2-Deoxy-d-glucose inhibits replication of novel coronavirus (SARS-CoV-2) with adverse effects on host cell metabolism

The treatment of viral infections is challenging owing to the intricate structure and metabolism of the viruses. In addition, they can highjack host cellular metabolism, mutate and adapt to harsh environmental conditions. The novel coronavirus (SARSCoV-2) displays further resilient attributes, making its eradication even more difficult. SARS-CoV-2 is an enveloped virus whose replication can be targeted by limiting the substrates available for structural incorporation. One such molecule that limits substrate availability and has received much attention lately is 2-Deoxy-d-glucose (2-DG). SARSCoV-2 infection induces glycolysis, impairs mitochondrial function and damages the infected cells. Administration of 2-DG can inhibit increased glycolytic flux and some other metabolic processes to cause cessation of viral replication. This article provides a review of the mechanism of action and safety concerns associated with administering 2DG in the treatment of COVID-19. The drug can have adverse effects on normal cell metabolism since it targets cells non-selectively, possibly in a dose-dependent manner. In addition, the drug has limited use in SARS-CoV-2 infection associated with stroke, hypoxic-ischemic encephalopathy, and critical illness.


Introduction
COVID-19 has emerged as the most rampant and deadliest pandemic of the 21 st century. As the numbers of infections continue to ramp up globally, research and drug approvals to find a definite cure for the disease have been expedited. However, there is a lack of drugs effective against the virus.
There is a bidirectional relationship between SARS-CoV-2 infection and increased blood glucose [1,2]. The cells infected with the virus, like cancer cells, have a high cellular uptake of glucose and its analogs [3,4] 2-Deoxy-d-glucose is another synthetic analog of glucose that acts primarily by inhibiting glycolysis. The novel coronavirus (SARS-CoV-2) upregulates glycolytic and other associated pathways to obtain substrates vital for its structure, function, and replication. It has proven effective in the treatment of cancer, seizures, and viral infections [5,6]. Since the drug causes complete cessation of replication of SARS-CoV-2 in monocytes infected with the virus, it could help treat COVID-19 along with the commonly used drugs [3]. However, its therapeutic use is limited due to adverse effects on the cellular metabolism noticed in pre-clinical trials and the limited safety data from clinical trials [7][8][9][10].

Mechanism of action
2-Deoxy-d-glucose (2-DG) is an analog of glucose ( Figure 1) that causes competitive inhibition of the rate-limiting enzyme -Hexokinase. This inhibition induces phosphorylation of glucose to 2-Deoxy-D-glucose-6-phosphate (2-DG-6-P). 2-DG-6-P acts as an allosteric and competitive inhibitor of Hexokinase, and its accumulation also inhibits the enzyme -Phosphoglucoisomerase [11,12]. Thus, 2-DG can inhibit glycolytic flux, although it can influence multiple other cellular pathways: The depletion of ATP causes catabolic switching while inhibiting the anabolic cellular processes (Figure 2a.). In addition, intracellular ATP depletion is also associated with necrosis as the major pathway of cell death due to membrane instability and extracellular ATP release. These events can result in over-activation of the immune system leading to cytokine storm and acute respiratory distress syndrome (ARDS) in COVID-19 [13].
Glycolysis and apoptosis are closely linked, but the association is poorly understood.
Localization of Hexokinase II (HKII) to mitochondria facilitates phosphorylation of glucose by ATP generated inside mitochondria and also has an anti-apoptotic effect by inhibiting the activity of some molecules (Figure 2b.) [14]. Hexokinase inhibition can thus induce apoptosis of the cells through the intrinsic pathway by destabilization of mitochondrial complexes. In addition, inhibition of glycolysis results in apoptosis through the extrinsic pathway and autophagy [12].

