Disclosing the Interaction of Carbonic Anhydrase IX with Cullin-Associated NEDD8-Dissociated Protein 1 by Molecular Modeling and Integrated Binding Measurements

Human Carbonic Anhydrase (hCA) TX is a membrane-associated member of the CA enzyme family, involved in solid tumor acidification. This enzyme is a marker of tumor hypoxia and a prognostic factor for Several human cancers. In a recent paper, we showed that CA IX interacts with cullin-associated NEDD8-dissociated protein 1 (CANDI), a nuclear protein involved in gene transcription and assembly of SCF ubiquitin ligase complexes. A functional role for this interaction was also identified, since lower CA IX levels were observed in cells with decreased CANDI expression via shRNA-mediated interference. In this paper, we describe the identification of the structural determinants responsible for the CA IX/CAND1 interaction by means of a multidisciplinary approach, consisting of binding assay measurements, molecular docking, arid site-directed mutagenesis. These data open a novel scenario in the design of anticancer drugs targeting CA IX. Indeed, the knowledge of the structural determinants responsible for the CANDI/CA IX interaction provides the molecular basis to design molecriles able to destabilize it. Due to the proposed function of CANDI. in stabilizing CA DC, these molecules could represent an efficient to-of-to lower the amount of CA IX in hypoxic cancer cells, thus limiting its action in survival and the metastatic spread of tumors.