{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,4]],"date-time":"2026-10-04T05:53:56Z","timestamp":1791093236410,"version":"4.1.0"},"reference-count":56,"publisher":"Oxford University Press (OUP)","issue":"11","license":[{"start":{"date-parts":[[2004,11,1]],"date-time":"2004-11-01T00:00:00Z","timestamp":1099267200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2004,11,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalities. Here, we used mutagenesis, kinetic analysis, and computer modeling to identify the residues important for the binding of known ALP inhibitors to the TNALP active site. These data will enable drug design efforts aimed at developing improved specific TNALP inhibitors for therapeutic use.<\/jats:p>\n               <jats:p>Introduction: We have shown previously that the genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) function leads to amelioration of soft-tissue ossification in mouse models of osteoarthritis and ankylosis (i.e., Enpp1\u2212\/\u2212 and ank\/ank mutant mice). We surmise that the pharmacologic inhibition of TNALP activity represents a viable therapeutic approach for these diseases. As a first step toward developing suitable TNALP therapeutics, we have now clarified the residues involved in binding well-known uncompetitive inhibitors to the TNALP active site.<\/jats:p>\n               <jats:p>Materials and Methods: We compared the modeled 3D structure of TNALP with the 3D structure of human placental alkaline phosphatase (PLALP) and identified the residues that differ between these isozymes within a 12 \u00c5 radius of the active site, because these isozymes differ significantly in inhibitor specificity. We then used site-directed mutagenesis to substitute TNALP residues to their respective homolog in PLALP. In addition, we mutagenized most of these residues in TNALP to Ala and the corresponding residues in PLALP to their TNALP homolog. All mutants were characterized for their sensitivity toward the uncompetitive inhibitors l-homoarginine (L-hArg), levamisole, theophylline, and L-phenylalanine.<\/jats:p>\n               <jats:p>Results and Conclusions: We found that the identity of residue 108 in TNALP largely determines the specificity of inhibition by L-hArg. The conserved Tyr-371 is also necessary for binding of L-hArg. In contrast, the binding of levamisole to TNALP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. The main determinant of sensitivity to theophylline is His-434. Thus, we have clarified the location of the binding sites for all three TNALP inhibitors, and we have also been able to exchange inhibitor specificities between TNALP and PLALP. These data will enable drug design efforts aimed at developing improved, selective, and drug-like TNALP inhibitors for therapeutic use.<\/jats:p>","DOI":"10.1359\/jbmr.040608","type":"journal-article","created":{"date-parts":[[2006,4,26]],"date-time":"2006-04-26T23:27:15Z","timestamp":1146094035000},"page":"1862-1872","source":"Crossref","is-referenced-by-count":86,"title":["Residues Determining the Binding Specificity of Uncompetitive Inhibitors to Tissue-Nonspecific Alkaline Phosphatase"],"prefix":"10.1093","volume":"19","author":[{"given":"Alexey","family":"Kozlenkov","sequence":"first","affiliation":[{"name":"Department of Medical Biosciences, Medical Genetics, Ume\u00e5 University, Ume\u00e5, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marie Helene","family":"Le Du","sequence":"additional","affiliation":[{"name":"Department d'Ingenierie et d'Etude des Proteines, Gif-sur-Yvette, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philippe","family":"Cuniasse","sequence":"additional","affiliation":[{"name":"Department d'Ingenierie et d'Etude des Proteines, Gif-sur-Yvette, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tor","family":"Ny","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry and Biophysics, Ume\u00e5 University, Ume\u00e5, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc F","family":"Hoylaerts","sequence":"additional","affiliation":[{"name":"Center for Molecular and Vascular Biology, University of Leuven\u2014Campus Gasthuisberg, Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9 Luis","family":"Mill\u00e1n","sequence":"additional","affiliation":[{"name":"The Burnham Institute, La