NF023 binding to XIAP‐BIR1: Searching drugs for regulation of the NF‐κB pathway

Abstract

Inhibitor of Apoptosis Proteins (IAPs) are the target of extensive research in the field of cancer therapy since they regulate apoptosis and cell survival. Smac-mimetics, the most promising IAP-targeting compounds specifically recognize the IAP-BIR3 domain and promote apoptosis, competing with caspases for IAP binding. Furthermore, Smac-mimetics interfere with the NF-κB survival pathway, inducing cIAP1 and cIAP2 degradation through an auto-ubiquitination process. It has been shown that the XIAP-BIR1 (X-BIR1) domain is involved in the interaction with TAB1, an upstream adaptor for TAK1 kinase activation, which in turn couples with the NF-κB survival pathway. Preventing X-BIR1 dimerization abolishes XIAP-mediated NF-κB activation, thus implicating a proximity-induced mechanism for TAK1 activation. In this context, in a systematic search for a molecule capable of impairing X-BIR1/TAB1 assembly, we identified the compound NF023. Here we report the crystal structure of the human X-BIR1 domain in the absence and in the presence of NF023, as a starting concept for the design of novel BIR1-specific compounds acting synergistically with existing pro-apoptotic drugs in cancer therapy.

Structure‐Activity Relationship of NF023 Derivatives Binding to XIAP‐BIR1

“…The published crystallographic data of XIAP‐BIR1 in complex with NF023 (PDB: 4MTZ) show a binding mode that is not congruent with the interference on the protein dimerization observed in solution . With the aim to select a new potential NF023 binding site in solution, we identified, through in silico docking, a favored conformation of NF023 along XIAP‐BIR1 dimerization surface (Figure A; docking energy of −9.13 kcal mol −1 ) . Such conformation allowed us to select a set of BIR1 mutants likely able to impair NF023 binding: R62S, D71A, R82S and V86E (known to prevent XIAP‐BIR1 dimerization) (Figure B).…”

“…Increasing concentrations of NF023 were used to titrate wild type XIAP‐BIR1 (100 nM) yielding an estimated dissociation constant ( K d ) of 25±5 μM. This value is consistent with that previously reported, indicating that protein labelling does not interfere with NF023 binding. Analogous experiments were therefore performed on all the mutant variants of XIAP‐BIR1 (Table ).…”

“…The binding mode of 5 a with the BIR1 domain is almost identical to that observed for NF023 (Figure ) with 5 a sitting above the association interface of the dimer and establishing nearly symmetrical interactions with both subunits.…”

“…Inhibition of XIAP‐BIR1 dimerization and the consequent assembly of the XIAP‐BIR1/TAB1 complex could in fact represent a novel strategy for the development of anti‐cancer drugs. In a previous work we identified NF023 as a promising XIAP‐BIR1 inhibitor . Here, the analysis of the interaction between NF023 and four XIAP‐BIR1 mutants (i. e. R62S, D71A, R82S and V86E) demonstrates that NF023 can bind to the domain in an additional site beyond the one observed in the crystal structure.…”

“…Here, the analysis of the interaction between NF023 and four XIAP‐BIR1 mutants (i. e. R62S, D71A, R82S and V86E) demonstrates that NF023 can bind to the domain in an additional site beyond the one observed in the crystal structure. The new binding site explains the perturbative effect of NF023 on the equilibrium between monomeric and dimeric forms of the protein in solution …”