AG-348 (Mitapivat), an allosteric activator of red blood cell pyruvate kinase, increases enzymatic activity, protein stability, and ATP levels over a broad range of PKLR genotypes
Minke A E Rab, Brigitte A Van Oirschot, Penelope A Kosinski, Jeffrey Hixon, Kendall Johnson, Victor Chubukov, Lenny Dang, Gerard Pasterkamp, Stephanie Van Straaten, Wouter W Van Solinge, Eduard J Van Beers, Charles Kung, Richard Van Wijk, Minke A E Rab, Brigitte A Van Oirschot, Penelope A Kosinski, Jeffrey Hixon, Kendall Johnson, Victor Chubukov, Lenny Dang, Gerard Pasterkamp, Stephanie Van Straaten, Wouter W Van Solinge, Eduard J Van Beers, Charles Kung, Richard Van Wijk
Abstract
Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red cell (RBC) glycolysis, causing changes in metabolism including a deficiency in ATP. This affects red cell homeostasis, promoting premature removal of RBCs from the circulation. In this study we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBCs and erythroid precursors from PK-deficient patients. In 15 patients ex vivo treatment with AG-348 resulted in increased enzymatic activity in all patient cells after 24 hours (mean increase 1.8-fold, range 1.2-3.4). ATP levels increased (mean increase 1.5-fold, range 1.0-2.2) similar to control cells (mean increase 1.6-fold, range, 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBCs. Ex vivo treatment with AG-348 increased residual activity 1.4 to >10-fold than residual activity of vehicle-treated samples. Protein analyses suggests that a sufficient level of PK protein is required for cells to respond to AG-348 treatment ex-vivo, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients, ex vivo treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG-348 effectively upregulates PK enzymatic activity and increases stability in PK-deficient RBCs over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. ClinicalTrials.gov: NCT02476916, NCT03853798, NCT03548220, NCT03559699).
Figures
References
- Koralkova P, van Solinge WW, van Wijk R. Rare hereditary red blood cell enzymopathies associated with hemolytic anemia - pathophysiology, clinical aspects, and laboratory diagnosis. Int J Lab Hematol. 2014;36(3):388-397.
- Beutler E, Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene frequency in the general white population. Blood. 2000;95(11):3585-3588.
- Carey PJ, Chandler J, Hendrick A, et al. Prevalence of pyruvate kinase deficiency in a northern European population in the north of England. Blood. 2000;96(12):4005-4007.
- de Medicis E, Ross P, Friedman R, et al. Hereditary nonspherocytic hemolytic anemia due to pyruvate kinase deficiency: a prevalence study in Quebec (Canada). Hum Hered. 1992;42(3):179-183.
- Kanno H, Fujii H, Miwa S. Structural analysis of human pyruvate kinase L-gene and identification of the promoter activity in erythroid cells. Biochem Biophys Res Commun. 1992;188(2):516-523.
- Kanno H, Fujii H, Hirono A, Miwa S. cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384 --> Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary hemolytic anemia. Proc Natl Acad Sci U S A. 1991;88(18):8218-8221.
- Zanella A, Fermo E, Bianchi P, Chiarelli LR, Valentini G. Pyruvate kinase deficiency: the genotype-phenotype association. Blood Rev. 2007;21(4):217-231.
- van Wijk R, Huizinga EG, van Wesel ACW, van Oirschot BA, Hadders MA, van Solinge WW. Fifteen novel mutations in PKLR associated with pyruvate kinase (PK) deficiency: structural implications of amino acid substitutions in PK. Hum Mutat. 2009;30(3):446-453.
- Canu G, De Bonis M, Minucci A, Capoluongo E. Red blood cell PK deficiency: an update of PK-LR gene mutation database. Blood Cells Mol Dis. 2016;57:100-109.
- Glader B. Salicylate-induced injury of pyruvate- kinase-deficient erythrocytes. N Engl J Med. 1976;294(17):916-918.
- Mentzer WC, Baehner RL, Schmidt- Schönbein H, Robinson SH, Nathan DG. Selective reticulocyte destruction in erythrocyte pyruvate kinase deficiency. J Clin Invest. 1971;50(3):688-699.
