RESUMEN
Zinc fingers have rarely been regarded as drug targets. On the contrary, the zinc-binding site of enzymes has often been considered a target of inhibitors. We previously developed a dithiol compound called SN-1 that binds to the zinc finger protein tumor necrosis factor receptor-associated factor 6 (TRAF6) and suppresses downstream nuclear factor-κB (NF-κB) signaling. To determine the minimal structure requirements of TRAF6 inhibitors based on SN-1, NF-κB inhibitory activity and cytotoxicity of its derivatives including new compounds were examined. SN-2, an oxidative type of prodrug of SN-1 with 2-nitrophenylthio groups via disulfide, has the minimum structure for an inhibitor of TRAF6, as seen with cellular experiments. The importance of two side chains with a thiol group was shown with molecular modelling. This study may lead to development of selective TRAF6 inhibitors in the near future.
Asunto(s)
Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Dedos de Zinc/genética , Humanos , Estructura MolecularRESUMEN
Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, A3G), is a human anti-virus restriction protein which works deaminase-dependently and -independently. A3G is known to be ubiquitinated by HIV-1 viral infectivity factor (Vif) protein, leading to proteasomal degradation. A3G contains two zinc ions at the N-terminal domain and the C-terminal domain. Four lysine residues, K(297), K(301), K(303), and K(334), are known to be required for Vif-mediated A3G ubiquitination and degradation. Previously, we reported compound SN-1, a zinc chelator that increases steady-state expression level of A3G in the presence of Vif. In this study, we prepared Biotin-SN-1, a biotinylated derivative of SN-1, to study the SN-1-A3G interaction. A pull-down assay revealed that Biotin-SN-1 bound A3G. A zinc-abstraction experiment indicated that SN-1 binds to the zinc site of A3G. We carried out a SN-1-A3G docking study using molecular operating environment. The calculations revealed that SN-1 binds to the C-terminal domain through Zn(2+), H(216), P(247), C(288), and Y(315). Notably, SN-1-binding covers the H(257), E(259), C(288), and C(291) residues that participate in zinc-mediated deamination, and the ubiquitination regions of A3G. The binding of SN-1 presumably perturbs the secondary structure between C(288) and Y(315), leading to less efficient ubiquitination.
Asunto(s)
Desaminasa APOBEC-3G/química , Antivirales/farmacología , Zinc/química , Zinc/farmacología , Antivirales/química , Células HEK293 , Humanos , Modelos Moleculares , Peso MolecularRESUMEN
Despite various inhibitors targeting the zinc center(s) of enzymes, drugs that target zinc fingers have not been examined in detail. We previously developed a dithiol compound named SN-1 that has an inhibitory effect on the function of zinc finger transcription factors, but its mechanism of action has not yet been elucidated. To establish a general principle for new drugs, the details of the action of SN-1 against a zinc finger protein were examined. As a zinc-finger-containing protein, we focused on TRAF6, which is related to cancer and inflammation. Binding of SN-1 to TRAF6 and its effect on TRAF6 ubiquitination were examined inâ vitro, and the binding mode was calculated by computational methodology. Furthermore, ubiquitination of TRAF6 and downstream signaling was examined by cell-based experiments. The results show that SN-1 binds to TRAF6, inhibiting its auto-ubiquitination and downstream NF-κB signaling. Docking studies indicate that SN-1 binds directly to the first zinc finger of TRAF6. This binding disrupts the neighboring structure, that is, the RING finger domain, to suppress the ubiquitin ligase activity of TRAF6. Taken together, this study provides a platform for developing new small molecules that target zinc finger proteins.
Asunto(s)
Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Tolueno/análogos & derivados , Sitios de Unión/efectos de los fármacos , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Simulación del Acoplamiento Molecular , Estructura Molecular , Factor 6 Asociado a Receptor de TNF/química , Factor 6 Asociado a Receptor de TNF/metabolismo , Tolueno/química , Tolueno/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
Human apoplipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) 3G (A3G) is an antiviral protein that blocks HIV-1 replication. However, the antiviral activity of A3G is overcome by the HIV-1 protein Vif. This inhibitory function of Vif is related to its ability to degrade A3G in the proteasome. This finding prompted us to examine the activities of 4-(dimethylamino)-2,6-bis[(N-(2-[(2-nitrophenyl)dithio]ethyl)amino)methyl]pyridine (SN-2) and SN-3. We found that 5 µM SN-2 increases the expression of A3G to a level much higher than that observed in the absence of Vif, without affecting the level of Vif expression. The proteasome inhibitor MG-132 increased the level of both A3G and Vif expression. These results demonstrate that A3G is ubiquitinated and degraded in the proteasome by a factor other than Vif, and that SN-2 selectively inhibits these processes. Furthermore, 5 µM SN-2 significantly inhibited the MAGI cell infectivity of wild-type HIV-1. These findings may contribute to the development of a novel anti-HIV-1 drug.