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- Title
Alchemical Design of Pharmacological Chaperones with Higher Affinity for Phenylalanine Hydroxylase.
- Authors
Conde-Giménez, María; Galano-Frutos, Juan José; Galiana-Cameo, María; Mahía, Alejandro; Victor, Bruno L.; Salillas, Sandra; Velázquez-Campoy, Adrián; Brito, Rui M. M.; Gálvez, José Antonio; Díaz-de-Villegas, María D.; Sancho, Javier
- Abstract
Phenylketonuria (PKU) is a rare metabolic disease caused by variations in a human gene, PAH, encoding phenylalanine hydroxylase (PAH), and the enzyme converting the essential amino acid phenylalanine into tyrosine. Many PKU-causing variations compromise the conformational stability of the encoded enzyme, decreasing or abolishing its catalytic activity, and leading to an elevated concentration of phenylalanine in the blood, which is neurotoxic. Several therapeutic approaches have been developed to treat the more severe manifestations of the disorder, but they are either not entirely effective or difficult to adhere to throughout life. In a search for novel pharmacological chaperones to treat PKU, a lead compound was discovered (compound IV) that exhibited promising in vitro and in vivo chaperoning activity on PAH. The structure of the PAH-IV complex has been reported. Here, using alchemical free energy calculations (AFEC) on the structure of the PAH-IV complex, we design a new generation of compound IV-analogues with a higher affinity for the enzyme. Seventeen novel analogues were synthesized, and thermal shift and isothermal titration calorimetry (ITC) assays were performed to experimentally evaluate their stabilizing effect and their affinity for the enzyme. Most of the new derivatives bind to PAH tighter than lead compound IV and induce a greater thermostabilization of the enzyme upon binding. Importantly, the correspondence between the calculated alchemical binding free energies and the experimentally determined ΔΔGb values is excellent, which supports the use of AFEC to design pharmacological chaperones to treat PKU using the X-ray structure of their complexes with the target PAH enzyme.
- Subjects
ESSENTIAL amino acids; PHENYLALANINE; ENZYME stability; ISOTHERMAL titration calorimetry; BINDING energy; MOLECULAR chaperones
- Publication
International Journal of Molecular Sciences, 2022, Vol 23, Issue 9, p4502
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms23094502