Scouting new molecular targets for CFTR therapy: the HSC70/BAG-1 complex. A computational study
- First Online:
- Cite this article as:
- Cichero, E., Basile, A., Turco, M.C. et al. Med Chem Res (2012) 21: 4430. doi:10.1007/s00044-012-9985-1
- 146 Views
HSC70 has been identified as an important molecular target involved in the ΔF508-CFTR cystic fibrosis. HSC70 associates ΔF508-CFTR to a much greater extent than WT-CFTR and after this step, it recruits other co-chaperones (BAG1, CHIP) and performs the ubiquitination and proteosomal degradation of the protein. Up to now, several X-ray data concerning the HSC70:BAG1 complexes are available. Thus, we performed an “in silico” investigation focused to explore which different amino acid residues are involved in the binding of ATP, the natural substrate, and the co-crystallized ligands at the HSC70/BAG-1 interface. The study allowed us to evaluate sildenafil and KM11060, which proved to be also CFTR correctors, as potential HSC70:BAG1 inhibitors, and also let us derive interesting perspectives for the development of new CFTR correctors.
Cystic fibrosis (CF) is a multi-organ genetic disorder caused by the loss of function of the CF transmembrane conductance regulator (CFTR) (Riordan, 2008). CF is the most common autosomal recessive disease in people of European ancestry, with more than 1,700 different mutations identified in the CFTR gene. The CFTR protein is expressed in epithelial cells and is a member of the ATP-binding cassette (ABC) transporter superfamily, which also includes the multidrug-resistance protein. CFTR consists of two membrane-spanning domains, two nucleotide-binding domains (NBDs), and a regulatory domain, which controls channel activity. CFTR is a chloride channel and regulator of other transporters (Quinton, 1983; Quinton and Bijman, 1983).
Loss of function of CFTR results in viscous secretions of the exocrine glands in multiple organ systems and elevated sweat chloride levels. The most prevalent symptoms develop in the gastrointestinal and respiratory tracts. While administration of pancreatic enzyme supplements has significantly reduced the gastrointestinal complications and extended the lifespan of patients to more than 35 years, the chronic bacterial airway infections lead to respiratory failure. At present, the respiratory complications are responsible for the majority of mortality associated with this disease. In spite of the fact that symptomatic combination therapies have been employed, including antibiotic, anti-inflammatory, and mucolytic medications, the lung function often declines to less than 30%, and transplantation remains the last treatment option. Since the discovery of the CFTR gene in 1989 (Riordan et al., 1989), a major effort has been directed to develop causative therapies for CF. These include gene-replacement strategies, based on both viral and non-viral vectors (Griesenbach and Alton, 2009), and a number of alternative approaches (Amaral and Kunzelmann, 2007) among which potentiators, compounds that stimulate pre-activated CFTR channel activity and correctors, agents that rescue cell-surface expression of mutated/defective CFTR, in particular ΔF508-CFTR, the most abundant protein mutation. However, despite these efforts, the current arsenal of CFTR “drugs” is limited, with only three compounds reaching the clinical trials and none on the market.
More recently, increasing attention has been devoted to those molecular chaperones which assist the protein folding processes within the cell and, among these, HSC70 has been identified as an important molecular target. HSC70, in fact, associates ΔF508-CFTR to a much greater extent than WT-CFTR and after this step, it recruits other co-chaperones (BAG1, CHIP) and performs the ubiquitination and proteosomal degradation of the protein (Alberti et al., 2004). Interestingly, the X-rays of HSC70 in complex with BAG-1 and ATP, or small inhibitors are available on PDB (PDB code: 3FZF and 3LDQ, respectively) (Williamson et al., 2009; Macias et al., 2011).
On these basis, we found it could be interesting to perform an “in silico” investigation focused to explore which different amino acid residues are involved in the binding of ATP, the natural substrate, and the co-crystallized ligands at the HSC70/BAG-1 interface, so as to define which interactions are specific for switching the activation and which ones are instead specific for switching the inhibition of the chaperone-co-chaperone assembly. This information could in fact represent the first feature able to discriminate between the two groups of substances and could subsequently be useful to select from large databases new potential CFTR agents to be preliminary tested in vitro.
Notably, these compounds are well-known type 5 phosphodiesterase inhibitors, wherein the pyrazolo pyrimidine scaffold is able to resemble the cAMP behavior, by properly fitting the enzyme catalytic site. Similarly, it could be probable that the same compounds could act as HSC70:BAG1 ligands, occupying the protein ATP domain.
The obtained results, being in agreement with the experimental data, allowed us to reasonably hypothesize (for the examined compounds) a binding mode able to suggest interesting perspectives for expedite the development of new CFTR correctors.
Materials and methods
For our studies, compounds I and II (Fig. 1) were selected as well-known HSC70:BAG1 inhibitors, while sildenafil and KM11060 (Fig. 2) were chosen since they have proved to be the most promising CFTR correctors, among the PDE5 inhibitors.
All the compounds were built, parameterised (Gasteiger–Huckel method) and energy minimized within MOE using MMFF94 force field (MOE: Chemical Computing Group Inc. Montreal. H3A 2R7 Canada. http://www.chemcomp.com).
The I and II sulphonic groups were considered in the undissociated (Iu and IIu) and in the dissociated (Id and IId) acid forms.
Successively docking studies were performed, using the HSC70:BAG1 complexes. The X-ray coordinates of HSC70 in complex with BAG-1 and ATP (PDB code: 3FZF) and those of HSC70 in complex with BAG-1 and a small inhibitor (PDB code: 2LDQ) were downloaded by the PDB protein data bank.
Each compound was docked into the HSC70:BAG1 assembly, by means of the Surflex docking module implemented in Sybyl-X 1.0 (Sybyl-X 1.0., Tripos Inc 1699 South Hanley Road, St Louis, Missouri, 63144, USA). Then, for all the compounds, the best docking geometries (selected on the basis of the SurFlex scoring functions) were refined by ligand–receptor complex energy minimisation (CHARMM27), by means of the MOE software. In order to verify the reliability of the derived docking poses, the obtained protein/ligand complexes were further investigated by docking calculations (10 run), using MOE-Dock (Genetic algorithm; applied on the poses already located into the protein-binding site). The conformers showing lower energy scoring functions and RMSD values (respect to the starting poses) were selected as the most stable.
Results and discussion
The ATP-binding mode into the HSC70:BAG1 assembly
The binding mode of a small inhibitor into the HSC70:BAG1 assembly
A comparison between HSC70:BAG1 in complex with ATP and in complex with the small inhibitor
Taking into account all the experimental data previously discussed, the agonist behavior seems to be determined by several hydrophilic contacts with the P1 region, which is unoccupied by the compound showing inhibitory activity. Notably, the agonist and the inhibitor share H-bonds with Ser275, Glu268, and Lys271 residues, suggesting that the three amino acids could become an interesting starting point to design new HSC70:BAG1 ligands.
Docking studies on compounds I and II
With the aim of optimizing the compound II scaffold, we here propose some structural modifications which could be made.
Interestingly, the introduction of a flexible linker between the ring bearing the sulphonic group and the subsequent one, could be beneficial for the inhibitory activity, by enhancing the formation of cation–π contacts with Arg342. Furthermore, the substitution of the two quaternary nitrogen atoms with an H-bond acceptor function could allow the establishment of H-bonds with the key residue Arg272.
Docking studies on sildenafil and KM11060
Accordingly, KM11060 proves to have greater corrector activity than sildenafil.
On the basis of these data, the introduction of an indole ring between the quinoline one and the sulfonyl group (instead of a piperazine), could be useful to properly occupy the protein-binding pocket. On the other hand, the quinoline ring could be substituted by a flexible linker, with an H-bond acceptor function, which could be engaged in H-bond with the key residue Arg272.
Key interactions observed in the derived HSC70:BAG1/ligand complexes
Thr14 (3.02 Å), Tyr15 (2.95 Å), Gly339 (3.39 Å),
Glu268 (2.33 Å), Lys271 (2.93 Å), Ser275 (2.51 Å)
2LDQ small inhibitor
Glu268 (2.73 Å), Lys271 (3.37 Å), Ser275 (2.68 Å)
Glu268 (2.82 Å)
Asp366 (2.78 Å),
Glu231 (2.83 Å), Ser275 (2.75 Å)
Asp366 (2.85 Å)
Arg272 (2.75 Å), Glu268 (3.02 Å)
Lys271 (2.73 Å), Ser340 (2.84 Å)
Lys271 (3.06 Å),
The docking studies here presented highlight the key structural features impacting the behavior of compounds which act as HSC70:BAG1 agonists or inhibitors. In particular, the agonist behavior seems to be determined by several hydrophilic contacts with the P1 region, which is unoccupied by the 2LDQ small inhibitor. On the other hand, the agonist and the inhibitor share H-bonds with the Ser275, Glu268, and Lys271 residues, suggesting that these three amino acids could become an interesting starting point to design new HSC70:BAG1 ligands. Accordingly, on the basis of our docking studies, the NSC71948 mixture (I and II) which acts as HSC70:BAG1 inhibitor, interacts with (at least) one of the three key residues previously described.
Furthermore, the derived key interactions have been employed to evaluate two PDE5 inhibitors, sildenafil and KM11060, which proved to be also CFTR correctors. The results obtained allowed us to reasonably hypothesize the binding mode of the two compounds onto the HSC70:BAG1 complex, and also to derive interesting perspectives for the development of new CFTR correctors.
This research was supported by Italian Cystic Fibrosis Foundation (Grant FFC#5/2010), with the contribution of Philip Watch–Morellato & Sector Group. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. E.C. was financially supported by a post-doc fellowship, Area Chimica, University of Genova.