Dendrimers as Potential Inhibitors of the Dimerization of the Capsid Protein of HIV-1 Rosa Dome ´ nech, † Olga Abian, ‡,§ Rebeca Bocanegra, | Juan Correa, ⊥ Ana Sousa-Herves, ⊥ Ricardo Riguera, ⊥ Mauricio G. Mateu, | Eduardo Fernandez-Megia, ⊥ Adria ´n Vela ´ zquez-Campoy,* ,‡,# and Jose ´ L. Neira* ,†,‡ Instituto de Biologı ´a Molecular y Celular, Universidad Miguel Herna ´ ndez, Elche, Alicante, Spain, Instituto de Biocomputacio ´n y Fı ´sica de Sistemas Complejos, Universidad de Zaragoza, Spain, I+CS (Aragon Health Sciences Institute), CIBERehd, Zaragoza, Spain, Centro de Biologı ´a Molecular Severo Ochoa (CSIC-UAM), Universidad Auto ´ noma de Madrid, Cantoblanco, Madrid, Spain, Departamento de Quimica Orga ´nica, Facultad de Quı ´mica, and Unidad de RMN de Biomole ´ culas Asociada al CSIC, Universidad de Santiago de Compostela, Santiago de Compostela, La Corun ˜ a, Spain, and Fundacio ´ n ARAID, Diputacio ´n General de Arago ´ n, Zaragoza, Spain Received April 21, 2010; Revised Manuscript Received June 9, 2010 Assembly of the mature human immunodeficiency virus type 1 capsid involves the oligomerization of the capsid protein, CA. The C-terminal domain of CA, CTD, participates both in the formation of CA hexamers and in the joining of hexamers through homodimerization. Intact CA and the isolated CTD are able to homodimerize in solution with similar affinity (dissociation constant in the order of 10 µM); CTD homodimerization involves mainly an R-helical region. In this work, we show that first-generation gallic acid-triethylene glycol (GATG) dendrimers bind to CTD. The binding region is mainly formed by residues involved in the homodimerization interface of CTD. The dissociation constant of the dendrimer-CTD complexes is in the range of micromolar, as shown by ITC. Further, the affinity for CTD of some of the dendrimers is similar to that of synthetic peptides capable of binding to the dimerization region, and it is also similar to the homodimerization affinity of both CTD and CA. Moreover, one of the dendrimers, with a relatively large hydrophobic moiety at the dendritic branching (a benzoate), was able to hamper the assembly in vitro of the human immunodeficiency virus capsid. These results open the possibility of considering dendrimers as lead compounds for the development of antihuman immunodeficiency virus drugs targeting capsid assembly. Introduction Trying to inhibit protein-protein interactions with a small ligand for therapeutic purposes is an attractive, but challenging idea. This is probably due to the relatively large and stere- ochemically complex protein-protein interfaces that make it difficult for a small molecule to encompass the whole binding site. 1,2 Entropic considerations may also aggravate the problem of achieving enough affinity and specificity between the inhibitor molecule and the protein surface. Recently, however, specifically designed small molecules have been shown to disrupt such large and complex interfaces by binding to “hotspots”. 2 A promising example is that of the B-cell lymphoma protein (Bcl-XL) and the pro-apoptotic molecule Bcl-2 antagonist of cell death (BAK), currently in phases 1 and 2 clinical trials. 3 Virus capsids provide other biomedically relevant targets for the design of protein- protein interfacial inhibitors. Because of the omnipresent nature of protein-protein interactions in virion assembly and matura- tion, it could be possible to design antiviral strategies based on the inhibition of those macromolecular complexes. 4 Human immunodeficiency virus (HIV), the agent responsible for AIDS, belongs to the retrovirus family. The HIV genome is a model of economic packaging because the virus uses complex processing (both in the production and cleavage of mRNAs and in the final viral polypeptides) to generate several proteins that can work in the host cell. Among them, one of the most fascinating examples is perhaps the Gag-polyprotein. Assembly of the immature HIV type 1 (HIV-1) capsid occurs through the controlled polymerization of the Gag-polyprotein, which is transported to the plasma membrane of infected cells, where morphogenesis of the immature, noninfectious virion occurs. Immediately after budding of the immature virion, maturation is initiated by the cleavage of the Gag protein by a viral protease, which yields the MA (matrix), CA (capsid), NC (nucleocapsid), and p6 proteins, as well as two spacer peptides. This maturation process induces conformational changes in CA, which reassembles as an independent protein to form a capsid with a distinctive conical shape. 5,6 Because of its critical role during HIV-1 morphogenesis, CA has emerged as a promising target for the development of new anti-HIV drugs based on disruption of capsid assembly. 7,8 The CA protein is formed by two independently folded domains: the N-terminal domain, NTD, and the C-terminal domain, CTD, separated by a flexible linker. 9-12 The NTD (residues 1-146 in the numbering of the whole intact protein) is composed of five coiled-coil R-helices (corresponding to helices 1-5 of CA), with two additional short R-helices (helices 6 and 7) following an extended proline-rich loop. The CTD * To whom correspondence should be addressed. Tel.: +34 976762996 (A.V.-C.); +34 966658459 (J.L.N.). Fax: +34 976762990 (A.V.-C.); +34 966658758 (J.L.N.). E-mail: adrianvc@unizar.es (A.V.-C.); jlneira@umh.es (J.L.N.). † Universidad Miguel Herna ´ndez. ‡ Universidad de Zaragoza. § I+CS (Aragon Health Sciences Institute). | Universidad Auto ´noma de Madrid. ⊥ Universidad de Santiago de Compostela. # Fundacio ´n ARAID. Biomacromolecules 2010, 11, 2069–2078 2069 10.1021/bm100432x  2010 American Chemical Society Published on Web 07/13/2010