- Koller CA, Orringer EP, Parker JC. Quinine protects pyruvate‐kinase deficient red cells from dehydration. Am J Hematol. 1979; 7(3):193-199.
- Park Y, Best CA, Badizadegan K, et al. Measurement of red blood cell mechanics during morphological changes. Proc Natl Acad Sci U S A. 2010;107(15):6731-6736.
- Weed RI, LaCelle PL, Merrill EW. Metabolic dependence of red cell deformability. J Clin Invest. 1969;48(5):795-809.
- Aizawa S, Harada T, Kanbe E, et al. Ineffective erythropoiesis in mutant mice with deficient pyruvate kinase activity. Exp Hematol. 2005;33(11):1292-1298.
- Aizawa S, Kohdera U, Hiramoto M, et al. Ineffective erythropoiesis in the spleen of a patient with pyruvate kinase deficiency. Am J Hematol. 2003;74(1):68-72.
- Aisaki K, Aizawa S, Fujii H, Kanno J, Kanno H. Glycolytic inhibition by mutation of pyruvate kinase gene increases oxidative stress and causes apoptosis of a pyruvate kinase deficient cell line. Exp Hematol. 2007; 35(8):1190-1200.
- Grace RF, Bianchi P, van Beers EJ, et al. The clinical spectrum of pyruvate kinase deficiency: data from the Pyruvate Kinase Deficiency Natural History Study. Blood. 2018;131(20):2183-2192.
- Grace RF, Layton DM, Barcellini W. How we manage patients with pyruvate kinase deficiency. Br J Haematol. 2019;184(5):721-734.
- van Straaten S, Bierings M, Bianchi P, et al. Worldwide study of hematopoietic allogeneic stem cell transplantation in pyruvate kinase deficiency. Haematologica. 2018; 103(2):e82-e86.
- Garcia-Gomez M, Calabria A, Garcia-Bravo M, et al. Safe and efficient gene therapy for pyruvate kinase deficiency. Mol Ther. 2016;24(7):1187-1198.
- Kung C, Hixon J, Kosinski PA, et al. AG-348 enhances pyruvate kinase activity in red blood cells from patients with pyruvate kinase deficiency. Blood. 2017;130(11):1347-1356.
- Yang H, Merica E, Chen Y, et al. Phase 1 single- and multiple-ascending-dose randomized studies of the safety, pharmacokinetics, and pharmacodynamics of AG-348, a firstin- class allosteric activator of pyruvate kinase R, in healthy volunteers. Clin Pharmacol Drug Dev. 2019;8(2):246-259.
- Grace RF, Rose C, Layton M, et al. Safety and efficacy of Mitapivat in pyruvate kinase deficiency. N Engl J Med. 2019;381(10):933-944.
- Allen EL, Ulanet DB, Pirman D, et al. Differential aspartate usage identifies a subset of cancer cells particularly dependent on OGDH. Cell Rep. 2016;17(3):876-890.
- Clasquin MF, Melamud E, Rabinowitz JD. LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine. Curr Protoc Bioinformatic. 2012; 14:14.11.
- Kim H, Kosinski P, Kung C, et al. A fit-forpurpose LC–MS/MS method for the simultaneous quantitation of ATP and 2,3-DPG in human K2EDTA whole blood. J Chromatogr B Analyt Biomed Life Sci. 2017; 1061-1062:89-96.
- Beutler E, Blume K, Kaplan J, Lohr G, Ramot B, Valentine W. International Committee for Standardization in Haematology: recommended methods for red-cell enzyme analysis. Br J Haematol. 1977;35(2):331-340.
- Beutler E. Red cell metabolism. A manual of biochemical methods. 3rd edition. Grune & Stratton Inc., Orlando FL, 1984.
- Blume KG, Arnold H, Lohr GW, Beutler E. Additional diagnostic procedures for the detection of abnormal red cell pyruvate kinase. Clin Chim Acta. 1973;43(3):443-446.
- van Oirschot BA, Francois JJJM, van Solinge WW, et al. Novel type of red blood cell pyruvate kinase hyperactivity predicts a remote regulatory locus involved in PKLR gene expression. Am J Hematol. 2014;89(4):380-384.
