Aroylhydrazone and thiosemicarbazone iron (Fe) chelators have potent antitumor activity. The aim of the current study was to examine the antitumor effects and mechanisms of action of a novel series of Fe chelators, the di-2-pyridyl thiosemicarbazones. Of 7 new chelators synthesized, 4 showed pronounced antiproliferative effects. The most active chelator was Dp44mT, which had marked and selective antitumor activity-for example, an IC 50 of 0.03 M in neuroepithelioma cells compared with more than 25 M in mortal fibroblasts. Indeed, this antiproliferative activity was the greatest yet observed for an Fe chelator. Efficacy was greater than it was for the cytotoxic ligand 311 and comparable to that of the antitumor agent doxorubicin. Strikingly, Dp44mT significantly (P < .01) decreased tumor weight in mice to 47% of the weight in the control after only 5 days, whereas there was no marked change in animal weight or hematologic indices. IntroductionIron (Fe) is essential for proliferation, and many studies have shown that tumor cells are more sensitive to Fe deprivation than normal cells. [1][2][3][4][5][6][7][8][9][10][11] This sensitivity probably exists because cancer cells have greater Fe requirements than their normal counterparts [12][13][14][15] and because cancer cells express higher levels of the Fe-containing enzyme, ribonucleotide reductase (RR), which is the critical rate-limiting step in DNA synthesis. [16][17][18][19][20][21] Many in vitro 2,4,[7][8][9][21][22][23][24][25][26][27][28] and in vivo 6,29,30 studies and clinical trials 3,5,[31][32][33][34][35][36] have demonstrated that chelators are effective antiproliferative agents (for reviews, see Hershko,10 Lovejoy and Richardson, 11 and Richardson 37 ). The most well-studied chelator is desferrioxamine (DFO; Figure 1). 10,11 However, its short half-life and low efficacy at permeating membranes limit its antiproliferative activity. 22,38 Indeed, these factors probably resulted in its failure to inhibit tumor growth in some studies. 39,40 One group of ligands with high Fe chelation efficacy is the pyridoxal isonicotinoyl hydrazone (PIH) class. [41][42][43][44] We characterized PIH analogs that show far greater Fe chelation efficacy and antiproliferative activity than DFO. 23,24,45 Some of these ligands, such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311; Figure 1) inhibit RR activity 9 and affect the expression of molecules vital for cell cycle control. [46][47][48] One mechanism by which chelators and other factors (eg, oxidative stress) cause tumor cell death may be through the induction of apoptosis. 22,24,[49][50][51][52][53][54][55][56] However, the precise mechanisms involved in chelator-mediated apoptosis remain unclear, particularly for aroylhydrazone ligands.The caspase enzymes are common executors of apoptosis. 52,57,58 Two caspase-activating cascades that regulate apoptosis have been described; one is initiated through death receptors (eg, CD95), and the other is triggered by changes in mitochondrial integrity. 52,59,60 In the ...

Table S1. Crystal data and structure refinement for HDp4mT. Identification code ds2369 Empirical formula C 13 H 13 N 5 S Formula weight 271.34 Temperature 293(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group C2/c Unit cell dimensions a = 19.740(2) Å α= 90°. b = 11.694(1) Å β= 91.391(9)°. c = 11.514(2) Å γ = 90°. Volume 2657.1(6) Å 3 Z 8 Density (calculated) 1.357 Mg/m 3 Absorption coefficient 0.237 mm -1 F(000) 1136 Crystal size 0.5 x 0.5 x 0.3 mm 3 Theta range for data collection 2.02 to 24.96°. Index ranges 0<=h<=23, 0<=k<=13, -13<=l<=13 Reflections collected 2399 Independent reflections 2329 [R(int) = 0.0491] Completeness to theta = 24.96° 99.7 % Absorption correction Psi-scan Max. and min. transmission 0.9317 and 0.8896 Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 2329 / 0 / 172 Goodness-of-fit on F 2 1.039 Final R indices [I>2sigma(I)] R1 = 0.0382, wR2 = 0.0926 R indices (all data) R1 = 0.0623, wR2 = 0.1029 Largest diff. peak and hole 0.202 and -0.209 e.Å -3

The metal-chelating compound Dp44mT is a di-2-pyridylketone thiosemicarbazone (DpT) which displays potent and selective antitumor activity. This compound is receiving translational attention, but its mechanism is poorly understood. Here, we report that Dp44mT targets lysosome integrity through copper binding. Studies using the lysosomotropic fluorochrome acridine orange established that the copper-Dp44mT complex (Cu[Dp44mT]) disrupted lysosomes. This targeting was confirmed with pepstatin A-BODIPY FL, which showed redistribution of cathepsin D to the cytosol with ensuing cleavage of the proapoptotic BH3 protein Bid. Redox activity of Cu[Dp44mT] caused cellular depletion of glutathione, and lysosomal damage was prevented by cotreatment with the glutathione precursor N-acetylcysteine. Copper binding was essential for the potent antitumor activity of Dp44mT, as coincubation with nontoxic copper chelators markedly attenuated its cytotoxicity. Taken together, our studies show how the lysosomal apoptotic pathway can be selectively activated in cancer cells by sequestration of redox-active copper. Our findings define a novel generalized strategy to selectively target lysosome function for chemotherapeutic intervention against cancer. Cancer Res; 71(17);