Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients

Research output: Contribution to journalArticle

Authors

  • Erica J. Fratz-Berilla
  • Leonid Breydo
  • Laurent Gouya
  • Hervé Puy
  • Vladimir N. Uversky
  • Gloria C. Ferreira

External Institution(s)

  • University of South Florida
  • Institut national de la santé et de la recherche médicale
  • Laboratory of Excellence
  • Hôpital Louis Mourier

Details

Original languageEnglish (US)
Pages (from-to)428-439
Number of pages12
JournalBiochimica et Biophysica Acta - Molecular Basis of Disease
Volume1863
Issue number2
StatusPublished - Feb 1 2017
Peer-reviewedYes

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 Ki of 1.2 μM. 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 (5 mg·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.

    Research areas

  • 5-Aminolevulinate synthase, Anemia, Enzyme inhibitors, Enzyme stuctures, Heme, Porphyria, Pyridoxal 5′-phosphate

Citation formats

APA

Fratz-Berilla, E. J., Breydo, L., Gouya, L., Puy, H., Uversky, V. N., & Ferreira, G. C. (2017). Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients. Biochimica et Biophysica Acta - Molecular Basis of Disease, 1863(2), 428-439. https://doi.org/10.1016/j.bbadis.2016.11.011

Harvard

Fratz-Berilla, EJ, Breydo, L, Gouya, L, Puy, H, Uversky, VN & Ferreira, GC 2017, 'Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients', Biochimica et Biophysica Acta - Molecular Basis of Disease, vol. 1863, no. 2, pp. 428-439. https://doi.org/10.1016/j.bbadis.2016.11.011