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

Figure 1.
Figure 1.
Baseline levels of pyruvate kinase (PK) activity, PK/Hexokinase (HK) ratio, PK thermostability, and PK-R protein levels in PK-deficient patients and controls. (A) PK activity of PK deficient patients (red) and healthy controls (gray). (B) PK/HK ratio in patients (red) and healthy controls (gray), as a means to evaluate PK activity in the presence of high number of reticulocytes, reflected by increased HK activity. (C) PK thermostability of patients (red) and controls (gray). (D) PK-R protein levels of patients (P#) and controls (HC) measured by western blot. (E) PK-R protein levels of patients (red) and controls (gray) measured by Meso Scale Discovery. ECL: electrochemiluminescence.
Figure 2.
Figure 2.
Pyruvate kinase (PK)- deficient patients show glycolytic intermediates levels that are consistent with decreased PK activity. Upstream intermediates of PK of PK-deficient patients (red) are significantly increased compared to values of health controls (gray), with, for example, an almost 2-fold increase in 2,3-DPG. Downstream targets, pyruvate and adenosine triphosphate (ATP), are significantly decreased compared to healthy controls. PKD: pyruvate kinase deficiency; G6P: glucose- 6-phosphate; DHAP: dihydroxyacetone phosphate; 2,3- DPG: 2,3-diphosphoglycerate; PEP: phosphoenol pyruvate; FBP: fructose biphosphate; 2PG: 2-phosphoglycerate; 3PG: 3-phosphoglycerate; GAP: glyceraldehyde phosphate; 1,3- DPG: 1,3-diphosphoglycerate; HK: hexokinase; PGI: phosphogluco isomerase; PFK: phosphofructokinase; FBA: fructose biphosphate aldolase; TPI: triosephosphate isomerase; GAPDH: glyceraldehyde phosphate dehydrogenase; BPGM: bisphosphoglycerate mutase; PGK: phosphoglycerate kinase; BPKG: bisphosphoglycerate kinase; PGM: phosphoglycerate mutase; ENO: enolase; LDH: lactate dehydrogenase.
Figure 3.
Figure 3.
Ex vivo AG-348 treatment of red blood cells (RBC) increase pyruvate kinase (PK) activity and adenosine triphosphate (ATP) levels in a dose dependent manner. (A and B) Representative patient dose response curves of PK activity, expressed as fold activation compared to incubation without AG-348, as measured in RBC of patients 12 (A) and 9 (B) after 6- (turquoise) and 24-hour (h) (dark green) incubation with AG-348. (C and D) representative patient ATP dose response curves, expressed as fold activation compared to incubation without AG- 348, as measured in RBC of patients 12 (C) and 9 (D) after 6- (turquoise) and 24- h (dark green) incubation with AG-348. (E) PK activation in RBC of 15 PK deficient patients after 6- (turquoise) and 24-h (dark green) incubation with 10 mM AG-348. Mean PK fold activation of healthy controls after 6- (light gray) and 24-h (dark gray) incubation with AG-348 10μM is shown on the left of the graph. (F) ATP response (fold activation) of 15 PK deficient patients after 6- (turquoise) and 24-h (dark green) incubation with 10 mM AG-348. Mean ATP response of healthy controls is shown on the left of the graph (gray bars). Error bars represent standard deviation. Dashed turquoise line represents 6-h incubation with AG-348, solid dark green line represents 24-h incubation.
Figure 4.
Figure 4.
Pyruvate kinase (PK) thermostability is restored upon ex vivo treatment with AG-348. (A and B) Representative PK-deficient (PKD) patients' PK thermostability of red blood cell (RBC) lysates of patients 12 (A) and 9 (B) pre-incubated in absence (dashed line) or presence (solid line) of 2 mM AG-348 for 2 hours (h) at 37°C. For PK thermostability, residual PK activity (%) is measured after 5, 10, 20, 40 and 60 minutes (min) of heat treatment at 53°C. (C) Residual PK activity (%) after 60 min of heat treatment of 15 PKD pre-incubated in absence (light turquoise) and presence (dark turquoise bars) of 2 mM AG-348. Levels are normalized to baseline levels of PK-activity before heat treatment. Absence of bars indicate that PK residual activity was below the detection limit. Error bars represent standard deviation.
Figure 5.
Figure 5.
Red blood cell (RBC) deformability in pyruvate kinase (PK)-deficient patients is slightly decreased. (A) Osmoscan curve of patient 7 (red) and 15 healthy controls (HC) (gray). (B) Maximum deformability (EImax) is slightly decreased in PK deficiency compared to HC. When grouped according to genotype, i.e., missense/ missense M/M versus missense/ non-missense (M/NM), there is a significant difference, indicating that a more severe genotype results in lower maximal RBC deformability. (C) Omin is slightly increased in PKD patients compared to HC. (D) EImax is slightly decreased in PKD patients compared to HC. (E) Ohyper is slightly increased in PKD patients compared to HC. (F) After 24 hours of ex vivo treatment with 20 mM AG- 348 in about half of the PKD patients an improvement in RBC deformability was observed (patients 1, 3, 4, 6, 7, 8, and 9) compared to their untreated RBC. Error bars represent standard deviation. **P<0.01, *P<0.05.
Figure 6.
Figure 6.
Ex vivo treatment with AG-348 does not evidently affect pyruvate kinase (PKD) ex vivo erythroid proliferation and differentiation. (A) Morphology of ex vivo cultured erythroid cells of a PKD patient (P3) and a healthy control (HC) at the final stage of proliferation (day 10) and on various days during differentiation (days 0, 4 and 10). (B) Ex vivo erythroid proliferation (cell numbers in %) was slightly higher when cells were cultured in presence of 2 mM AG-348 (dark purple) compared to 0 mM AG-348 (light purple). This was also observed for HC. (C) PK/HK ratio of erythroid cells of PKD patients (purple) and HC (gray) cultured in presence (dark) or absence (light) of 2 mM AG-348 respectively was increased compared to cells cultured without AG-348. W/o: with/without. Error bars represent standard deviation.
Figure 7.
Figure 7.
Pyruvate kinase (PK)-deficient red cells with higher PK-R protein levels are more likely to show an increase in adenosine triphosphate (ATP) levels and/or PK-R activity after ex vivo treatment with AG-348. Base line PK-R protein levels in patients showing a response in PK activity or ATP levels (i.e., >1.5- fold response vs. <1.5-fold response, red bars) upon ex vivo treatment with AG- 348 PK-R levels of healthy controls are shown in gray. ECL: electrochemiluminescense; gHb: gram hemoglobin. Error bars represent standard deviation.

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