Distribution of furamidine analogues in tumor cells: targeting of the nucleus or mitochondria depending on the amidine substitution.
Diphenylfuran diamidines represent an important class of DNA minor groove binders of high therapeutic interest as antiparasitic or antitumor agents depending on the compounds structures. To exert their cytotoxic action, the compounds must first get into the cell and reach the nuclear compartment where the main target, DNA, is located. The forces that drive the drugs into cell nuclei, as well as the influence of the molecular structures on the cell distribution, are not known. To address these issues, we took advantage of the fluorescence of the molecules to analyze their intracellular distribution profiles in tumor cells of different origins (B16 melanoma, MCF7 mammary adenocarcinoma, A549 lung carcinoma, HT29 colon carcinoma, LNCaP, and PC3 prostatic carcinoma) by epifluorescence and confocal microscopy. A homogeneous series of synthetic bis-substituted alkyl or phenyl amidine and reverse amidine derivatives of furamidine was used to dissect the molecular mechanisms that control the distribution of the drugs into the cytoplasm or the nucleus of the cells. The amidine (DB75) and the various N-alkyl derivatives were found to accumulate selectively in the cell nuclei. This is also the case for a guanidine derivative but not for the phenyl-substituted compound DB569, which essentially localizes in cytoplasmic granules. Similar cytoplasmic patterns were observed with a reverse amidine analogue and a pyridine-substituted compound indicating that the presence of aromatic rings on the terminal side chain is the limiting factor that restricts the uptake of the compounds in the nuclear compartment. The use of different organelle-selective fluorescent probes, such as JC-1 and chloromethyl-X-rosamine, both specific to mitochondria and neutral red considered as a lysosome-selective probe, suggests that DB569 preferentially accumulates in mitochondria. Competition experiments with the antitumor drug daunomycin reveal that the diphenylfurans are attracted into the nuclei by the DNA. The DNA minor groove-drug interactions provide the driving force that permits massive accumulation of the fluorescent molecules in the nuclei. The DNA binding properties of the diphenylfuran derivatives were investigated by DNase I footprinting and surface plasmon resonance biosensor experiments to measure sequence selectivity and binding affinities, respectively. Furamidine and its phenyl-substituted analogue that accumulate in the cell nuclei and mitochondria, respectively, share a common selectivity for AT sites and bind equally tightly to these sites. Therefore, it is possible to modulate the intracellular distribution of the furamidine derivatives without affecting their DNA binding and sequence recognition properties. The introduction of aromatic substituents on diphenylfuran diamidines represents a novel strategy to control the intracellular compartmentalization of these DNA binding agents and directs them to mitochondria. This drug design strategy may prove useful to trigger drug-induced apoptosis.
Lansiaux
Amélie
A
Institut National de la Santé et de la Recherche Médicale U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, 59045 Lille, France.
Tanious
Farial
F
Mishal
Zohar
Z
Dassonneville
Laurent
L
Kumar
Arvind
A
Stephens
Chad E
CE
Hu
Qiyue
Q
Wilson
W David
WD
Boykin
David W
DW
Bailly
Christian
C
D000230:Adenocarcinoma / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics* / Q000494:pharmacology; D001483:Base Sequence; D001550:Benzamidines / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D008297:Male; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000378:metabolism; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D001550:Benzamidines; D004273:DNA, Neoplasm; C033286:furamidine