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J. Biol. Chem., Vol. 280, Issue 33, 29765-29770, August 19, 2005

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Interdomain Communication in Hepatitis C Virus Polymerase Abolished by Small Molecule Inhibitors Bound to a Novel Allosteric Site^*

Stefania Di Marco, Cinzia Volpari, Licia Tomei, Sergio Altamura, Steven Harper, Frank Narjes, Uwe Koch, Michael Rowley, Raffaele De Francesco, Giovanni Migliaccio, and Andrea Carfí

From the Istituto di Ricerche di Biologia Molecolare P. Angeletti, 00040 Pomezia (Rome), Italy

The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have determined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3- and 2.4-Å resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small -helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen.

Received for publication, May 17, 2005

The atomic coordinates and structure factors (code 2BRK and 2BRL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a grant from the Ministero dell'Istruzione, dell'Università e della Ricerca. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 6 and supplemental Table II.

To whom correspondence may be addressed: Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km 30.600, 00040 Pomezia, Rome, Italy. Tel.: 39-06-91093238; Fax: 39-06-91093654; E-mail: stefania_dimarco{at}merck.com. To whom correspondence may be addressed: Istituto di Ricerche Biologia Molecolare P. Angeletti, Via Pontina Km 30.600, 00040 Pomezia, Rome, Italy. Tel.: 39-06-91093550; Fax: 39-06-91093654; E-mail: andrea_carfi{at}merck.com.

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