BINDING OF RUTHENIUM(III) ANTITUMOR DRUGS TO HUMAN LACTOFERRIN PROBEDBY HIGH-RESOLUTION X-RAY CRYSTALLOGRAPHIC STRUCTURE ANALYSES

The binding to human lactoferrin of three Ru(III) complexes with anti- tumor activity has been investigated by X-ray crystallography in order to gain insights into how such complexes might be carried during tran sferrin-mediated delivery to cells. The complexes, HIm[RuIm(2)Cl(4)], HInd[RuInd(2)Cl(4)] and (HInd)(2) [RuIndCl(5)], where Im = imidazole a nd Ind = indazole, were diffused into crystals of apo-lactoferrin (apo Lf). X-ray diffraction data were collected to 2.6 Angstrom, 2.2 Angstr om and 2.4 Angstrom respectively. The binding sites for the Ru complex es were determined from difference Fouriers, in comparison with native apoLf; the two indazole-apoLf complexes were also refined crystallogr aphically to final R factors of 0.202 (for 8.0 to 2.3 Angstrom data) a nd 0.192 (for 8.0 to 2.4 Angstrom data) respectively. Two types of bin ding site were identified, a high-affinity site at His 253 in the open N-lobe iron-binding cleft of apoLf (and by analogy a similar one at H is 597 in the C-lobe), and lower-affinity sites at surface-exposed His residues, primarily His 590 and His 654. The exogenous heterocyclic l igands remain bound to Ru, at least at the His 253 site, and modelling suggests that the nature and number of these ligands may determine wh ether the closed structure that is required for receptor binding could be formed or not. The results also highlight the importance of His re sidues for binding such complexes and the value of heavy atom binding studies from crystallographic analyses for identifying non-specific bi nding sites on proteins.