- Rijksen G, Veerman AJP, Schipper-Kester GPM, Staal GEJ. Diagnosis of pyruvate kinase deficiency in a transfusion-dependent patient with severe hemolytic anemia. Am J Hematol. 1990;35(3):187-193.
- Lazarova E, Gulbis B, van Oirschot B, van Wijk R. Next-generation osmotic gradient ektacytometry for the diagnosis of hereditary spherocytosis: interlaboratory method validation and experience. Clin Chem Lab Med. 2017;55(3):394-402.
- Da Costa L, Suner L, Galimand J, et al. Diagnostic tool for red blood cell membrane disorders: assessment of a new generation ektacytometer. Blood Cells Mol Dis. 2016;56(1):9-22.
- van den Akker E, Satchwell TJ, Pellegrin S, Daniels G, Toye AM. The majority of the in vitro erythroid expansion potential resides in CD34- cells, outweighing the contribution of CD34+ cells and significantly increasing the erythroblast yield from peripheral blood samples. Haematologica. 2010;95(9):1594-1598.
- Jansen G, Koenderman L, Rijksen G, Cats BP, Staal GEJ. Characteristics of hexokinase, pyruvate kinase, and glucose‐6‐phosphate dehydrogenase during adult and neonatal reticulocyte maturation. Am J Hematol. 1985;20(3):203-215.
- Wang C, Chiarelli LR, Bianchi P, et al. Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia. Blood. 2001; 98(10):3113-3120.
- Diez A, Gilsanz F, Martinez J, Pérez-Benavente S, Meza NW, Bautista JM. Lifethreatening nonspherocytic hemolytic anemia in a patient with a null mutation in the PKLR gene and no compensatory PKM gene expression. Blood. 2005;106(5):1851-1856.
- Rogers SC, Ross JGC, D’Avignon A, et al. Sickle hemoglobin disturbs normal coupling among erythrocyte O2 content, glycolysis, and antioxidant capacity. Blood. 2013; 121(9):1651-1662.
- Karger R, Lukow C, Kretschmer V. Deformability of red blood cells and correlation with atp content during storage as leukocyte-depleted whole blood. Transfus Med Hemother. 2012;39(4):277-282.
- Fischer DJ, Torrence NJ, Sprung RJ, Spence DM. Determination of erythrocyte deformability and its correlation to cellular ATP release using microbore tubing with diameters that approximate resistance vessels in vivo. Analyst. 2003;128(9):1163-1168.
- Clark R, Rossi E. Osmotic gradient ektacytometry: comprehensive characterization of red cell volume and surface maintenance. Blood. 1983;61(5):899-911.
- Nijhof W, Wierenga PK, Staal GEJ, Jansen G. Changes in activities and isozyme patterns of glycolytic enzymes during erythroid differentiation in vitro. Blood. 1984;64(3):607-613.
- Takegawa S, Fujii H, Miwa S. Change of pyruvate kinase isozymes from M2- to Ltype during development of the red cell. Br J Haematol. 1983;54(3):467-474.
- Zaninoni A, Fermo E, Vercellati C, et al. Use of laser assisted optical rotational cell analyzer (LoRRca MaxSis) in the diagnosis of RBC membrane disorders, enzyme defects, and congenital dyserythropoietic anemias: a monocentric study on 202 patients. Front Physiol. 2018;9:451.
- Llaudet-Planas E, Vives-Corrons JL, Rizzuto V, et al. Osmotic gradient ektacytometry: a valuable screening test for hereditary spherocytosis and other red blood cell membrane disorders. Int J Lab Hematol. 2018;40(1):94-102.
- Leblond PF, Coulombe L, Lyonnais J. Erythrocyte populations in pyruvate kinase deficiency anaemia following splenectomy. Br J Haematol. 1978;39(1):63-70.
- Cahalan SM, Lukacs V, Ranade SS, Chien S, Bandell M, Patapoutian A. Piezo1 links mechanical forces to red blood cell volume. Elife. 2015;4.
Source: PubMed