Jolla, California, USA"},{"name":"Department of Medical Biosciences, Medical Genetics, Ume\u00e5 University, Ume\u00e5, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2009,12,2]]},"reference":[{"key":"2024013107472912800_bib1","doi-asserted-by":"crossref","DOI":"10.1007\/978-1-4613-2970-1","volume-title":"Alkaline Phosphatases","author":"McComb","year":"1979"},{"key":"2024013107472912800_bib2","doi-asserted-by":"crossref","first-page":"1996","DOI":"10.1073\/pnas.47.12.1996","article-title":"Phosphate incorporation into alkaline phosphatase of E. coli","volume":"47","author":"Schwartz","year":"1961","journal-title":"Proc Natl Acad Sci USA"},{"key":"2024013107472912800_bib3","first-page":"137","article-title":"The human alkaline phosphatases: What we know and what we don't know","volume":"186","author":"Harris","year":"1989","journal-title":"Clin Chim Acta"},{"key":"2024013107472912800_bib4","doi-asserted-by":"crossref","first-page":"7666","DOI":"10.1073\/pnas.85.20.7666","article-title":"A missense mutation in the human liver\/bone\/kidney alkaline phosphatase gene causing a form of lethal hypophosphatasia","volume":"85","author":"Weiss","year":"1988","journal-title":"Proc Natl Acad Sci USA"},{"key":"2024013107472912800_bib5","doi-asserted-by":"crossref","first-page":"2501","DOI":"10.1093\/clinchem\/38.12.2501","article-title":"Missense mutations of the tissue-nonspecific alkaline phosphatase gene in hypophosphatasia","volume":"38","author":"Henthorn","year":"1992","journal-title":"Clin Chem"},{"key":"2024013107472912800_bib6","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1002\/(SICI)1098-1004(200004)15:4<309::AID-HUMU2>3.0.CO;2-C","article-title":"Hypophosphatasia: The mutations in the tissue-nonspecific alkaline phosphatase gene","volume":"15","author":"Mornet","year":"2000","journal-title":"Hum Mutat"},{"key":"2024013107472912800_bib7","first-page":"5313","volume-title":"The Metabolic and Molecular Bases of Inherited Disease","author":"Whyte","year":"2001"},{"key":"2024013107472912800_bib8","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1093\/hmg\/8.6.1039","article-title":"Correlations of genotype and phenotype in hypophosphatasia","volume":"8","author":"Zurutuza","year":"1999","journal-title":"Hum Mol Genet"},{"key":"2024013107472912800_bib9","doi-asserted-by":"crossref","first-page":"1383","DOI":"10.1359\/jbmr.2002.17.8.1383","article-title":"Kinetic characterization of hypophosphatasia mutations with physiological substrates","volume":"17","author":"Di Mauro","year":"2002","journal-title":"J Bone Miner Res"},{"key":"2024013107472912800_bib10","doi-asserted-by":"crossref","first-page":"31171","DOI":"10.1074\/jbc.M102788200","article-title":"Structural evidence for a functional role of human tissue: On-specific alkaline phosphatase in bone mineralization","volume":"276","author":"Mornet","year":"2001","journal-title":"J Biol Chem"},{"key":"2024013107472912800_bib11","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1083\/jcb.41.1.59","article-title":"Vesicles associated with calcification in the matrix of epiphyseal cartilage","volume":"41","author":"Anderson","year":"1969","journal-title":"J Cell Biol"},{"key":"2024013107472912800_bib12","doi-asserted-by":"crossref","first-page":"1513","DOI":"10.1073\/pnas.67.3.1513","article-title":"Isolation and characterization of calcifying matrix vesicles from epiphyseal cartilage","volume":"67","author":"Ali","year":"1970","journal-title":"Proc Natl Acad Sci USA"},{"key":"2024013107472912800_bib13","doi-asserted-by":"crossref","first-page":"56P","DOI":"10.1042\/bj1220056Pa","article-title":"Enzymic and electron-microscopic analysis of extracellular matrix vesicles associated with calcification in cartilage","volume":"122","author":"Ali","year":"1971","journal-title":"Biochem J"},{"key":"2024013107472912800_bib14","first-page":"218","article-title":"Ultrastructural localization of alkaline phosphatase in initial membranous osteogenesis","volume":"135","author":"Bernard","year":"1978","journal-title":"Clin Orthop"},{"key":"2024013107472912800_bib15","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1016\/0169-6009(92)90877-G","article-title":"Immunolocalization of alkaline phosphatase in osteoblasts and matrix vesicles of human fetal bone","volume":"19","author":"Morris","year":"1992","journal-title":"Bone