Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19.

Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including ß-hydroxybutyrate (BHB)1-6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with  influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.

Inhibition Mechanism of SARS-CoV-2 Main Protease with Ketone-Based Inhibitors Unveiled by Multiscale Simulations: Insights for Improved Designs*.

We present the results of classical and QM/MM simulations for the inhibition of SARS-CoV-2 3CL protease by a hydroxymethylketone inhibitor, PF-00835231. In the noncovalent complex the carbonyl oxygen atom of the warhead is placed in the oxyanion hole formed by residues 143 to 145, while P1-P3 groups are accommodated in the active site with interactions similar to those observed for the peptide substrate. According to alchemical free energy calculations, the P1' hydroxymethyl group also contributes to the binding free energy. Covalent inhibition of the enzyme is triggered by the proton transfer from Cys145 to His41. This step is followed by the nucleophilic attack of the Sγ atom on the carbonyl carbon atom of the inhibitor and a proton transfer from His41 to the carbonyl oxygen atom mediated by the P1' hydroxyl group. Computational simulations show that the addition of a chloromethyl substituent to the P1' group may lower the activation free energy for covalent inhibition.

A new class of α-ketoamide derivatives with potent anticancer and anti-SARS-CoV-2 activities.

Inhibitors of the proteasome have been extensively studied for their applications in the treatment of human diseases such as hematologic malignancies, autoimmune disorders, and viral infections. Many of the proteasome inhibitors reported in the literature target the non-primed site of proteasome's substrate binding pocket. In this study, we designed, synthesized and characterized a series of novel α-keto phenylamide derivatives aimed at both the primed and non-primed sites of the proteasome. In these derivatives, different substituted phenyl groups at the head group targeting the primed site were incorporated in order to investigate their structure-activity relationship and optimize the potency of α-keto phenylamides. In addition, the biological effects of modifications at the cap moiety, P1, P2 and P3 side chain positions were explored. Many derivatives displayed highly potent biological activities in proteasome inhibition and anticancer activity against a panel of six cancer cell lines, which were further rationalized by molecular modeling analyses. Furthermore, a representative α-ketoamide derivative was tested and found to be active in inhibiting the cellular infection of SARS-CoV-2 which causes the COVID-19 pandemic. These results demonstrate that this new class of α-ketoamide derivatives are potent anticancer agents and provide experimental evidence of the anti-SARS-CoV-2 effect by one of them, thus suggesting a possible new lead to develop antiviral therapeutics for COVID-19.

The dark side of the spoon - glucose, ketones and COVID-19: a possible role for ketogenic diet?

The novel coronavirus disease (COVID-19) is posing a serious challenge to the health-care systems worldwide, with an enormous impact on health conditions and loss of lives. Notably, obesity and its related comorbidities are strictly related with worse clinical outcomes of COVID-19 disease. Recently, there is a growing interest in the clinical use of ketogenic diets (KDs), particularly in the context of severe obesity with related metabolic complications. KDs have been proven effective for a rapid reduction of fat mass, preserving lean mass and providing an adequate nutritional status. In particular, the physiological increase in plasma levels of ketone bodies exerts important anti-inflammatory and immunomodulating effects, which may reveal as precious tools to prevent infection and potential adverse outcomes of COVID-19 disease. We discuss here the importance of KDs for a rapid reduction of several critical risk factors for COVID-19, such as obesity, type 2 diabetes and hypertension, based on the known effects of ketone bodies on inflammation, immunity, metabolic profile and cardiovascular function. We do believe that a rapid reduction of all modifiable risk factors, especially obesity with its metabolic complications, should be a pillar of public health policies and interventions, in view of future waves of SARS-CoV-2 infection.

Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19.

The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.

COVID-19: Proposing a Ketone-Based Metabolic Therapy as a Treatment to Blunt the Cytokine Storm.

