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 K of 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·day) 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.

Iron chelation rescues hemolytic anemia and skin photosensitivity in congenital erythropoietic porphyria

“…34 The antituberculosis agent isoniazid (INH) is a potent inhibitor of the ALAS2 enzyme and may induce sideroblastic anemia in some patients. 35 It was recently shown that INH reduces PPIX accumulation in cellular and murine models of protoporphyria. 36 However, pilot clinical studies failed to confirm the therapeutic effect in patients with XLP and EPP after administration of INH at the standard treatment dose (5 mg/kg per day, up to 300 mg/d) for Mycobacterium tuberculosis.…”

Toxicoproteomic Profiling of hPXR Transgenic Mice Treated with Rifampicin and Isoniazid

“…ABCB7 exports [2Fe–2S] clusters out of the mitochondria and into the cytosol, and ABCB7 deficiency causes an iron-deficient phenotype and mitochondrial iron accumulation [ 79 , 80 ]. Indeed, sideroblastic anemia responsive to vitamin B 6 administration is reported with antitubercular therapy [ 8 , 9 , 10 ], and multiple types of anemia (including iron-deficiency anemia) occur with antitubercular therapy [ 81 , 82 , 83 ]. These data and previous reports suggest that isoniazid and rifampicin lead to an iron-deficient state, with concomitant changes in heme biosynthesis regulation.…”

“…Hepatotoxicity is a leading cause of TB therapy treatment failure, with up to 28% of all anti-TB treatment failure attributed to toxicity [ 3 , 4 ]. Antitubercular therapy may also be associated with acute intermittent porphyria [ 5 , 6 ], sideroblastic anemia [ 7 , 8 , 9 , 10 ], hypercoagulability [ 11 , 12 , 13 ], peripheral neuropathy [ 14 , 15 , 16 ], porphyria cutanea tarda [ 17 ], vitamin B 3 deficiency [ 18 , 19 , 20 ], and vitamin B 6 deficiency [ 8 , 14 , 21 , 22 ].…”

Current and innovative emerging therapies for porphyrias with hepatic involvement

“…Some patients with EPP have described worsening of photosensitivity after iron administration, but associated changes in protoporphyrin levels have not been studied prospectively. D) Alternatively, reducing haem and haemoglobin synthesis through erythrocyte transfusion, haem infusion, hydroxyurea, iron suppression or direct inhibition of ALAS2 95 might ameliorate all erythropoietic porphyrias by reducing synthesis of porphyrin intermediates. 5 In this regard, improvement was reported using haem transfusion and hydroxycarbamide (hydroxyurea) in ADP 11 and congenital erythropoietic porphyria, 96 and iron reduction in a patient with congenital erythropoietic porphyria.…”