Innovative Strategy toward Mutant CFTR Rescue in Cystic Fibrosis: Design and Synthesis of Thiadiazole Inhibitors of the E3 Ligase RNF5

Abstract

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CF transmembrane conductance regulator (CFTR) is associated to misfolding and defective gating of the mutant channel. One of the most promising CF drug targets is the ubiquitin ligase RNF5, which promotes F508del-CFTR degradation. Recently, the first ever reported inhibitor of RNF5 was discovered, i.e., the 1,2,4-thiadiazol-5-ylidene inh-2. Here, we designed and synthesized a series of new analogues to explore the structure−activity relationships (SAR) of this class of compounds. SAR efforts ultimately led to compound 16, which showed a greater F508del-CFTR corrector activity than inh-2, good tolerability, and no toxic side effects. Analogue 16 increased the basal level of autophagy similar to what has been described with RNF5 silencing. Furthermore, co-treatment with 16 significantly improved the F508del-CFTR rescue induced by the triple combination elexacaftor/tezacaftor/ivacaftor in CFBE41o − cells. These findings validate the 1,2,4-thiadiazolylidene scaffold for the discovery of novel RNF5 inhibitors and provide evidence to pursue this unprecedented strategy for the treatment of CF.

Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states

“…Our functional genomics platform permitted rapid, quantitative gene-drug interaction experiments that define the relationship between ERAD and corrector drugs, confirming structural models (Fiedorczuk and Chen, 2022b) and biochemical insights (Tomati et al, 2015;Sondo et al, 2018;Brusa et al, 2023). Our data support a model in which the equilibrium between CFTR-F508del folding intermediates in the ER is shifted away from ERAD susceptibility by binding to correctors.…”

“…Despite these remarkable advances in our understanding of CFTR structure, folding, and correction, our understanding of the mechanism by which CFTR-F508del is triaged and degraded by ERAD remains rudimentary because of the lack of a systematic, genome-wide investigation. Since 2001, at least seven ubiquitin E3 ligases, CHIP/STUB1 (Meacham et al, 2001), FBXO2 (Ramachandran et al, 2016), NEDD4L (Caohuy et al, 2009), RNF185 (Khouri et al, 2013), GP78/AMFR (Ballar et al, 2010), RMA1/RNF5 (Younger et al, 2006;Tomati et al, 2015;Sondo et al, 2018;Brusa et al, 2023), and HRD1 (Ballar et al, 2010;Ramachandran et al, 2016) have been implicated in CFTR-F508del degradation, yet the relative contribution of each to CFTR-F508del degradation is unknown. Because disruption of CFTR-F508del ERAD can synergize with and/or complement pharmacological correction (Sondo et al, 2018;Borgo et al, 2022;Brusa et al, 2022Brusa et al, , 2023, a detailed and comprehensive mechanistic understanding of CFTR ERAD is warranted.…”

RNF5: inhibiting antiviral immunity and shaping virus life cycle

“…The original discovery of FX12, a small molecule that acts as both an inhibitor and degrader of the RNF5 ubiquitin ligase, provides a novel strategy for RNF5 inhibition. Rather than solely suppressing RNF5 E3 activity, FX12 directly binds to RNF5 and hijacks ERAD to initiate degradation of RNF5, possibly by altering RNF5's normal structure to be recognized as a misfolded ER protein (75). However, while these studies have focused on the potential use of RNF5 inhibitors in the treatment of cystic fibrosis, limited research to date has explored their application in antiviral therapy (63).…”

Small-molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states

“…, 2018 ; Borgo et al. , 2022 ; Brusa et al. , 2023 ), a detailed and comprehensive mechanistic understanding of CFTR ERAD is warranted.…”