A metallopeptide assembly of the HIV-1 gp41 coiled coil is an ideal receptor in fluorescence detection of ligand binding.

Angewandte Chemie Stabilizing the gp41 Trimer in HIV-1 A Metallopeptide Assembly of the HIV-1 gp41 Coiled Coil Is an Ideal Receptor in Fluorescence Detection of Ligand Binding** Miriam Gochin,* Rodney Kiplin Guy, and Martin A. Case The transmembrane subunit, gp41, of the envelope glycopro- tein of HIV-1 plays a crucial role in the fusion of the virus to the target cell during infection. [1–3] During the conformational changes that accompany virus–cell binding, the coiled-coil domain of three HR1 helices of trimeric gp41 becomes exposed and vulnerable to attack by antiviral drugs that could prevent fusion and infection of cells. [4] To date, the only successful drug against fusion is the peptide T-20 (DP178), derived from the HR2 region of gp41. [5–8] T-20 binds tightly to the HR1 coiled coil, thus interfering with formation of the fusion-competent six-helix bundle structure of gp41, compris- ing three HR1 and three HR2 helices. There is considerable interest in the discovery and development of nonproteolytic alternatives to T-20, which is very expensive to produce and not orally bioavailable. A key challenge to screening compound libraries for drugs that target the three stranded coiled coil of gp41 arises from the fact that it is unstable when excised from full-length gp41 and buried in intact gp41. Peptides from gp41 HR1 are hydrophobic and tend to aggregate in solution. Previous approaches for accessing the coiled coil domain have involved stabilizing it as part of a larger soluble protein construct. Prior work has included the addition of GCN4 trimeric helices which extend the coiled coil by several heptad repeats (IQN17), [9] the preparation of 5-helix, a gp41 protein lacking one HR2 domain, [10] and the use of mutant 6-helix protein in which the HR1–HR2 interaction is destabilized, thus allowing for transient exposure of the HR1 coiled coil. [11] We report herein on the design of a stable fragment of the HR1 coiled- coil domain that does not involve extra protein domains, and that can be used in a fluorescence assay for rapid screening of potential fusion inhibitors. The basis of the design is depicted in Figure 1. A metal- ion-binding bidentate ligand, 2,2’-bipyridyl (bpy) is attached to the N-terminus of a 31 residue peptide that contains residues 565–584 of gp41. The region selected contains a prominent cavity within the grooves of the coiled coil. [12] Addition of a metal ion such as Fe II or Ni II should result in formation of a tris-bipyridyl metal complex, which stabilizes the coiled-coil structure, as has been shown previously for several designed peptides. [13–18] [*] Prof. M. Gochin Dept. Microbiology, University of the Pacific School of Dentistry, San Francisco, CA 94115 (USA) and Dept. Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 (USA) Fax (+ 1) 415-929-6564 E-mail: miriam@picasso.ucsf.edu, mgochin@uop.edu R. Kiplin Guy Dept. Pharmaceutical Chemistry University of California San Francisco, CA 94143 (USA) M. A. Case Department of Chemistry University of Vermont Burlington, VT 0540 [**] This work was supported by grants from the University-wide AIDS research project of the State of California (to M.G.), the American Foundation for AIDS Research (to M.G.), the National Science Foundation (to M.A.C.) and from the Sandler Research Foundation (to R.K.G). Angew. Chem. Int. Ed. 2003, 42, 5325 –5328 Figure 1. Model of the expected Fe-env2.1—dansylated C-peptide inter- action, derived by homology modeling from pdb2ez0. Residues of Fe env2.1 corresponding to the native sequence of the gp41 coiled coil are shown in gray, and all other coiled coil residues in black. Iron(ii) is shown in black, coordinated to the bipyridine groups in light gray. The C-peptide residues are off-white, with the dansyl group shown in light gray attached to the cysteine side chain in an arbitrary conformation. For clarity, side chains of coiled coil residues not in direct contact with the C-peptide are omitted. The distance between iron(ii) and the dansyl group is estimated from the model to be about DOI: 10.1002/anie.200352006 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim