{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T08:42:39Z","timestamp":1776328959713,"version":"3.50.1"},"reference-count":55,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2022,4,19]],"date-time":"2022-04-19T00:00:00Z","timestamp":1650326400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003329","name":"Ministry of Economy, Industry and Competitiveness","doi-asserted-by":"publisher","award":["BFU2016-78232-P"],"award-info":[{"award-number":["BFU2016-78232-P"]}],"id":[{"id":"10.13039\/501100003329","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003329","name":"Ministry of Economy, Industry and Competitiveness","doi-asserted-by":"publisher","award":["PID2019-107293GB-I00"],"award-info":[{"award-number":["PID2019-107293GB-I00"]}],"id":[{"id":"10.13039\/501100003329","id-type":"DOI","asserted-by":"publisher"}]},{"name":"European Union","award":["Interreg-SUDOE (NEUROMED)"],"award-info":[{"award-number":["Interreg-SUDOE (NEUROMED)"]}]},{"DOI":"10.13039\/501100011100","name":"Spanish Foundation for Science and Technology","doi-asserted-by":"publisher","award":["Precipita"],"award-info":[{"award-number":["Precipita"]}],"id":[{"id":"10.13039\/501100011100","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Gobierno de Arag\u00f3n","award":["LMP30_18"],"award-info":[{"award-number":["LMP30_18"]}]},{"name":"Gobierno de Arag\u00f3n","award":["E45_20R"],"award-info":[{"award-number":["E45_20R"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 \u0394\u0394Gb 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.<\/jats:p>","DOI":"10.3390\/ijms23094502","type":"journal-article","created":{"date-parts":[[2022,4,19]],"date-time":"2022-04-19T22:07:26Z","timestamp":1650406046000},"page":"4502","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Alchemical Design of Pharmacological Chaperones with Higher Affinity for Phenylalanine Hydroxylase"],"prefix":"10.3390","volume":"23","author":[{"given":"Mar\u00eda","family":"Conde-Gim\u00e9nez","sequence":"first","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"},{"name":"Biocomputation and Complex Systems Physics Institute (BIFI)-GBsC-CSIC Joint Unit, Universidad de Zaragoza, 50018 Zaragoza, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1896-7805","authenticated-orcid":false,"given":"Juan Jos\u00e9","family":"Galano-Frutos","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"},{"name":"Biocomputation and Complex Systems Physics Institute (BIFI)-GBsC-CSIC Joint Unit, Universidad de Zaragoza, 50018 Zaragoza, Spain"}]},{"given":"Mar\u00eda","family":"Galiana-Cameo","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"},{"name":"Biocomputation and Complex Systems Physics Institute (BIFI)-GBsC-CSIC Joint Unit, Universidad de Zaragoza, 50018 Zaragoza, Spain"}]},{"given":"Alejandro","family":"Mah\u00eda","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"},{"name":"Biocomputation and Complex Systems Physics Institute (BIFI)-GBsC-CSIC Joint Unit, Universidad de Zaragoza, 50018 Zaragoza, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6472-5700","authenticated-orcid":false,"given":"Bruno L.","family":"Victor","sequence":"additional","affiliation":[{"name":"Coimbra Chemistry Center-Institute of Molecular Sciences (CQC-IMS), Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0195-5434","authenticated-orcid":false,"given":"Sandra","family":"Salillas","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"},{"name":"Biocomputation and Complex Systems Physics Institute (BIFI)-GBsC-CSIC Joint Unit, Universidad de Zaragoza, 50018 Zaragoza, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5702-4538","authenticated-orcid":false,"given":"Adri\u00e1n","family":"Vel\u00e1zquez-Campoy","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"},{"name":"Biocomputation and Complex Systems Physics Institute (BIFI)-GBsC-CSIC Joint Unit, Universidad de Zaragoza, 50018 Zaragoza, Spain"},{"name":"Aragon Health Research Institute (IIS Arag\u00f3n), 50009 Zaragoza, Spain"},{"name":"CIBER de Enfermedades Hep\u00e1ticas y Digestivas CIBERehd, Instituto de Salud Carlos III, 28029 Madrid, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9128-2557","authenticated-orcid":false,"given":"Rui M. M.","family":"Brito","sequence":"additional","affiliation":[{"name":"Coimbra Chemistry Center-Institute of Molecular Sciences (CQC-IMS), Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4972-7476","authenticated-orcid":false,"given":"Jos\u00e9 Antonio","family":"G\u00e1lvez","sequence":"additional","affiliation":[{"name":"Instituto de S\u00edntesis Qu\u00edmica y Cat\u00e1lisis Homog\u00e9nea (ISQCH), Departamento de Qu\u00edmica Org\u00e1nica, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9033-8459","authenticated-orcid":false,"given":"Mar\u00eda D.","family":"D\u00edaz-de-Villegas","sequence":"additional","affiliation":[{"name":"Instituto de S\u00edntesis Qu\u00edmica y Cat\u00e1lisis Homog\u00e9nea (ISQCH), Departamento de Qu\u00edmica Org\u00e1nica, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2879-9200","authenticated-orcid":false,"given":"Javier","family":"Sancho","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"},{"name":"Biocomputation and Complex Systems Physics Institute (BIFI)-GBsC-CSIC Joint Unit, Universidad de Zaragoza, 50018 Zaragoza, Spain"},{"name":"Aragon Health Research Institute (IIS Arag\u00f3n), 50009 Zaragoza, Spain"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,19]]},"reference":[{"key":"ref_1","unstructured":"Scriver, C.R., Beaudet, A.L., and Slyetal, W.S. 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