Deciphering the mode of action of clinically relevant next generation c2 corrector compounds GLPG2737 and GLPG3221

Abstract

The current therapeutic strategy to repair cystic fibrosis-causing defects in the chloride channel CFTR is to develop novel and better correctors (to improve folding) and potentiators (to improve function). Galapagos- AbbVie identified C2 correctors by high-throughput compound screening and Med Chem optimization for cell surface rescue of F508del-CFTR. These C2 correctors are acting synergistically with a type I corrector such as ABBV/GLPG2222. Two C2 correctors, ABBV/GLPG2737 and ABBV/ GLPG3221 were optimized for drug like properties and are in clinical and pre-clinical evaluation, respectively. From both the functional halide efflux assays and pulse chase analysis we showed that the rescue efficiency of F508del-CFTR after combination treatment (C1 + C2) is markedly higher (≥50% of wild-type levels) than the sum of C1 and C2 correction. These strong synergistic effects show not only that C1 and C2 have a different mode of action, but also highlight the benefit of the triple-combination treatment with addition of a potentiator. To investigate how, when and where these C2 correctors act on CFTR we use radiolabeling approaches in combination with protease susceptibility assays. We first evaluated C1 corrector ABBV/GLPG2222 using in vitro translation and translocation assays in the presence of semi-intact HEK293 cells as source for endoplasmic reticulum (ER) membranes. We found that ABBV/GLPG2222, but not the C2 correctors, acted on transmembrane domain 1 (TMD1) in an identical fashion as lumacaftor by promoting its cytoplasmic loop packing important for domain folding. Varying the time of drug addition in pulse chase experiments showed that, like C1 corrector, both C2 correctors reached maximal rescue efficiency when present during, and shortly after the 15-minute pulse labelling. The C2 correctors acted additively with all F508del suppressors (I539T, G550E and R1070W) and did not restore nucleotide binding domain 1 (NBD1) folding in the F508del-CFTR background. Although we did not identify yet where the C2 correctors act, these compounds restored trafficking of the NBD2-less F508del-CFTR (F508del-1219X) construct very well. Our results show that the C2 correctors promote the earliest folding events of the ER-export competent CFTR molecule lacking NBD2, ruling out all possible NBD2 inter-domain assembly events (TMD1/NBD2; NBD1/NBD2; TMD2/NBD2) as target candidate. The triple-combination treatment that includes these C2 correctors significantly raises the F508del-CFTR rescue ceiling, with the aim to reach sufficient clinical benefit for most CF patients in the near future.