A Low-Molecular-Weight Inhibitor against the Chemokine Receptor CXCR4: A Strong Anti-HIV Peptide T140 Hirokazu Tamamura,* ,1 Younong Xu,† Toshio Hattori,† Xiaoyan Zhang,† Rieko Arakaki,‡ Kenji Kanbara,‡ Akane Omagari,* Akira Otaka,* Toshiro Ibuka,* Naoki Yamamoto,§ Hideki Nakashima,‡ and Nobutaka Fujii* ,1 *Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan; †Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan; ‡Kagoshima University Dental School, Sakuragaoka, Kagoshima 890-8544, Japan; and §Tokyo Medical and Dental University, School of Medicine, Bunkyo-ku, Tokyo 113-8519, Japan Received November 27, 1998 T22 ([Tyr 5,12 , Lys 7 ]-polyphemusin II) is an 18-resi- due peptide amide, which has strong anti-HIV ac- tivity. T22 inhibits the T cell line-tropic (T-tropic) HIV-1 infection through its specific binding to a che- mokine receptor CXCR4, which serves as a coreceptor for the entry of T-tropic HIV-1 strains. Herein, we re- port our finding of novel 14-residue CXCR4 inhibitors, T134 and T140, on the basis of the T22 structure. In the assays we examined, T140 showed the highest inhibi- tory activity against HIV-1 entry and the strongest inhibitory effect on the binding of an anti-CXCR4 monoclonal antibody (12G5) to CXCR4 among all the CXCR4 inhibitors that have been reported up to now. © 1998 Academic Press Nowadays, the life cycle of HIV-1 has been inten- sively investigated and many anti-HIV agents are available. While combination therapy using multi- types of drugs, such as protease inhibitors and re- verse transcriptase inhibitors, has improved the clin- ical treatment of AIDS patients (1,2), further development of new effective drugs is desired for multiple drug-combination regimens that affect dif- ferent stages of HIV-replication. In 1996, coreceptors (second receptors) for HIV-1 entry were identified: A CXC-chemokine receptor, CXCR4/fusin, is a corecep- tor for the entry of T cell line-tropic (T-tropic) HIV-1 (3), and a CC-chemokine receptor, CCR5, is a core- ceptor for the entry of macrophage-tropic (M-tropic) HIV-1 (4 – 8). With the identification of coreceptors, agents targeting chemokine receptors have been thought to be useful as one choice of the multi-drugs for combination therapy. In such situation, we have searched for effective anti-HIV compounds derived from self-defense peptides of horseshoe crabs, tachyplesin and polyphemusin. By chemical modifi- cations of these peptides, we have found a strong anti-HIV peptide, T22 ([Tyr 5,12 , Lys 7 ]-polyphemusin II) (9). T22 is an 18-residue peptide amide (Fig. 1), which takes an antiparallel -sheet structure with a type II -turn, that is maintained by two intramo- lecular disulfide bridges (10). Based on the structure- activity relationship study (11), we designed and synthesized several downsized analogs, such as T134 (a 14-residue peptide) (12), which has high anti-HIV activity and significantly less cytotoxicity when com- pared to T22 (Fig. 1). Our previous mechanistic stud- ies showed that T22 affects an HIV-cell fusion pro- cess (13), and that it inhibits the T-tropic HIV-1 infection through its specific binding to CXCR4 (14). T22 is about 4-fold smaller than stromal cell-derived factor (SDF)-1, which is the natural ligand for CXCR4 and a 67-residue large peptide (15–18). Thus, a search for stronger and smaller CXCR4 inhibitors on the basis of the T22 structure is worthwhile. Up to date, three small-size CXCR4 inhibitors (T22, AMD3100 (bicyclam) (19,20) and ALX40-4C (nona-D- Arg) (21)) have been found, and one broad-spectrum inhibitor (a distamycin analog (22)), directed to sev- eral chemokine receptors (CXCR4, CCR5, CCR3 and CCR1), has also been reported. In this connection, two analogs of RANTES have been reported as CCR5 antagonists with inhibitory activity against the M-tropic HIV entry (23,24). In this paper, we report development of a novel, small-sized effective CXCR4 inhibitor, T140. We also discuss its comparative study with other CXCR4 inhibitors (AMD3100 and ALX40-4C). 1 To whom correspondence should be addressed. Fax: 81-75-753- 4570. E-mail: tamamura@pharm.kyoto-u.ac.jp/nfujii@pharm.kyoto- u.ac.jp. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 253, 877– 882 (1998) ARTICLE NO. RC989871 877 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.