GRP78 is a novel target of AZM
To clarify the molecular basis underlying how AZM imposed its effects on RA FLSs, we aimed to isolate protein binding partners of AZM. By implementing DARTS assay and mass spectrometry, glucose-regulated protein 78 (GRP78) was identified as potential candidates (Fig. 4A). Then, we performed Western blot of DARTS samples with which a series of protease to cell lysate ratios were implemented, and found that AZM protected GRP78 (Fig. 4B). Subsequently, we conducted CETSA, which allows for quantification of the change in thermal denaturation temperature of a target protein under different conditions, including those of varying temperature and concentrations of drug (X. Wang et al., 2013). AZM increased the resistance of GRP78 in RA FLSs to denaturation caused by heating from 52 °C to 58 °C compared with DMSO (Fig. 4C). Furthermore, AZM increased stability of GRP78 in a dose-dependent manner (Fig. 4D).
In order to identify the domain by which AZM targets to GRP78, recombinant molecules were produced and the DARTS assay revealed that AZM retained protective effects on mutant GRP78 lacking peptide-binding region while showed no protective effects on mutant lacking ATPase domain (Fig. 4E). Then, molecular docking of AZM towards GRP78 was carried out using DOCK6 (Hu et al., 2012) and their potential interacting sites were presented in Fig. 4F. AZM showed a salt-bridge interaction with residue Asp178 of the GRP78 protein. It also formed H-bond interactions with residue Arg181, Ile199, Val241 and Gln401. Importantly, DARTS assay clearly demonstrated that AZM lost its protective effect upon substituting Asp178 of GRP78 into Ala (Fig. 4G), demonstrating that Asp178 is the critical amino acid required for AZM targeting to GRP78.
Since the ATPase domain of GRP78 is required for its activity upon binding with ATP (Galligan et al., 2014), AZM might competitively bind GRP78 with ATP. As expected, the enzyme activity of GRP78 was competitively inhibited by AZM in the presence of ATP (Fig. 4H). HA15 is able to inhibit the BiP activity via directly suppressing its ATPase activity (Cerezo et al., 2016). Importantly, AZM could inhibit GRP78 activity as efficiently as HA15 (Fig. 4I). Thus, GRP78 serves as a novel substrate for AZM in RA FLSs.