Structural insights into the dual-targeting mechanism of Nutlin-3 Jae-Sun Shin a , Ji-Hyang Ha a , Fahu He b , Yutaka Muto b , Kyoung-Seok Ryu c , Ho Sup Yoon d , Sunghyun Kang a , Sung Goo Park a , Byoung Chul Park a , Sang-Un Choi e,⇑ , Seung-Wook Chi a,⇑ a Medical Proteomics Research Center, KRIBB, Daejeon 305-806, Republic of Korea b RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan c Division of Magnetic Resonance, Korea Basic Science Institute, Chungcheongbuk-Do 363-883, Republic of Korea d Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637511, Singapore e Research Center for Drug Discovery Technology, Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600, Republic of Korea article info Article history: Received 15 February 2012 Available online 28 February 2012 Keywords: Apoptosis Multi-targeting drug NMR Nutlin-3 Bcl-2 family proteins abstract Multi-targeting therapy is an emerging strategy of drug discovery to improve therapeutic efficacy, safety and resistance profiles. In this study, we monitored the binding of a potent MDM2 inhibitor Nutlin-3 with anti-apoptotic Bcl-2 family proteins using NMR spectroscopy. Our results showed the universal binding of Nutlin-3 with diverse anti-apoptotic Bcl-2 family proteins. Taken together with the binding data for Nutlin-3 analogs, the structural model of the Bcl-X L /Nutlin-3 complex showed that the binding mode of Nutlin-3 resembles that of the Bcl-X L /Bcl-2 inhibitors, suggesting the molecular mechanism of tran- scription-independent mitochondrial apoptosis by Nutlin-3. Finally, our structural comparison provides structural insights into the dual-targeting mechanism of how Nutlin-3 can bind to two different target proteins, MDM2 and anti-apoptotic Bcl-2 family proteins in a similar manner. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Multi-target-based therapy is an emerging strategy of drug development to improve therapeutic efficacy, safety and resistance profiles [1–3]. Many human diseases such as cancer arise from var- ious factors, and thus single target-directed therapeutics show lower than desired clinical efficacies. Multi-targeting is based on the rekindled concept of ‘‘poly-pharmacology’’ in which one drug can bind to more than one target proteins [4]. Noteworthy exam- ples of clinically successful multi-targeting drugs are anti-cancer multi-kinase inhibitors sorafenib, sunitnib, dasatinib and lapatinib [4,5]. Recently, targeting multiple arms of the apoptotic regulatory machinery could also provide new opportunities for apoptosis- promoting cancer therapy [6]. Nutlin-3 is one of the most potent MDM2 antagonists that serves as a competitive inhibitor of p53–MDM2 interaction [7,8]. Currently, Nutlins are being evaluated as cancer therapeutics in clinical trials. As an imidazoline-based compound, the structure of Nutlin mimics the structure of p53 transcriptional activation domain (p53TAD) peptide bound to MDM2 [9]. Initially, anti-cancer activity of Nutlin was thought to be attributed only to the inhibition of MDM2 func- tion by blocking the p53–MDM2 interaction. However, Kojima et al. demonstrated that high levels of MDM2 do not prevent Nutlin from inducing apoptosis and that Nutlin exerts its effect not only in a transcription-dependent pathway but also a transcription-indepen- dent pathway for inducing p53-mediated apoptosis [10]. More re- cently, it was shown that the transcription-independent apoptotic mechanism of p53 provides an alternative pathway contributing to the Nutlin-induced apoptosis in tumor cells [11,12]. However, the detailed molecular mechanism underlying the transcription- independent mitochondrial apoptosis by Nutlin remains unclear. In our previous studies, we showed that MDM2 and anti-apoptotic Bcl-2 family proteins share a remarkably similar mode of binding to p53TAD [13–15]. Based on this finding, we hypothesized that a p53TAD-mimetic MDM2 antagonist can directly bind to anti- apoptotic Bcl-2 family proteins in a manner analogous to that with p53TAD. Through NMR binding experiments, we provided a ‘‘proof- of-concept’’ evidence that the MDM2 antagonist Nutlin-3 binds to the anti-apoptotic Bcl-2 family proteins Bcl-X L and Bcl-2 [14]. As Bcl-2 is an essential player in the apoptotic regulation, frequently overexpressed in many type of cancers, and is closely associated with the tumor progression and resistance to chemotherapeutic agents, targeting the anti-apoptotic Bcl-2 family of proteins is an attractive strategy for cancer therapy [16,17]. An inhibitor of Bcl- 2 family proteins, ABT-737, was shown to induce regression of solid tumors [18]. However, ABT-737 can bind to only a few Bcl-2 family members such as Bcl-2 and Bcl-X L , which confers resistance to apoptosis induced by ABT-737 [19,20]. Thus, neutralization of mul- tiple anti-apoptotic Bcl-2 family proteins is required for efficient cancer therapy. 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.113 ⇑ Corresponding authors. Fax: +82 42 861 4246 (S.-U. Choi), +82 42 879 8596 (S.-W. Chi). E-mail addresses: suchoi@krict.re.kr (S.-U. Choi), swchi@kribb.re.kr (S.-W. Chi). Biochemical and Biophysical Research Communications 420 (2012) 48–53 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc