaAPA-Orn, and 5,8-dideazaAPA-Orn, as well as those of N(α)-pteroyl-L-ornithine (PteOrn) and its deaza analogues 5-deazaPteOrn and 5,8-dideazaPteOrn. The inhibition constant K(i) of dAPA-Orn was 7-fold greater than that of APA-Orn, indicating that the 2-amino group plays a role in binding to the active site. The binding affinity of the 2,4-diamino compounds increased in the order 5-deazaAPA < APA-Orn < 5,8-dideazaAPA-Orn < 8-deazaAPA-Orn, and that of the 2-amino-4(3H)-oxo compounds increased in the order 5-deazaPteOrn < Pte-Orn < 5,8-dideazaPteOrn. The most potent inhibitor of both groups was 8-deazaAPA-Orn, with a K(i) of 0.018 μM, corresponding to an 8-fold and 15-fold increase in affinity relative to APA-Orn and 5-deazaAPA-Orn, respectively. The results suggest (a) that the binding of Orn-containing folylpolyglutamate synthetase inhibitors is affected to a greater degree by replacement of N⁸ by a carbon atom than it is by the corresponding change at N⁵, (b) that the effect of carbon for nitrogen replacement is greater in the 2,4-diamino derivatives than in the 2-amino-4(3H)-oxo compounds, and (c) that the 2,4-diamines are the better inhibitors. Comparison of the K(i) values of the Orn-containing inhibitors with the K(m) values of the corresponding glutamate-containing substrates revealed that K(m)/K(i) ratio can vary as much as 100-fold depending on the nature of the heterocyclic moiety, suggesting that caution should be exercised in using K(m) values of known substrates to predict K(i) values of putative inhibitors.