Conformational Study of a Highly Speci®c CXCR4 Inhibitor, T140, Disclosing the Close Proximity of Its Intrinsic Pharmacophores Associated with Strong Anti-HIV Activity Hirokazu Tamamura, a, * Makiko Sugioka, b Yoshihiko Odagaki, b Akane Omagari, a Yukiko Kan, c Shinya Oishi, a Hideki Nakashima, d Naoki Yamamoto, e Stephen C. Peiper, f Nobuyuki Hamanaka, b, * Akira Otaka a and Nobutaka Fujii a, * a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan b Minase Research Institute, Ono Pharmaceutical Co., Ltd., Osaka 618-8585, Japan c Suntory Institute for Bioorganic Research, Osaka 618-8503, Japan d Department of Microbiology and Immunology, Kagoshima University Dental School, Sakuragaoka, Kagoshima 890-8544, Japan e Tokyo Medical and Dental University, School of Medicine, Bunkyo-ku, Tokyo 113-8519, Japan f University of Louisville, Louisville, KY 40202, USA Received 21 September 2000; accepted 16 November 2000 AbstractÐWe report the solution structure of T140, a truncated polyphemusin peptide analogue that eciently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 through its speci®c binding to a chemokine receptor, CXCR4. Nuclear magnetic resonance analysis and molecular dynamic calculations revealed that T140 has a rigidly structured conformation constituted by an antiparallel b-sheet and a type II 0 b-turn. A protuberance is formed on one side of the b-sheet by the side-chain functional groups of the three amino acid residues (l-3-(2-naphthyl)alanine 3 , Tyr 5 and Arg 14 ), each of which is indispensable for strong anti-HIV activity. These ®ndings provide a rationale to dissect the structural basis for the ability of this compound to block the interaction between CXCR4 and envelope glycoproteins from T-tropic strains of HIV-1. # 2001 Elsevier Science Ltd. All rights reserved. A perfect therapy has not yet been established for HIV- 1 infection. Although ``highly active anti-retroviral therapy (HAART)'', which involves a combination of reverse transcriptase/protease inhibitors, has dramati- cally improved the clinical treatment of individuals with AIDS, there are several serious problems with this regi- men. 1 These include the emergence of viral strains with multi-drug resistance, signi®cant side eects and a high cost. An ideal therapeutic approach would block an early stage of HIV-infection, such as viral entry. The discovery that a subset of chemokine receptors function as coreceptors for membrane fusion mediated by the envelope glycoprotein and the identi®cation of CCR5 and CXCR4 as the front line coreceptors for entry by macrophage (M-) and T-cell line (T-) tropic strains of HIV-1 provide molecular targets for such a strategy. 2 5 This approach is supported by the relative resistance to infection of commonly transmitted forms of HIV-1 by individuals carrying a protective allele that encodes a non-functional form of CCR5. 3f We have previously demonstrated that a peptide with anti-HIV-1 activity, T22 ([Tyr 5,12 , Lys 7 ]-polyphemusin II), is an inhibitor of CXCR4 that blocks T-tropic HIV-1 entry mediated by this coreceptor. T22 is an 18-residue peptide amide, which was previously found by us based on analysis of the structure±activity relationships (SARs) of tachyple- sins and polyphemusins, which function as autoprotec- tive peptides of horseshoe crabs. 6 Determination of the solution structure of T22 by proton nuclear magnetic resonance (NMR) spectroscopy revealed that it assumes an antiparallel b-sheet conformation connected by a type II b-turn that is maintained by two disul®de bridges. 7 A downsized analogue, TW70 (des-[Cys 8,13 ,Tyr 9,12 ]-[d- Lys 10 ,Pro 11 ]-T22), which is a 14-residue peptide amide with one intrachain disul®de bond, was also found to have an antiparallel b-sheet structure with a type II 0 b-turn by NMR. 8 SAR studies of TW70 enabled us to discover additional analogues, T134 ([l-citrulline (Cit) 12 ]-TW70 lacking the C-terminal amide) and T140 {[ l-3-(2-naphthy- l)alanine (Nal) 3 ]-T134}, which have increased anti-HIV-1 activity and diminished cytotoxicity, in comparison with 0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(00)00664-8 Bioorganic & Medicinal Chemistry Letters 11 (2001) 359±362 *Corresponding authors. Tel.: +81-75-753-4551; fax: +81-75-753-4570 (Tamamura and Fujii); Tel.: +81-75-961-1151; fax: +81-75-962-9314 (Hamanaka); e-mail: tamamura@pharm.kyoto-u.ac.jp; hamanaka@ ono.co.jp; nfujii@pharm.kyoto-u.ac.jp