Figure 2 2-DG impacts cell metabolism and induces apoptosis
a. 2-DG causes glucose deprivation to decrease ATP and increase ROS production. As a result, AMPK is activated to increase cell metabolism in favor of increased catabolic activity. It is achieved through increased glycolysis by activating PFKFB and decreased fatty acid synthesis by inhibiting ACC. In addition, activated AMPK increases autophagy through ULK1. It also increases the expression of the tumor suppressor gene p53 to cause apoptosis.
b. Hexokinase is mobilized from the cytosol to mitochondria to associate with VDAC. This association inhibits the activity of pro-apoptotic molecules-Bak, Bad, Bax. Activation of these pro-apoptotic molecules can cause apoptosis through the release of cytochrome c into the mitochondria. 2-DG induces apoptosis by inhibiting the anti-apoptotic function of Hexokinase. Monocyte-macrophages at the sites of inflammation are metabolically analogous to the cancer cells. They have impaired mitochondrial function and switch to glycolysis to sustain the increased metabolic demand of their pro-inflammatory phenotype (M1) [17].
In contrast, the anti-inflammatory phenotype (M2) of monocyte-macrophage lineage predominantly depends on oxidative phosphorylation for ATP generation and reparation of the inflammatory damage. However, this phenotype is prevented from activation by mitochondrial dysfunction, AMPK downregulation, and p53 inactivation induced by inflammatory mediators produced in COVID-19 [4].
Aerobic glycolysis is also increased in the virally infected endothelial cells and pneumocytes of the alveolar-capillary barrier. Moreover, alveolar epithelial cells are increasingly predisposed to infection with SARS-CoV-2 than endothelial cells or monocytes, especially in the early phase of the infection [18].
Increased glycolytic activity is a major pathway driving viral replication and the inflammatory response in COVID-19 [3,19]. Evidence suggests that monocytes infected consequently, electron transport chain and citric acid cycle [3].
According to a recent study, most of the monocytes derived from peripheral blood of patients with COVID-19 pneumonia were redistributed in favor of increased intermediate/pro-inflammatory cells. They had decreased oxidative phosphorylation and glycolysis [20]. It is unclear whether these blood cells were harboring SARS-COV-2.
These metabolic effects could be exerted distantly by the mediators produced at the sites of inflammation since COVID-19 is associated with low viral detection rates in the blood [21][22][23].

Association between blood glucose and viral infection
There is a bidirectional relationship between diabetes and COVID-19. COVID-19 is more common and severe in obese people with insulin resistance and type II diabetes.
It is also associated with new-onset diabetes and exacerbation of metabolic complications of pre-existing diabetes -diabetic ketoacidosis and hyperosmolarity [1].
Hyperglycemia associated with SARS-CoV-2 infection is multifactorial -insulin resistance, the stress response to severe illness, pancreatic beta-cell dysfunction, use of steroids for treatment. Amongst these, insulin resistance probably induced by adipose tissue dysfunction is the primary factor underlying hyperglycemia in COVID-19 [24].
Golgi protein 73 (GP73) is a trans-membrane protein of the Golgi apparatus.
Hepatocyte injury is associated with increased expression of the protein and it could serve as a diagnostic and prognostic marker of liver injury in chronic HBV infection [25,26]. The circulating levels of the protein are approximately 2x elevated in SARS-CoV-2-infected patients relative to the reference population (p<0.0001) and its serum concentration correlates with the severity of COVID-19 [27]. It exerts its effect primarily through upregulation of hepatic and renal gluconeogenesis with minimal effect on glycogenolysis. Increased expression of GP73 could be responsible for new onset increase in blood glucose levels associated with SARS-CoV-2 infection [27]. However, since evidence of raised liver enzymes is present in only 14-53% cases with COVID-19 [28], GP73 mediated gluconeogenesis can't explain the increased blood glucose levels associated with SARS-COV-2 infection with no hepatocyte injury.
Elevated blood sugar levels cause dysfunction and cell death in diabetes due to increased cellular uptake of glucose. Hexokinase-2 (HK2) mediated glycolytic flux in people with diabetes can impair cellular functions without altering transcription.

ERK/MAPK and PI3K/AKT/mTOR signaling pathways modulate replication of MERS-
CoV [30]. Evidence suggests that SARS-CoV-2 infection is associated with increased blood glucose and activation of these pathways, which provides the substrates for viral replication and affects host cell metabolism ( Figure 3) [4,31]. Thus, impairment of the glycolytic pathway can cause a complete cessation of viral replication in cells infected with SARS-CoV-2 [4,32].

Effect of 2-DG on viruses
Since the structure and multiplication of the viruses need substrates and energy derived from the host, exploiting the increased glycolytic flux for these functions could be a  SARS-CoV2 induces the PI3K/Akt pathway through its effects on the host cell. PI3K/Akt pathway can stimulate glycolysis through increased glucose uptake via GLUT and activation of enzymes-HK, PGI, LDH. Due to activation of LDH and inhibition of PDH, pyruvate is preferentially converted to lactate. Upregulation of the pentose phosphate pathway through increased precursors and fatty acid synthesis through activated ATP citrate lyase provides substrates for viral replication and envelope formation. respiratory syncytial virus, and measles virus [33]. Limited data from human studies show that it is well tolerated [8][9][10]. However, since the drug can interfere with the normal cellular metabolism, its use in humans is concerning due to possible associated adverse effects that can arise after extrapolating the data obtained from the pre-clinical studies. The drug can adversely affect the neurological, cardiovascular, endocrinological, and hematological systems. trauma, meningitis, epilepsy. However, the neuroprotection is not absolute, and increased neuronal death is observed in pathological states associated with hypoxiastroke, vascular dementia, and trauma [40].

Cytomegalovirus (CMV) causes activation of intracellular calmodulin
The prevalence of neurological involvement in severe COVID-19 varies from 3.5% to 84% in different studies [41][42][43]. In a prospective observational study on 4,491 patients with COVID-19, 606 (13.5%) developed neurological symptoms [44]. The most common neurological diagnoses were -metabolic encephalopathy (6.8%), seizure (1.6%), stroke (1.9%), and hypoxic injury (1.4%). There were no cases of meningitis or myelitis, and RT-PCR tests conducted on CSF were negative. This indicates that CNS involvement with COVID-19 infection is an indirect consequence of systemic impairment caused by SARS-CoV-2. COVID-19 associated seizures can be theoretically treated by the administration of 2-DG [5]. However, the drug can have adverse effects in case of stroke and hypoxia-mediated brain injury.
The drug can have adverse effects on the peripheral nervous system. A clinical trial administering the glycolysis inhibitor dichloroacetate was prematurely terminated due to increased mortality and adverse effects of the drug on the peripheral nervous system [45]. This adverse effect could be attenuated by providing an alternate source of ATP via ketogenic or Atkin's diet [5,46]. However, since 2-DG administration results in a similar metabolic profile as caloric restriction, it is associated with poor clinical outcomes if used for treating critically ill patients for whom caloric restriction is absolutely contraindicated [46].

b. Cardiovascular effects
Chronic ingestion of 20 to 300 mg/kg 2-DG increases the risk of cardiotoxicity, pheochromocytoma, and mortality in rats [7]. The cardiotoxicity was reversible after 60 days, and a decline in cardiac function could be predicted by monitoring the trend of biomarkers like NT-proBNP and BNP levels [38]. Furthermore, it was associated with abnormal ECG changes like T-wave flattening and QT prolongation, which were nonserious and reversible within 24 hours [8].

c. Endocrinological effects
Hypoglycemia-associated autonomic failure: Administration of 2-DG is associated with cellular glucose deprivation (glucoprivation) and can impair resulting central and autonomic compensatory mechanisms ( Figure 4). The stress response induced secretion of glucocorticoids possibly mediates this hypoglycemia-associated autonomic failure, which may even be fatal [47]. 2-DG induced compensatory hyperglycemia can be prevented by the administration of beta-blockers.
Administration of 2-DG was associated with increased prevalence of pheochromocytoma in a pre-clinical study conducted in rats [7]. Alteration of the HIFα pathway and mitochondrial impairment induced by the administration of the drug can underlie this tumorigenesis.

d. Hematological effects
Platelet activation is associated with increased glycolysis and glycogenolysis. The 2-DG associated decrease in glucose uptake and metabolism can impair platelet aggregation and degranulation in vitro [48]. from the mitochondria to cytosol and eventual cell death.  Funding: This research received no external funding.