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Accelerated erythrocyte senescence causes dose-limiting anemia of antimalarial enolase inhibitors

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are being investigated for obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a key dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase – a critical enzyme in glycolysis – and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage that induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity.


INTRODUCTION
Infection with Plasmodium spp. malaria parasites contributes to over 500,000 deaths annually.
Chemotherapeutic resistance remains a central roadblock to malaria control, and novel antimalarials are urgently required. One approach to circumvent resistance is through targeting host, rather than parasitic, processes essential for parasite survival. As the pathogenic stage of Plasmodium spp. develops within erythrocytes, the red cell niche presents a unique set of possible host targets essential for erythrocyte metabolic homeostasis. The promise of erythrocyte-targeted antimalarial therapies is underscored by the prevalence of hereditary erythrocyte metabolic disorders in humans. These include glucose-6-phosphate dehydrogenase deficiency (G6PDd, the most common enzymopathy in humans), pyruvate kinase deficiency (PKd), and sickle cell trait, all of which are under positive selection in malaria-endemic geographic regions. [1][2][3][4][5] These prevalent genetic disorders reveal an evolutionary pressure on hosts to modulate the metabolic state of the red cell to improve survival during malaria, suggesting that acute, specific targeting of red cell glycolysis, the sole energy-generating pathway in mature erythrocytes, is a promising antimalarial approach.
Previous studies on hereditary erythrocyte enzymopathies suggest a complex interplay with malaria survival and do not provide definitive evidence as to whether acute, rather than chronic, metabolic disruption in erythrocytes indeed improves malaria clinical outcomes. For example, while reduced enzyme activity in G6PDd and PKd individuals correlates with protection from severe malaria, no specific genetic variants are associated with survival. 1 In addition, severe malaria has diverse clinical presentations; while G6PDd may protect during cerebral malaria, it appears to worsen malaria-associated severe anemia, although this model is itself controversial. 6,7 Finally, acute glycolytic inhibition is distinct from the phenotype arising from hereditary enzymopathies and their complex long-term physiologic adaptations, including increased red cell production, decreased half-life of circulating erythrocytes, and sometimes an overall increase in metabolic robustness in circulating red cells (due to reticulocytosis). Similarly, caloric restriction has been proposed as a sub-acute "anti-glycolytic" state that is malariaprotective, but this intervention has pleiotropic physiologic and immunologic effects. 8 Efforts to study red cell glycolytic homeostasis using a chemical approach have been similarly complicated due to a lack of specificity. Several compounds, including sodium fluoride, arsenic, 6-aminonicatinomide, oxythiamine, and 3-bromopyruvate, are used experimentally as glycolytic inhibitors despite known off-target cellular effects. 9-11 While commonly deployed as a metabolic inhibitor, 2-deoxyglucose (2-DG) is likewise complex. First, 2-DG does not completely recapitulate low glucose conditions or glycolytic inhibition, because, like glucose itself, 2-DG is processed in erythrocytes (through hexokinase and glucose 6-phosphate dehydrogenase) toward NADPH production. [12][13][14][15] Second, use of 2-DG in animal models impacts all cell types, including immunocytes. Metabolic reprogramming is essential to the function of immune cells, including macrophages and natural killer cells, which themselves are well-recognized mediators of malaria-induced anemia. [16][17][18][19][20][21] Enolase (phosphopyruvate hydratase; E.C. 4.2.1.11) catalyzes the eighth step in glycolysis.
Humans express three isozymes of enolase (α, β, γ) that comprise multiple dimer configurations: ENO1 (αα), found in all cell types; ENO2 (γγ), primarily in neurons; and ENO3 (ββ), a muscle-specific isoform. 22 Like neurons, erythrocytes particularly rely on ENO2 for glycolytic function. 23 While red bloods cells predominantly express the α isozyme they rely on the γ isozyme to a larger extent than any other non-nervous tissue. 24 Thus, enolase presents a unique opportunity to achieve cellular specificity of glycolytic inhibition. In recent years, enolase inhibitors have been developed with compelling subtype specificity and enhanced potency via prodrug strategies. [25][26][27] This newly developed class of phosphonate enolase inhibitors (some of which are ENO2-specific) and their cognate pro-drugs represent novel reagents to evaluate the role of specific and acute glycolytic disruption in erythrocytes during malaria infection. Neurons, which express both ENO1 and ENO2, withstand ENO2 specific inhibition, while erythrocytes do not. 27 Here we query and define the signature of disrupted red cell glycolytic homeostasis, validate tools to investigate antiglycolytic approaches to human disease states, and use these tools to establish the impact of acute and specific glycolytic inhibition of erythrocytes on Plasmodium spp. infection.
Importantly, the observed metabolic profiles (Supplemental Tables 1,4) are consistent with acute and specific inhibition of glycolysis at the level of enolase, such that metabolites immediately upstream of enolase accumulate and metabolites immediately downstream are depleted ( Figure 1A). Hierarchical clustering yields two prominent clusters: in the first, glycolytic inhibition slows or prevents metabolite accumulation, and in the second, glycolytic inhibition accelerates metabolite depletion ( Figure 1B).
Altogether, these metabolite characterizations indicate that acute glycolytic inhibition leads to a collapse in cellular reductive capacity. As glutathione synthesis is an ATP-dependent phenomenon, inhibition of glycolysis thus decreases total red cell antioxidant capacity. In corroboration, we also find a reduction in the total glutathione pool (reduced [GSH] + oxidized [GSSG]), driven by a depletion of GSH upon enolase inhibitor treatment ( Figure 1A, 2C). It is likely that the reduced availability of glutathione component amino acids further compounds this effect. As GSH is the most abundant low molecular weight cellular antioxidant, acute glycolytic disruption in erythrocytes likely causes a profound susceptibility to oxidative insults.

Acute glycolytic inhibition causes erythrocyte senescence
To evaluate oxidative susceptibility during acute glycolytic inhibition in erythrocytes, we quantified methemoglobin, in which the heme iron of hemoglobin is oxidized to the ferric (Fe 3+ ), rather than ferrous (Fe 2+ ) form. While methemoglobin forms spontaneously under normoxic conditions, it is typically reduced by cellular protective mechanisms, some of which are dependent on reduced glutathione. 29,30 As methemoglobin reductase is fueled by NADH (and, to a lesser extent, NADPH), we hypothesized that inhibition of late glycolysis would result in a metabolic bottleneck causing decreased NADH availability and thus impaired capacity to reduce ferric iron ( Figure 4A). In keeping with this hypothesis, 1,2,3-13 C3-glucose tracing highlights significant decreases in 13 C3-pyruvate labeling and the ratio of 13 C3-pyruvate/ 13 C3-lactate, while flux through glycolysis versus the pentose phosphate pathway is unaltered -as gleaned by the ratios of isotopologues M+2/M+3 of lactate ( Figure 4B). We observed a consistent dose-dependent increase in methemoglobin formation upon inhibitor treatment ( Figure 3A), confirming a functional loss of reductive capacity. Cellular insults, including oxidative damage, cause erythrocytes to be marked for removal from circulation. Senescent erythrocytes are characterized by a loss of membrane asymmetry that results in extracellular exposure of phosphatidylserine (PS), detected by annexin-V staining. [31][32][33] While normal cells would respond by promoting PS internalization by ATP-dependent flippases, inhibition of glycolysis is expected to disrupt such repair. As with methemoglobin formation, we find that glycolytic inhibition of human erythrocytes leads to a dose-dependent increase in annexin V positivity ( Figure 3B). Together, these results establish that glycolysis is essential to erythrocyte homeostasis. In addition, these studies reveal a likely mechanism by which genetic erythrocyte enzymopathies, which impair central carbon metabolism, reduce the circulatory halflife of erythrocytes. [34][35][36] Acute glycolytic inhibition does not directly cause erythrocyte lysis Our data support a mechanism by which glycolytic inhibition marks erythrocytes for macrophage-mediated destruction in vivo. In parallel, we evaluated whether acute glycolytic inhibition also leads directly to hemolysis. To our surprise, continuous exposure to high concentrations of enolase inhibitors does not cause lysis in static culture ( Figure 3C). These results indicate that senescence and premature splenic clearance and destruction, rather than direct hemolysis, lead to the decreased circulating half-life and anemia associated with interrupted glycolysis in erythrocytes in vivo.

Antimalarial effects of acute inhibition of erythrocyte glycolysis
Plasmodium spp. induce oxidative stress in infected erythrocytes. 37-39 Therefore, inhibitors that impair erythrocyte reductive capacity may result in malaria protection. Erythrocyte-targeted antimalarials would be highly desirable as "resistance-proof" antiparasitics. [40][41][42] We therefore evaluated whether acute inhibition of erythrocyte glycolysis was directly antiparasitic to cultured P. falciparum ( Figure 3D; Table 1). Phosphonates (SF-2312 and HEX) had poor antimalarial activity, with half maximal effective concentrations (EC50s) > 15M, likely due to poor cellular penetration. 43-45 However, lipophilic ester pro-drugs exhibit 20-to 160-fold increased antimalarial potencies, compared to their cognate phosphonates ( Figure 3D; Table 1). The most potent of these, POM-SF (EC50 = 25010nM), exhibits an in vitro potency in the submicromolar range as compared to other antimalarial compounds under development, such as the dihydroorotate dehydrogenase inhibitor DSM265 (EC50 = 53nM) and the recently FDA-approved tafenoquine (EC50 = 436nM). 46,47 Related phosphonate and pro-drug enolase inhibitors were evaluated for their antimalarial potency and their erythrotoxicity. We find that the antimalarial potency of each compound was highly correlated (r 2 =0.94) to its capacity to disrupt erythrocyte redox balance (methemoglobin EC50) ( Figure 3E). This strict correlation indicates that both effects are likely mediated through a single common target, erythrocyte enolase. In addition, the degree of inhibitor-induced anemia in vivo in mice is also correlated with the antimalarial potency in vitro (r 2 =0.67) ( Figure 3F).
POM-SF and POM-HEX are effective against multi-drug resistant P. falciparum We further evaluated the antimalarial activity of POM-SF and POM-HEX against parasites with reduced sensitivity to multiple classes of antimalarials, including chloroquine, sulfadoxinepyrimethamine, mefloquine, and artemisinin. As expected when targeting erythrocyte biology, while there is a modest decrease in sensitivity to POM-SF, multi-drug-resistant parasite lines are not cross-resistant to POM-HEX (Supplemental Table 8).

Chemical complementation with pyruvate improves antiparasitic selectivity
We evaluated strategies to rescue the erythrotoxicity of enolase inhibition. Pyruvate is a metabolic product immediately downstream of enolase and has been successfully and safely used in humans as a clinical antioxidant, including as a cardio-and neuro-protectant. 48-50 We find that exogenous pyruvate ameliorates methemoglobin formation caused by glycolytic inhibition of erythrocytes. In contrast, we find pyruvate potentiates parasite growth inhibition ( Figure 4). As a result, pyruvate supplementation markedly improves the selectivity index of glycolytic inhibitors (Table 1).

Parasite-selective enolase inhibition ameliorates cerebral malaria
Previous studies indicate that the glucose However, our in vitro data demonstrate that POM-HEX non-selectively harms both uninfected and parasitized erythroctyes, with similar erythrotoxic and antimalarial potencies (Table 1). In contrast, its phosphonate parent compound, HEX, is less erythrotoxic, likely due to its poor cellular penetration, although it retains modest antiparasitic activity. In murine Plasmodium infection, HEX treatment reduced overall parasite burden [d4, control parasitemia 7.83.3%

Non-human primates tolerate high doses of HEX with longer half life
Because HEX treatment improves survival in mice with cerebral malaria, we sought to investigate the pharmacodynamic and pharmacokinetic properties of Hex in non-human primates as a model for human application. We determined the serum half-life of HEX in cynomolgus monkeys and mice following sub-cutaneous injections. Although mice were treated with higher doses [400mg/kg], HEX-treated non-human primates [100mg/kg] display a higher serum concentration throughout the experiment and a markedly prolonged serum half-life [mice, 6 minutes vs. non-human primates, 1 hour] ( Figure 6). Compellingly, we find that the serum concentrations of HEX remain above the in vitro EC50 concentrations against Plasmodium for at least four hours. Longer serum parasite killing concentrations in non-human primates bodes well for improved antimalarial efficacy in humans.

DISCUSSION
As the central backbone of cellular metabolism, glycolysis controls energy production, redox balance, and biosynthesis. Antiglycolytic inhibitors-so-called "starvation mimetics"-are under increasing consideration as potential therapies for conditions including obesity, cancer, aging, autoimmunity, and infectious diseases. [53][54][55][56][57] While glycolytic disruption is generally well tolerated, glycolysis is critical to metabolic homeostasis in erythrocytes, which exclusively rely on the Embden-Meyerhof-Parnas pathway for ATP generation. As such, anemia is a key dose-limiting side effect and presents a major threat to otherwise promising antiglycolytic therapies. 27, 58 Our work provides a model through which to understand acute inhibition of erythrocyte glycolysis.
Inhibition of erythrocyte glycolysis is a particularly compelling target for antimalarial therapies.
Not only are the symptomatic asexual stages of Plasmodium spp. housed within the red cell niche, but long-standing evidence suggests that the outcome of clinical malaria is intertwined with red cell metabolism. Most notably, heritable erythrocyte disorders that appear to reduce malaria severity, including enzymopathies such as G6PDd and PKd, dysregulate glycolytic function and antioxidant capacity. 1,2 Because hemoglobin binds and competes for band 3 sequestration of glycolytic enzymes, malaria-protective hemoglobinopathies such as sickle cell trait also disrupt glycolytic regulation and sensitize erythrocytes to oxidative stress. 2 These natural genetic experiments suggest that modulating erythrocyte metabolism successfully improves malaria outcomes, and underscore that long-standing disruptions in erythrocyte metabolic homeostasis can be reasonably well tolerated. As Plasmodium spp. have an remarkable capacity for drug resistance, targeting host factors for antimalarial discovery is highly attractive to slow emergence of resistance.
However, our data demonstrate that non-discriminately disrupting glycolysis in infected and uninfected erythrocytes neither drives antiparasitic activity nor improves the outcome of acute malaria infection. Potent enolase inhibition does not forestall a lethal parasite burden (POM-SF and POM-HEX) and does not prevent cerebral malaria (POM-HEX). Rather, our nonprodrugged phosphonates (e.g., HEX) reduce parasite burden, extend survival, and decrease clinical symptoms of malaria. A possible explanation for the discrepancy in antimalarial efficacy of POM-HEX in vitro vs in vivo is that POM-HEX is rapidly converted to Hemi-POM-HEX in mouse plasma, such that effective plasma exposure to POM-HEX itself is quite low. 27 The high carboxylesterase activity in mouse (compared to human) plasma has been a recurrent complication for modeling the effects of POM-containing pro-drugs and suggests that the performance of POM-HEX in mice likely underestimates its efficacy in humans. 59 The discrepancy between mouse pharmacokinetics and primates is further highlighted in our study, in which the half-life of POM-HEX in primates is 10-fold longer than that in mice.
Because our compounds exhibit a tight correlation between antiparasitic and erythrotoxic potencies, both phenotypes are likely due to inhibition of a single common cellular target, human enolase. Our studies cannot unequivocally rule out other potential secondary targets, including the P. falciparum enolase, which is likely essential for intraerythrocytic parasite development. Because Plasmodium falciparum is an obligate intracellular parasite, it is challenging to evaluate cellular parasite enolase inhibition in isolation. In support of human enolase as the primary antimalarial target of these compounds, however, we also find little or no antiparasitic selectivity for most inhibitors in this series. Nonetheless, multiple-target agents that simultaneously inhibit the host and parasite enzymes would be expected to have durable, resistance-resistant antiparasitic activity. 60 Among our enolase inhibitors, two compounds are notably selective for antiparasitic activity over erythrocyte toxicity, the unmodified parent phosphonates, specifically HEX and DeoxySF-2312.
During intraerythrocytic development, Plasmodium spp. parasites establish new permeability pathways on the host erythrocyte membrane and are thus relatively permissive to small anions, specifically including phosphonates. 61-63 Compared to uninfected red cells that lack these transporters, Plasmodium-infected red cells are thus more susceptible to enolase inhibition with charged phosphonate inhibitors. Therefore, we suggest that small molecule inhibitors designed to specifically target glycolysis in Plasmodium-infected-but not uninfected-erythrocytes may represent a novel, successful, "resistance-proof" antimalarial strategy. Rapid compound clearance likely ameliorates the therapeutic effect of phosphonates such as HEX in mice, and our studies suggest that primates may represent a better pre-clinical model due to increased circulating half-life.
Our studies also establish a mechanistic model for anemia caused by acute antiglycolytic therapeutics through rapid premature erythrocyte senescence, rather than direct hemolysis.
Targeted enolase inhibitor treatment of erythrocytes leads to a metabolic signature characterized by a collapse in glutathione homeostasis. The antioxidant capacity of erythrocytes is dependent on energy production, as erythrocytes, lacking mitochondria, are uniquely dependent on aerobic glycolysis. Enolase inhibition reduces the net ATP per glucose molecule by half, thus hampering production of the energy-expensive cellular antioxidant, glutathione.
The production of this tripeptide (glutamate, cysteine, and glycine) requires ATP at each assembly step. Thus, enolase inhibition globally disturbs amino acid metabolism and leads to a state of energy depletion and the onset of oxidative stress.
Depletion of reduced glutathione establishes the molecular mechanism of methemoglobin formation and senescence in inhibitor-treated erythrocytes. Lacking organelles, erythrocytes undergo a distinct cell death known as eryptosis. Like apoptosis, eryptosis causes cell shrinkage, membrane blebbing, protease activation, and loss of membrane asymmetry.
Changing membrane phospholipids (e.g., phosphatidylserine) serves as a removal signal for circulating macrophages and splenic clearance. Our investigations thus explain the features of anemia observed in enolase-inhibitor-treated mice, who develop profound splenomegaly but not circulating extracellular hemoglobin (as would be expected by hemolysis).
As antiglycolytic therapies proceed through development, strategies to ameliorate erythrotoxicity will be required. We find that exogenous pyruvate supplementation restores reductive capacity to inhibitor-treated erythrocytes. This agrees with previous findings that pyruvate alone maintains erythrocyte ATP levels and 2,3-diphosphoglycerate (2,3-DPG) levels during long-term storage. 64,65 In contrast, pyruvate potentiates the antiparasitic effect of enolase inhibition, perhaps through a feed-back inhibition of parasite glycolysis. These data suggest a profound biological difference in glycolytic regulation between Plasmodium-infected and uninfected erythrocytes that may be harnessed for future antimalarial therapeutics. In addition, welltolerated oral pyruvate supplementation may represent a useful erythroprotective strategy alongside antiglycolytic "starvation-mimetic" therapies-to have one's cake, and eat it, too. 66-68 data analysis, experimental design, data interpretation and manuscript preparation. R. C-H.

Plasmodium falciparum growth inhibition assays
Cultures diluted to 1% parasitemia in 100μL wells were subjected to drug dilutions, solvent controls, and untreated uninfected erythrocyte controls in technical duplicates. Following a three-day incubation, DNA content was quantified using Picogreen (Life Technologies), as previously described. 71 Half-maximal effective concentrations (EC50) were determined from nonlinear regression of three biological replicates (GraphPad Prism).

Detection and quantification of methemoglobin formation
Methemoglobin (MetHb) was detected by measuring absorbance in the 630-640nm range, which is specific from other hemoglobin subforms (oxyhemoglobin, deoxyhemoglobin, and carboxyhemoglobin). Supernatants were removed and erythrocyte pellets were fully lysed and diluted, as described above. The absorption peak for oxyhemoglobin (575nm) and methemoglobin (630nm) were measured and the EC50 of MetHb formation was determined using non-linear regression analysis (GraphPad Prism).

Retro-orbital bleeding and hematocrit determination
Mice were anesthetized using isoflurane. Blood was collected using heparinized microhematocrit capillary tubes (Fisherbrand #22-362574) via retro-orbital approach and was sealed with hemato-seal capillary tube sealant (Fisherbrand #02-678). After spinning at 1,500xg for 10 min, hematocrit was calculated by dividing the measured height of red blood cell layer by the total height of the blood. Metabolite data processing and analysis Data were auto-scaled and normalized to zero-hour time points. Metabolites were removed from the dataset if greater than 50% of the samples contained missing values or the relative standard deviation among replicates exceeded 25%. Additionally, data was filtered using the interquartile range to reduce near-constant values from the experiment by 5%.

Sample collection for untargeted metabolomics
A principal component analysis of preprocessed data was plotted with the 95% confidence interval minimum volume enclosing ellipsoid for each respective group (MetaboAnalyst, cluster, plotly). A two-way repeated measures analysis of variance (ANOVA) was performed with multiple comparisons corrected for using a false discovery rate p-value cutoff of 0.05.
Metabolite-Pathway analysis was performed using the R package of MetaboAnalyst, as previously described. 75 Metabolite-Protein nodal analysis was performed using omicsnet.ca and as previously described. 28 Determining in vivo efficacy of enolase inhibitors against blood stage parasite burden

Determining in vivo efficacy of enolase inhibitors against cerebral malaria
The glucose mimetic 2-DG and enolase inhibitors POM-HEX, and HEX were evaluated in the P.
berghei mouse model of cerebral malarial, as previously described. 52 Briefly, in addition to groups of mice dosed as described above for POM-HEX and HEX, 2-DG was administered to an additional group at 200mg/kg twice daily and all were monitored for survival, parasitemia

hr after SC injection) by comparing integrals of HEX and TSP (reference) in 1H
spectra. To calculate HEX concentration for other time points, we obtained the 1D projections of 2D 1H-31P HSQC spectrum using the "proj" commend in TopSpin. In the projected spectra, the peak at 16 ppm chemical shift appeared only in HEX treated samples which we used to calculate HEX concentration. We took the ratio of integral of 16 ppm peak for samples with unknown concentration of HEX versus one with known concentration.

Lead Contact and Materials Availability
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Audrey Odom John (johna3@email.chop.edu).

20.
Stegmann    The respective EC50s are calculated using non-linear regression from each of the independent biological replicates (GraphPad Prism). Hb = hemoglobin, met-Hb = methemoglobin.     Figure 1A as a three-dimensional projection in twodimensional space.