Human SARS-CoV-2 infection is characterized by a high mortality rate due to some patients developing a large innate immune response associated with a cytokine storm and acute respiratory distress syndrome (ARDS). This is characterized at the molecular level by decreased energy metabolism, altered redox state, oxidative damage, and cell death. Therapies that increase levels of (R)-beta-hydroxybutyrate (R-BHB), such as the ketogenic diet or consuming exogenous ketones, should restore altered energy metabolism and redox state. R-BHB activates anti-inflammatory GPR109A signaling and inhibits the NLRP3 inflammasome and histone deacetylases, while a ketogenic diet has been shown to protect mice from influenza virus infection through a protective γδ T cell response and by increasing electron transport chain gene expression to restore energy metabolism. During a virus-induced cytokine storm, metabolic flexibility is compromised due to increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that damage, downregulate, or inactivate many enzymes of central metabolism including the pyruvate dehydrogenase complex (PDC). This leads to an energy and redox crisis that decreases B and T cell proliferation and results in increased cytokine production and cell death. It is hypothesized that a moderately high-fat diet together with exogenous ketone supplementation at the first signs of respiratory distress will increase mitochondrial metabolism by bypassing the block at PDC. R-BHB-mediated restoration of nucleotide coenzyme ratios and redox state should decrease ROS and RNS to blunt the innate immune response and the associated cytokine storm, allowing the proliferation of cells responsible for adaptive immunity. Limitations of the proposed therapy include the following: it is unknown if human immune and lung cell functions are enhanced by ketosis, the risk of ketoacidosis must be assessed prior to initiating treatment, and permissive dietary fat and carbohydrate levels for exogenous ketones to boost immune function are not yet established. The third limitation could be addressed by studies with influenza-infected mice. A clinical study is warranted where COVID-19 patients consume a permissive diet combined with ketone ester to raise blood ketone levels to 1 to 2 mM with measured outcomes of symptom severity, length of infection, and case fatality rate.

Michael Acceptors Tuned by the Pivotal Aromaticity of Histidine to Block COVID-19 Activity.

The question of whether COVID protease (SARS-CoV-2 Mpro) can be blocked by inhibitors has been examined, with a particularly successful performance exhibited by α-ketoamide derivative substrates like 13b of Hilgenfeld and co-workers (Zhang, L., et al. Science 2020, 368, 409-412). After the biological characterization, here density functional theory calculations explain not only how inhibitor 13b produces a thermodynamically favorable interaction but also how to reach it kinetically. The controversial and unprovable concept of aromaticity here enjoys being the agent that rationalizes the seemingly innocent role of histidine (His41 of Mpro). It has a hydrogen bond with the hydroxyl group and is the proton carrier of the thiol of Cys145 at almost zero energy cost that favors the interaction with the inhibitor that acts as a Michael acceptor.

Induction of ketosis as a potential therapeutic option to limit hyperglycemia and prevent cytokine storm in COVID-19.

The severe form of coronavirus disease 19 (COVID-19) is characterized by cytokine storm syndrome (CSS) and disseminated intravascular coagulation (DIC). Diabetes, obesity, and hypertension have, as minor common denominators, chronic low-grade inflammation and high plasma myeloperoxidase levels, which could be linked to pulmonary phagocytic hyperactivation and CSS. The hyperactivation of M1 macrophages with a proinflammatory phenotype, which is linked to aerobic glycolysis, leads to the recruitment of monocytes, neutrophils, and platelets from circulating blood and plays a crucial role in thrombo-inflammation (as recently demonstrated in COVID-19) through the formation of neutrophil extracellular traps and monocyte-platelet aggregates, which could be responsible for DIC. The modulation of glucose availability for activated M1 macrophages by means of a eucaloric ketogenic diet (EKD) could represent a possible metabolic tool for reducing adenosine triphosphate production from aerobic glycolysis in the M1 macrophage phenotype during the exudative phase. This approach could reduce the overproduction of cytokines and, consequently, the accumulation of neutrophils, monocytes, and platelets from the blood. Second, an EKD could be advantageous for the metabolism of anti-inflammatory M2 macrophages because these cells predominantly express oxidative phosphorylation enzymes and are best fed by the oxidation of fatty acids in the mitochondria. An EKD could guarantee the availability of free fatty acids, which are an optimal fuel supply for these cells. Third, an EKD, which could reduce high lactate formation by macrophages due to glycolysis, could favor the production of interferon type I, which are inhibited by excessive lactate production. From a practical point of view, the hypothesis, in addition to being proven in clinical studies, must obviously take into account the contraindications of an EKD, particularly type 1 or 2 diabetes treated with drugs that can cause hypoglycemia, to avoid the risk for side effects of the diet.

Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2*.

The global pandemic caused by SARS-CoV-2 calls for the fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high-throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including unnatural amino acids that targets the nonprimed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within a human proteome without background. We expect that these reagents will be useful in the drug-development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses.