2,2'-DITHIOBIS(N-ETHYL-SPERMINE-5-CARBOXAMIDE) IS A HIGH-AFFINITY, MEMBRANE-IMPERMEANT ANTAGONIST OF THE MAMMALIAN POLYAMINE TRANSPORT-SYSTEM

We have synthesized 2,2'-dithiobis(N-ethyl-spermine-5-carboxamide) (DE SC), its thiol monomer (MESC), and the mixed MESC-cysteamine disulfide (DEASC) as potential inhibitors of polyamine transport in mammalian c ells, DESC was the most potent antagonist; of spermine transport in ZR -75-1 human breast cancer cells, with K-i values of 5.0 +/- 0.7, 80 +/ - 31, and 16 +/- 3 mu m for DESC, MESC, and DEASC, respectively. DESC also strongly blocked putrescine and spermidine uptake in ZR-75-1 cell s (K-i = 1.6 +/- 0.5 and 2.7 +/- 1.1 mu M, respectively). While DESC a nd MESC were purely competitive inhibitors of putrescine transport, DE ASC was a mixed competitive/ noncompetitive antagonist, Remarkably, DE SC was virtually impermeant in ZR-75-1 cells despite its low K-i towar d polyamine transport, The marked difference in affinity between DESC and MESC was essentially due to the tail-to-tail juxtaposition of two spermine-like structures, suggesting that dimeric ligands of the polya mine transporter might simultaneously interact with more than one bind ing site. While DESC strongly decreased the initial rate of [H-3]sperm idine transport, even a 40-fold molar excess of antagonist could not c ompletely abolish intracellular spermidine accumulation Moreover, as l ittle as 0.3 mu M spermidine fully restored growth in ZR-75-1 cells tr eated with an inhibitor of polyamine biosynthesis in the presence of 5 0 mu M DESC, thus emphasizing the importance of uptake of trace amount s of exogenous polyamines. Thus, reducing the exogenous supply of poly amines with a patent competitive inhibitor mag be kinetically inadequa te to block; replenishment of the polyamine pool in polyamine-depleted tumor cells that display high transport capacity. These results demon strate that polyamine analogues cross-linked into a dimeric structure such as DESC interact with high affinity with the mammalian polyamine carrier without being used as substrates. These novel properties provi de a framework for the design of specific irreversible inhibitors of t he polyamine transporter, which should present advantages over competi tive antagonists for an efficient blockade of polyamine transport in t umor cells.