Inhibition of HIV protease by monoclonal antibodies † P. Rezacova 1 , J. Brynda 1 , M. Fabry 1 , M. Horejsi 1 , R. Stouracova 1 , J. Lescar 2 , V. Chitarra 3 , M. M. Riottot 3 , J. Sedlacek 1 and G. A. Bentley 3 * 1 Department of Gene Manipulation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 6, Prague, Czech Republic 2 School of Biological Sciences, Nanyang Walk, Blk 5, Level 3, Singapore 637616 3 Unite ´ d’Immunologie Structurale (CNRS URA 2185), Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation. We have produced monoclonal antibodies specific for the HIV-1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme’s activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterized two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognize epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms of protease inhibition by these antibodies. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: monoclonal antibodies; HIV protease; enzyme inhibition; crystal structure Received 2 May 2002; accepted 10 May 2002 INTRODUCTION The HIV protease, which belongs to the aspartate protease family, is responsible for maturation of the Gag and Gag/Pol polyprotein precursors of the structural proteins (matrix, capsid and nucleocapsid) and the three viral enzymes, including the protease, itself produced by auto-cleavage (reviewed by Katz and Skalka, 1994). Inhibition of HIV protease arrests maturation of the newly budded virion to the infectious form of the pathogen. The enzyme is therefore the object of active research for the development of anti- viral medicaments. The monomeric subunit of the protease, comprising 99 amino acids, carries the invariant catalytic triplet Asp25–Thr26–Gly27. The active protease is a homodimer in which the interface between the subunits is formed largely by interdigitation of the N-terminal (residues 1–4) and C-terminal (residues 96–99) regions from both monomers to create a four-stranded anti-parallel b sheet (Navia et al., 1989; Wlodawer et al., 1989). In the homodimer, the catalytic triplets of the subunits are placed adjacent to each other at the monomer–monomer interface, forming a pepsin-like catalytic site covered by two identical flexible loops or ‘flap’ regions (one from each subunit) that enclose the substrate in a hydrophobic environment during proteolysis. We have produced murine monoclonal antibodies (mAb) raised against recombinant HIV-1 protease and screened them for inhibition of proteolytic activity of the enzyme. Our objective was to use these antibodies as tools to probe proteolytic activity and as eventual aids in the design of inhibitors. Since the active site is inaccessible to antibodies, these protease-inhibiting mAbs should be directed to other regions of the enzyme. In this paper we describe two anti- HIV-1 protease mAbs, F11.2.32 (Lescar et al., 1996, 1997) and 1696 (Lescar, et al., 1999; Rezacova et al., 2001), which inhibit activity of the viral enzyme. Although we have not yet been able to crystallize these antibodies as complexes with the HIV protease itself, we have demonstrated their presence in solution. As an alternative approach to studying the antigenic recognition by these two mAbs at the structural level, we have crystallized them as complexes with the respective peptide fragments of the HIV protease that were determined as epitopes by mapping studies. The conforma- tion of the bound peptide in the crystal structure of both antibody–peptide complexes, however, implies that, although F11.2.32 and 1696 both recognize native HIV protease, the antigen is not in its native form when in the antibody-bound state. Here, we review our conclusions on these antibodies and their mechanism of HIV protease inhibition. JOURNAL OF MOLECULAR RECOGNITION J. Mol. Recognit. 2002; 15: 272–276 Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/jmr.587 Copyright # 2002 John Wiley & Sons, Ltd. *Correspondence to: G. A. Bentley, Unite ´ d’Immunologie Structurale (CNRS URA 2185), Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France. E-mail: bentley@pasteur.fr Contract/grant sponsor: Institut Pasteur. Contract/grant sponsor: Centre National de la Recherche Scientifique. Contract/grant sponsor: European Commission. Contract/grant sponsor: Grant Agency of the Academy of Sciences of the Czech Republic; contract/grant number: A5052502. Contract/grant sponsor: Grant Agency of the Czech Republic; contract/grant number: 203/98/K023. Abbreviations used: CDR, complementarity-determining region; HIV, human immunodeficiency virus; mAb, monoclonal antibody; scFv, single-chain Fv; TaqP, Thermus aquaticus DNA polymerase I. †This paper is published as part of a Special issue entitled Structure and function relationships in proteins: a tribute to Allen B. Edmundson.