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Structural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-Transferases

¹ Ce.S.I. Center of Excellence on Aging, "G. D'Annunzio" University Foundation and Department of Biomedical Sciences, University of Chieti, Chieti, Italy; ² Department of Biochemical Sciences, "Sapienza" University of Rome; ³ Department of Chemical Sciences and Technologies and ⁴ Departments of Internal Medicine and Laboratory Medicine, Policlinico di Tor Vergata, University of Rome "Tor Vergata", Rome, Italy

Requests for reprints: Luca Federici, Ce.S.I. Center of Excellence on Aging, University of Chieti "G. D'Annunzio", Via Colle dell'Ara, 66100 Chieti, Italy. Phone: 39-87154-1414; Fax: 39-87154-1598; E-mail: lfederici{at}unich.it.

Key Words: multidrug resistance • enzyme inhibition • cancer treatment

Glutathione S-transferases (GST) constitute a superfamily of enzymes with diversified functions including detoxification from xenobiotics. In many human cancers, Pi class GST (GSTP1-1) is overexpressed and contributes to multidrug resistance by conjugating chemotherapeutics. In addition, GSTP1-1 displays antiapoptotic activity by interacting with c-Jun NH₂-terminal kinase, a key regulator of apoptosis. Therefore, GSTP1-1 is considered a promising target for pharmaceutical treatment. Recently, a potent inhibitor of GSTs, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), was identified and tested on several tumor cell lines demonstrating high antiproliferative activity. To establish the structural basis of NBDHEX activity, we determined the crystal structure of NBDHEX bound to either GSTP1-1 or GSTM2-2 (mu class). NBDHEX in both cases binds to the H-site but occupies different positions. Furthermore, the compound is covalently attached to the GSH sulfur in the GSTM2-2 crystal, forming a -complex, although it is bound but not conjugated in the GSTP1-1 crystal. Several differences in the H-sites of the two isozymes determine the higher affinity of NBDHEX for GSTM2-2 with respect to GSTP1-1. One such difference is the presence of Ile¹⁰⁴ in GSTP1-1 close to the bound NBDHEX, whereas the corresponding position is occupied by an alanine in GSTM2-2. Mutation of Ile¹⁰⁴ into valine is a frequent GSTP1-1 polymorphism and we show here that the Ile¹⁰⁴Val and Ile¹⁰⁴Ala variants display a 4-fold higher affinity for the compound. Remarkably, the GSTP1-1/Ile¹⁰⁴Ala structure in complex with NBDHEX shows a considerable shift of the compound inside the H-site. These data might be useful for the development of new anticancer compounds. [Cancer Res 2009;69(20):8025–34]