Miner"},{"key":"2024013107472912800_bib16","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1042\/bj0170286","article-title":"The possible significance of hexosephosphoric esters in ossification","volume":"17","author":"Robison","year":"1923","journal-title":"Biochem J"},{"key":"2024013107472912800_bib17","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/0005-2744(75)90151-5","article-title":"Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase","volume":"391","author":"Majeska","year":"1975","journal-title":"Biochim Biophys Acta"},{"key":"2024013107472912800_bib18","first-page":"489","article-title":"Stereospecific inhibition of alkaline phosphatase by L-tetramisole prevents in vitro cartilage calcification","volume":"43","author":"Fallon","year":"1980","journal-title":"Lab Invest"},{"key":"2024013107472912800_bib19","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1042\/bj1020053","article-title":"Association of inorganic pyrophosphatase activity with human alkaline phosphatase preparations","volume":"102","author":"Moss","year":"1967","journal-title":"Biochem J"},{"key":"2024013107472912800_bib20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0003-9861(84)90356-4","article-title":"Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase","volume":"231","author":"Meyer","year":"1984","journal-title":"Arch Biochem Biophys"},{"key":"2024013107472912800_bib21","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1042\/bj3010517","article-title":"Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate","volume":"301","author":"Rezende","year":"1994","journal-title":"Biochem J"},{"key":"2024013107472912800_bib22","first-page":"1555","article-title":"Matrix vesicle in osteomalacic hypophosphatasia bone contain apatite-like mineral crystals","volume":"151","author":"Anderson","year":"1997","journal-title":"Am J Pathol"},{"key":"2024013107472912800_bib23","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1016\/S0002-9440(10)63172-0","article-title":"Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice","volume":"164","author":"Anderson","year":"2004","journal-title":"Am J Pathol"},{"key":"2024013107472912800_bib24","doi-asserted-by":"crossref","first-page":"R1365","DOI":"10.1152\/ajpregu.2000.279.4.R1365","article-title":"Tissue-nonspecific alkaline phosphatase (TNALP) and plasma cell membrane glycoprotein-1 (PC-1) act as selective and mutual antagonists of mineralizing activity by murine osteoblasts","volume":"279","author":"Johnson","year":"2000","journal-title":"Am J Physiol Regul Integr Comp Physiol"},{"key":"2024013107472912800_bib25","doi-asserted-by":"crossref","first-page":"9445","DOI":"10.1073\/pnas.142063399","article-title":"Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization","volume":"99","author":"Hessle","year":"2002","journal-title":"Proc Natl Acad Sci USA"},{"key":"2024013107472912800_bib26","doi-asserted-by":"crossref","first-page":"994","DOI":"10.1359\/jbmr.2003.18.6.994","article-title":"Linked deficiencies in extracellular inorganic pyrophosphate (PPi) and osteopontin expression mediate pathologic ossification in PC-1 null mice","volume":"18","author":"Johnson","year":"2003","journal-title":"J Bone Miner Res"},{"key":"2024013107472912800_bib27","doi-asserted-by":"crossref","first-page":"1199","DOI":"10.1016\/S0002-9440(10)63208-7","article-title":"Concerted regulation of inorganic pyrophosphate and osteopontin by Akp2. Enpp1 and Ank: An integrated model of the pathogenesis of mineralization disorders","volume":"164","author":"Harmey","year":"2004","journal-title":"Am J Pathol"},{"key":"2024013107472912800_bib28","first-page":"140","article-title":"Organ-specific inhibition of human alkaline phosphatase isoenzymes of liver, bone, intestine and placenta; L-phenylalanine, L-tryptophan and L-homoarginine","volume":"41","author":"Fishman","year":"1971","journal-title":"Enzymologia"},{"key":"2024013107472912800_bib29","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1016\/0002-9343(74)90631-7","article-title":"Perspectives on alkaline phosphatase isoenzymes","volume":"56","author":"Fishman","year":"1974","journal-title":"Am J Med"},{"key":"2024013107472912800_bib30","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1016\/0009-8981(77)90365-5","article-title":"Inhibition