It is now well established that polynuclear platinum compounds in which two or three platinum coordination units are linked in a linear fashion compose an important new class of anticancer drugs [1,2]. The first clinical compound, currently denoted BBR3464 (see Fig. 1A), is a bifunctional trinuclear DNA-binding agent with an overall 4ϩ charge (3). The phase I trials (4, 5) demonstrated a clear pattern of responses in cancers not normally treatable with cisplatin (cis-diamminedichloroplatinum(II)) (see Fig. 1A), including responses in melanoma and pancreatic and lung cancer. Objective responses in phase II were verified in relapsed ovarian cancer and non-small cell lung cancer (6, 7). Pre-clinical studies indicated activity in p53 mutant tumors and a minimum induction of p53 following BBR3464 treatment. The interactions of antitumor polynuclear platinum compounds with target DNA (for reviews, see Refs. 1, 2, and 8) are distinct from those of the mononuclear based cisplatin family and, indeed, unlike those of any DNA-damaging agent in clinical use. Hence, it is important to understand these novel interactions to the greatest extent possible and how they affect DNA structure and function to exploit the full clinical potential of these new agents.Bifunctional binding of BBR3464 to double-stranded DNA preferentially involves guanine residues and is characterized by the rapid formation of long-range intra-and interstrand cross-links (CLs) 1 in which the platinated sites are separated by 1 or more bp (9). Quantitation of DNA interstrand crosslinking in natural and linear DNAs indicated ϳ20% of the DNA to be interstrand cross-linked. This value is significantly higher than that for cisplatin (10, 11); on the other hand, an intriguing aspect of BBR3464 is that long-range delocalized intrastrand adducts are equally or even more probable than interstrand CLs (9). As the [Pt,Pt] intrastrand CLs of BBR3464 are analogs of the major adducts of cisplatin, which forms ϳ90% bifunctional intrastrand adducts between neighboring purine residues on DNA, we examined first in our recent work (12) how the structures of the various types of the intrastrand CLs of BBR3464 affect conformational properties of DNA and how these adducts are recognized by the HMGB1 protein and removed from DNA during in vitro nucleotide excision repair (NER) reactions. These studies were performed because some structures altered by platinum adducts, such as stable directional bending and unwinding, attract various damaged DNAbinding proteins such as those containing HMG domains (13)(14)(15). The binding of these proteins has been postulated to mediate the antitumor properties of the platinum drugs (14,15). In addition, several reports have demonstrated that intrastrand CLs of cisplatin are removed from DNA during NER reactions and that NER is also a major mechanism contributing to cisplatin resistance (16 -18). Recent work has revealed that intrastrand CLs of BBR3464 create a local conformational distortion, but that none of these intrastrand adducts results in

The new antitumor trinuclear platinum compound [{trans-PtCl(NH 3 ) 2 } 2 -trans-Pt(NH 3 ) 2 {H 2 N(CH 2 ) 6 NH 2 } 2 ] 4؉ (designated as BBR3464) is currently in phase II clinical trials. DNA is generally considered the major pharmacological target of platinum drugs. As such it is of considerable interest to understand the patterns of DNA damage. The bifunctional DNA binding of BBR3464 is characterized by the rapid formation of long range intra-and interstrand cross-links. We examined how the structures of the various types of the intrastrand crosslinks of BBR3464 affect conformational properties of DNA, and how these adducts are recognized by high mobility group 1 protein and removed from DNA during in vitro nucleotide excision repair reactions. The results have revealed that intrastrand cross-links of BBR3464 create a local conformational distortion, but none of these cross-links results in a stable curvature. In addition, we have observed no recognition of these crosslinks by high mobility group 1 proteins, but we have observed effective removal of these adducts from DNA by nucleotide excision repair. These results suggest that the processing of the intrastrand cross-links of BBR3464 in tumor cells sensitive to this drug may not be relevant to its antitumor effects. Hence, polynuclear platinum compounds apparently represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues.The trinuclear compound [{trans-PtCl(NH 3 ) 2 } 2 -transPt(NH 3 ) 2 {H 2 N(CH 2 ) 6 NH 2 } 2 ] 4ϩ ( Fig. 1) is currently in phase II clinical trials. The compound, designated as BBR3464, is the lead representative of an entirely new structural class of DNAmodifying anticancer agents based on the poly(di,tri)nuclear platinum structural motif (1-3). In phase I trials, objective partial responses in pancreatic and lung cancers as well as melanoma were observed (4, 5). These results suggest the potential for genuinely complementary clinical anticancer activity of BBR3464 in comparison to cis-diamminedichloroplatinum(II) (cisplatin).