Virtual Drug Repositioning as a Tool to Identify Natural Small Molecules That Synergize with Lumacaftor in F508del-CFTR Binding and Rescuing

Abstract

Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.

Keywords: F508del-CFTR; cystic fibrosis; drug repositioning; lumacaftor; molecular docking and dynamics; nicotinamide; surface plasmon resonance.

Figure 1

Schematic representation of the drug repositioning pipeline adopted in this work. Our previously published pipeline [25] was implemented to adapt it for the searching of small molecules able to combine with lumacaftor inside the DP1.

Figure 2

Docking pose of NAM in combination with the F508del-CFTR-lumacaftor structure. In blue is the NBD1 domain and in red is the ICL4.

Figure 3

RMSD analysis of F508del-CFTR four domains (A), and NAM and lumacaftor (B) for the three replicas.

Figure 4

Binding pose of NAM and lumacaftor in the representative conformations of the three replicas. Conformation 1 of replica 1 in teal, conformation 1 of replica 2 in coral, conformation 2 of replica 2 in brown, and conformation 1 of replica 3 in yellow.

Figure 5

NAM binding pose in the F508del-CFTR-lumacaftor-NAM complex. The H-bond is shown in yellow dotted lines. Residues involved in hydrophobic interactions with NAM are depicted with their hydrophobic surfaces.

Figure 6

Steady-state analysis of NAM injected onto the F508del-CFTR-containing biosensor. The results shown are representative of other seven analyses that gave similar results.

Figure 7

Binding pose of NAM in the ICL2:NBD2 interface when it binds to the apo F508del-CFTR. H-bond in yellow.

Figure 8

Effect of NAM-lumacaftor co-treatment on mutant F508del-CFTR rescue. The bar graphs show F508del-CFTR activity in CFBE41o-cells stably expressing the HS-YFP. CFTR activity was determined as a function of the YFP quenching rate following iodide influx in cells treated for 24 h with DMSO in the absence (vehicle) or in the presence of lumacaftor (3.0 µM) as a single agent or combined with the indicated concentrations of NAM. * p < 0.05.