Molecular dynamics unmasked two binding pockets (BPs) in NBD1. BP1 is smaller in wild type (WT) than in DF508-CFTR, due to two loops (drug site 1 and 2) making contact and closing the BPs. In DF508-NBD1, all the ligands tested bind BP1, VX809 and FCG in drug site 1, EN371B, EN277I and EN371A in drug site 2. VX809 binding to DF508- NBD1 is stable and samples the same space of the WT, indicating a conformational recovery. FCG association is unstable, indicating a different binding site. EN371A is inactive. SPR validated molecular modelling. When the compounds were assayed for their capacity to affect the DF508-NBD1/K8 interaction, EN277I was ineffective while EN371B prevented the interaction, indicating a competition with K8 for the same BP. SPR analysis validates and complements in silico predictions sustaining the integration of bioinformatics and SPR as effective in speeding up the discovery of new CFTR-targeted drugs.