Molecular Recognition A Tetraguanidinium Ligand Binds to the Surface of the Tetramerization Domain of Protein P53** Xavier Salvatella, Marc Martinell, Margarida Gair, Mauricio G. Mateu, Miguel Feliz, Andrew D. Hamilton, Javier de Mendoza, and Ernest Giralt* Proteins often perform their functions as part of networksregulated by protein–protein interactions [1] and the design of specific inhib- itors of these interactions will lead to new approaches for the treatmentof important diseases. [2] However,the successful design of such bindersto protein surface isdifficult because of the physicochemical properties of protein–protein interfaces,which arevery large,flat,and rich in well-solvated hydro- philic residues such as Asp,Glu,and Arg. [3] We showed previouslythat oligomersof chiral,bicyclic guanidines,which are known to interact effectively with anionic groups, [4] bind the surface of helical or partially helical tetraanionic peptides in alcoholic media. [5,6] We report herein that,in aqueous solution,the tetraguanidinium compound 1 (Scheme 1) binds to an anionic patch at the surface of the tetramerization domain of the tumor suppressor protein P53, a key therapeutic target for cancer treatment. [7] The shape and charge complementarity of the helical secondary structures of tetraguanidinium compounds such as 1 with those of helical peptidescontaining fouranionic residues atthe i and i+ 3 relative positions enables these molecules to bind in 10 % aqueous methanol. [5,6] To assess whether this supramolecular architecture can be applied to the recognition of such anionic sequences at the surface of a protein,we studied the binding ofcompound 1 with the tetramerization domain of P53. This particular domain was selected because it has two overlapping helical tetraanionic sequences on its surface,one formed by residues Glu 336, Glu 339,Glu 343,and Glu 346,and the other by Glu 343, Glu 346,Glu 349,and Asp 352 (Figure 1). [8] The main function of the tetramerization domain is to stabilize the overall structure of the protein. Since tetramerization is essential for P53 function, [7] small molecules that bind to this domain and affect its stability could potentially be used to modulate the antitumor activity of the protein. [9] We examined this system by both chemical shift pertur- bation (CSP) [10] and saturation transfer difference (STD) Figure 1. a) Ribbon representation of the tetramerization domain of protein P53. [8] For clarity, only the side chains of the residues Glu 336, Glu 339, Glu 343, Glu 346, Glu 349, and Asp 352 of one monomer are shown. b) Solvent-accessible surface of the domain. The surface corre- sponding to the side-chain oxygen atoms of the six anionic residues of one monomer is shown in red. The primary structure of the tetramerization domain in the region of interest (residues 335–355) is RERFEMFRELNEALELKDAQA. Scheme 1. Chemical structure of the tetraguanidinium ligand 1 and the model proposed for its interaction with aspartate (n = 1) or glutamate (n = 2) residues on peptide and protein surfaces. [*] Dr. X. Salvatella, + M. Martinell, Prof. E. Giralt Institut de Recerca Biom8dica de Barcelona Parc Cient9fic de Barcelona Josep Samitier 1–5, 08028 Barcelona (Spain) and Departament de Qu9mica Org>nica Universitat de Barcelona Mart9 i FranquAs 1–11, 08028 Barcelona (Spain) Fax: (+ 34) 93-403-7126 E-mail: egiralt@pcb.ub.es Dr. M. Gair9, Dr. M. Feliz Servei de Resson>ncia Magn8tica Nuclear Serveis Cientificot8cnics Universitat de Barcelona Josep Samitier 1–5, 08028 Barcelona (Spain) Prof. M. G. Mateu Centro de Biolog9a Molecular Universidad AutFnoma de Madrid Cantoblanco, 28049 Madrid (Spain) Prof. A. D. Hamilton Department of Chemistry, Yale University P.O. Box 208107, New Haven, CT 06520 (USA) Prof. J. de Mendoza Departamento de Qu9mica OrgInica Universidad AutFnoma de Madrid Cantoblanco, 28049 Madrid (Spain) [ + ] Current address: Chemical Laboratory, University of Cambridge Lensfield Road, Cambridge CB2 1EW (UK) [**] X.S. and M.M. acknowledge the Generalitat de Catalunya for a predoctoral fellowship. This work was partially supported by grants from the Ministerio de Ciencia y Technlog9a-FEDER (Grant no. BIO2002-2301) and the Generalitat de Catalunya (Grup Con- solidat 1999SGR0042 and Centre de Refer8ncia en Biotecnologia). Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Zuschriften 198 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/ange.200352115 Angew. Chem.2004,116, 198 –200