of human placental-type alkaline phosphatase variants by peptides containing L-leucine","volume":"75","author":"Doellgast","year":"1977","journal-title":"Clin Chim Acta"},{"key":"2024013107472912800_bib31","doi-asserted-by":"crossref","first-page":"972","DOI":"10.1093\/clinchem\/22.7.972","article-title":"Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans","volume":"22","author":"Van Belle","year":"1976","journal-title":"Clin Chem"},{"key":"2024013107472912800_bib32","doi-asserted-by":"crossref","first-page":"4680","DOI":"10.1016\/S0021-9258(19)85548-5","article-title":"Human skeletal alkaline phosphatase-kinetic studies including pH dependence and inhibition by theophylline","volume":"255","author":"Farley","year":"1980","journal-title":"J Biol Chem"},{"key":"2024013107472912800_bib33","volume-title":"Fundamentals of Enzyme Kinetics","author":"Cornish-Bowden","year":"1995"},{"key":"2024013107472912800_bib34","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1042\/bj2740091","article-title":"Gly429 is the major determinant of uncompetitive inhibition of human germ cell alkaline phosphatase by L-Leucine","volume":"274","author":"Hummer","year":"1991","journal-title":"Biochem J"},{"key":"2024013107472912800_bib35","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1111\/j.1432-1033.1991.tb16414.x","article-title":"Site-directed mutagenesis and epitope-mapped monoclonal antibodies define a specific important conformational difference between human placental and germ cell alkaline phosphatase","volume":"202","author":"Hoylaerts","year":"1991","journal-title":"Eur J Biochem"},{"key":"2024013107472912800_bib36","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1042\/bj2860023","article-title":"Molecular mechanism of uncompetitive inhibition of human placental and germ cell alkaline phosphatase","volume":"286","author":"Hoylaerts","year":"1992","journal-title":"Biochem J"},{"key":"2024013107472912800_bib37","doi-asserted-by":"crossref","first-page":"22992","DOI":"10.1074\/jbc.M202298200","article-title":"Function assignment to conserved residues in mammalian alkaline phosphatases","volume":"277","author":"Kozlenkov","year":"2002","journal-title":"J Biol Chem"},{"key":"2024013107472912800_bib38","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/0009-8981(82)90236-4","article-title":"Determination of human serum placental alkaline phosphatase using theophylline inhibition","volume":"118","author":"Ansari","year":"1982","journal-title":"Clin Chim Acta"},{"key":"2024013107472912800_bib39","doi-asserted-by":"crossref","first-page":"1020","DOI":"10.1002\/1097-0142(20010301)91:5<1020::AID-CNCR1093>3.0.CO;2-V","article-title":"Comparison of 5-fluorouracil alone, 5-flourouracil with levamisole, and 5-flourouracil with hepatic irradiation in the treatment of patients with residual, nonmeasurable, intra-abdominal metastasis after undergoing resection for colorectal carcinoma","volume":"91","author":"Witte","year":"2001","journal-title":"Cancer"},{"key":"2024013107472912800_bib40","doi-asserted-by":"crossref","first-page":"1437","DOI":"10.1054\/bjoc.2001.2117","article-title":"Adjuvant 5FU plus levamisole in colonic or rectal cancer; improved survival in stage II and III","volume":"85","author":"Taal","year":"2001","journal-title":"Br J Cancer"},{"key":"2024013107472912800_bib41","first-page":"588","article-title":"Periorchitis after tetramisole treatment in bulls implaned with Setaria labiatopapillos","volume":"186","author":"Powe","year":"1985","journal-title":"J Am Vet Med Assoc"},{"key":"2024013107472912800_bib42","first-page":"455","article-title":"Serum protein profile of mice during infection of Ancylostoma caninum and after the administration of tetramisole and levamisole","volume":"28","author":"Bhopale","year":"1984","journal-title":"J Hyg Epidemiol Microbiol Immunol"},{"key":"2024013107472912800_bib43","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1016\/0030-4220(77)90109-8","article-title":"Levamisole and hamster pouch carcinogenesis","volume":"43","author":"Eisenberg","year":"1977","journal-title":"Oral Surg Oral Med Oral Pathol Oral Radiol Endod"},{"key":"2024013107472912800_bib44","doi-asserted-by":"crossref","first-page":"52","DOI":"10.7326\/0003-4819-105-1-52","article-title":"Theophylline-induced