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

Abstract

AbstractNα‐[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 Nα‐pteroyl‐L‐ornithine (PteOrn) and its deaza analogues 5‐deazaPteOrn and 5,8‐dideazaPteOrn. The inhibition constant Ki 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 < PteOrn < 5,8‐dideazaPteOrn. The most potent inhibitor of both groups was 8‐deazaAPA‐Orn, with a Ki of 0.018 μM, coresponding 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 Ki values of the Orn‐containing inhibitors with the Km values of the corresponding glutamate‐containing substrates revealed that Km/Ki ratio can vary as much as 100‐fold depending on the nature of the heterocyclic moiety, suggesting that caution should be exercised in using Km values of known substrates to predict Ki values of putative inhibitors.