title: Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients.
abstract: Mutations in the C-terminus of human erythroid 5-aminolevulinate synthase (hALAS2), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are associated with two different blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA-causing mutations yield hALAS2 variants with decreased activity, while XLPP-causing mutations result in a gain-of-function of hALAS2. There are no specific treatments for XLPP. Isonicotinic acid hydrazide (isoniazid, INH), an antituberculosis agent, can cause sideroblastic anemia as a side-effect, by limiting PLP availability to hALAS2, via inhibition of pyridoxal kinase or reaction with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We hypothesized that INH also binds and directly inhibits hALAS2. Using fluorescence-activated cell sorting and confocal fluorescence microscopy, we demonstrate that INH reduces protoporphyrin IX levels in HeLa cells expressing either wild-type hALAS2 or XLPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) reversed the cellular inhibition of hALAS2 activity by INH. Steady-state kinetic analyses with purified hALAS2 indicated that INH directly inhibits the enzyme, noncompetitively or uncompetitively, with an apparent Kiof 1.2μM. Circular dichroism spectroscopy revealed that INH triggered tertiary structural changes in hALAS2 that altered the microenvironment of the PLP cofactor and hampered the association of PLP with apo-hALAS2. Treatment of four XLPP patients with INH (5mg·kg-1·day-1) over a six-month period was well tolerated but without statistically significant modification of PPIX levels. These results, taken together, permit us to further an INH inhibition kinetic mechanism for ALAS, which suggests the possible use of INH-derived drugs in treating patients with XLPP and potentially other protoporphyrin-accumulating porphyrias.
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA.
authors.forename: Erica J
authors.initials: EJ
authors.lastname: Fratz-Berilla
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA; USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA.
authors.forename: Leonid
authors.initials: L
authors.lastname: Breydo
authors.affiliation: INSERM U1149, Centre de Recherche sur l'inflammation, Université Paris Diderot, site Bichat, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France; AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Gouya
authors.affiliation: INSERM U1149, Centre de Recherche sur l'inflammation, Université Paris Diderot, site Bichat, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France; AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Puy
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA; USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA; Department of Chemistry, University of South Florida, Tampa, FL, USA. Electronic address: gferreir@health.usf.edu.
authors.forename: Gloria C
authors.initials: GC
authors.lastname: Ferreira
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000037:antagonists & inhibitors / Q000097:blood / Q000737:chemistry / Q000172:deficiency* / Q000378:metabolism; D000756:Anemia, Sideroblastic / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D040181:Genetic Diseases, X-Linked / Q000097:blood / Q000188:drug therapy* / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology* / Q000627:therapeutic use; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D046351:Protoporphyria, Erythropoietic / Q000097:blood / Q000188:drug therapy* / Q000201:enzymology; D011524:Protoporphyrins / Q000097:blood; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011736:Pyridoxine / Q000494:pharmacology; D014803:Vitamin B Complex / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D011524:Protoporphyrins; D014803:Vitamin B Complex; D011732:Pyridoxal Phosphate; C028025:protoporphyrin IX; D000624:5-Aminolevulinate Synthetase; C480079:ALAS2 protein, human; D011736:Pyridoxine; D007538:Isoniazid
keywords: 5-Aminolevulinate synthase; Anemia; Enzyme inhibitors; Enzyme stuctures; Heme; Porphyria; Pyridoxal 5′-phosphate