hypercalcemia","volume":"105","author":"McPherson","year":"1986","journal-title":"Ann Intern Med"},{"key":"2024013107472912800_bib45","doi-asserted-by":"crossref","first-page":"49808","DOI":"10.1074\/jbc.M207394200","article-title":"Structural evidence of functional divergence in human alkaline phosphatases","volume":"277","author":"Le Du","year":"2002","journal-title":"J Biol Chem"},{"key":"2024013107472912800_bib46","doi-asserted-by":"crossref","first-page":"2714","DOI":"10.1002\/elps.1150181505","article-title":"SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling","volume":"18","author":"Guex","year":"1997","journal-title":"Electrophoresis"},{"key":"2024013107472912800_bib47","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/0014-5793(88)80559-3","article-title":"Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms. Circumventing problems associated with folding","volume":"239","author":"Nilges","year":"1988","journal-title":"FEBS Lett"},{"key":"2024013107472912800_bib48","volume-title":"X-PLOR, version 3.1","author":"Br\u00fcnger","year":"1993"},{"key":"2024013107472912800_bib49","doi-asserted-by":"crossref","first-page":"3586","DOI":"10.1021\/jp973084f","article-title":"All-atom empirical potential for molecular modeling and dynamics studies of proteins","volume":"102","author":"MacKerell","year":"1998","journal-title":"J Phys Chem"},{"key":"2024013107472912800_bib50","author":"Frisch"},{"key":"2024013107472912800_bib51","doi-asserted-by":"crossref","first-page":"9158","DOI":"10.1074\/jbc.M009250200","article-title":"Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution. Implications for substrate specificity","volume":"276","author":"Le Du","year":"2001","journal-title":"J Biol Chem"},{"key":"2024013107472912800_bib52","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1007\/BF02124036","article-title":"Comparison of the inhibition of avian and mammalian bone alkaline phosphatases by levamisole and compound R8231","volume":"33","author":"Reynolds","year":"1977","journal-title":"Experientia"},{"key":"2024013107472912800_bib53","doi-asserted-by":"crossref","first-page":"3082","DOI":"10.1016\/S0021-9258(19)45215-0","article-title":"L-Homoarginine: An organ-specific, uncompetitive inhibitor of human liver and bone alkaline phosphohydrolases","volume":"247","author":"Lin","year":"1972","journal-title":"J Biol Chem"},{"key":"2024013107472912800_bib54","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1002\/j.1939-4640.2002.tb02334.x","article-title":"Inhibition of alkaline phosphatase activity of boar semen by pentoxifylline, caffeine, and theophylline","volume":"23","author":"Glogowski","year":"2002","journal-title":"J Androl"},{"key":"2024013107472912800_bib55","doi-asserted-by":"crossref","first-page":"21174","DOI":"10.1016\/S0021-9258(18)54836-5","article-title":"Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase","volume":"266","author":"Watanabe","year":"1991","journal-title":"J Biol Chem"},{"key":"2024013107472912800_bib56","doi-asserted-by":"crossref","first-page":"1227","DOI":"10.1016\/0006-2952(79)90334-4","article-title":"Inhibition of alkaline phosphatase by substituted xanthines","volume":"28","author":"Croce","year":"1979","journal-title":"Biochem Pharmacol"}],"container-title":["Journal of Bone and Mineral Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1359%2FJBMR.040608","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/academic.oup.com\/jbmr\/article-pdf\/19\/11\/1862\/56473783\/5650191115.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/jbmr\/article-pdf\/19\/11\/1862\/56473783\/5650191115.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,3,13]],"date-time":"2024-03-13T15:07:07Z","timestamp":1710342427000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jbmr\/article\/19\/11\/1862-1872\/7592713"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2004,11,1]]},"references-count":56,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2009,12,2]]},"published-print":{"date-parts":[[2004,11,1]]}},"URL":"https:\/\/doi.org\/10.1359\/jbmr.040608","relation":{},"ISSN":["0884-0431","1523-4681"],"issn-type":[{"value":"0884-0431","type":"print"},{"value":"1523-4681","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2004,11,1]]},"published":{"date-parts":[[2004,11,1]]}}}