Transcriptomic analysis in patients with SARS-CoV-2 virus infection. Identification of the association of risk/ protective profiles against the disease

Background/Objectives: SARS-CoV2 causes the COVID-19 disease, capable of producing a severe acute respiratory syndrome. Several clinical variables and genetic variants have been related to a worse prognosis. The aim of this study is to measure if difference in the gene expression are associated with COVID-19 severity. Method(s): We performed RNA-seq Transcriptome in RNA extracted from lymphoblastoid cell line in 20 patients who require hospitalization (10 from the intensive care unit) in a GeneStudio S5 Plus Sequencer (Ion Torrent Technology). FASTQ files were obtained and trimmed using BBtools, BBduk for cutting, filtering and masking the data, and Dedupe for the elimination of duplicates. Mapping and counting matrix was done in bash using the Salmon program. Differential expression analysis and subsequent functional enrichment was performed using Rstudio (DESeq2, ClusterProfiler, GO and KEGG). Result(s): We observed that 2042 differentially expressed genes (1996 overexpressed, LFC>0 and 406 underexpressed, LFC<0) were obtained between patients who require hospitalization versus those in the intensive care unit. We found some genes previously SARS-CoV-2 associated (PGLYRP1, HDAC9 and FUT4). Furthermore, genes involved in the activity of the immune system and in inflammatory processes showed significant differences between cohorts (ABCF1 (LFC = -25.14, padj = 1.05e-13), ABHD16A (LFC = 25.00, padj = 1.05e-13) and IER3 (LFC = -24.45, padj = 2.43e-13)). Conclusion(s): We described differential expression in genes of the immune system and inflammatory processes that might be have a role in the risk of develop severe symptoms of COVID-19, including admission in the intensive care unit. This results should be validated by additional functional studies.

Changes in the proteomics of exhaled breath condensate under the influence of inhaled hydrogen in patients with post-COVID syndrome.

Aim. To study the effect of inhalation therapy with an active hydrogen (AH) on the protein composition of exhaled breath condensate (EBC) in patients with post-COVID syndrome (PCS). Material and methods. This randomized controlled parallel prospective study included 60 patients after coronavirus disease 2019 (COVID-19) with PCS during the recovery period and clinical manifestations of chronic fatigue syndrome who received standard therapy according to the protocol for managing patients with chronic fatigue syndrome (CFS). The patients were divided into 2 groups: group 1 (main) - 30 people who received standard therapy and AH inhalations (SUISONIA, Japan) for 10 days, and group 2 (control) - 30 medical workers who received only standard therapy. Patients in both groups were comparable in sex and mean age. All participants in the study were sampled with EBC on days 1 and 10. Samples were subjected to tryptic digestion and high-performance liquid chromatography combined with tandem mass spectrometry analysis using a nanoflow chromatograph (Dionex 3000) in tandem with a high-resolution time-of-flight mass spectrometer (timsTOF Pro). Results. A total of 478 proteins and 1350 peptides were identified using high resolution mass spectrometry. The number of proteins in samples after AH therapy, on average, is 12% more than before treatment. An analysis of the distribution of proteins in different groups of patients showed that only half of these proteins (112) are common for all groups of samples and are detected in EBC before, after, and regardless of hydrogen therapy. In addition to the qualitative difference in the EBC protein compositions in different groups, quantitative changes in the concentration of 36 proteins (mainly structural and protective) were also revealed, which together made it possible to reliably distinguish between subgroups before and after treatment. It is worth noting that among these proteins there are participants of blood coagulation (alpha-1-antitrypsin), chemokine- and cytokine-mediated inflammation, and a number of signaling pathways (cytoplasmic actin 2), response to oxidative stress (thioredoxin), glycolysis (glyceraldehyde-3- phosphate dehydrogenase), etc. Conclusion. The use of hydrogen therapy can contribute to the switching of a number of physiological processes, which may affect the success of recovery in PCS patients. In particular, the obtained results indicate the activation of aerobic synthesis of adenosine triphosphate in mitochondria by hydrogen therapy, which correlates well with the decrease in the blood lactate level detected by laboratory studies. At the same time, this therapy can inhibit pro-inflammatory activity, negatively affecting the coagulation and signaling pathways of integrins and apoptosis, and, in addition, activate protective pathways, tricarboxylic acid cycle, FAS signaling, and purine metabolism, which may be essential for effective recovery after COVID-19.Copyright © 2023 Vserossiiskoe Obshchestvo Kardiologov. All rights reserved.

Production of Modified Nucleosides in a Continuous Enzyme Membrane Reactor.

Nucleoside analogues are important compounds for the treatment of viral infections or cancers. While (chemo-)enzymatic synthesis is a valuable alternative to traditional chemical methods, the feasibility of such processes is lowered by the high production cost of the biocatalyst. As continuous enzyme membrane reactors (EMR) allow the use of biocatalysts until their full inactivation, they offer a valuable alternative to batch enzymatic reactions with freely dissolved enzymes. In EMRs, the enzymes are retained in the reactor by a suitable membrane. Immobilization on carrier materials, and the associated losses in enzyme activity, can thus be avoided. Therefore, we validated the applicability of EMRs for the synthesis of natural and dihalogenated nucleosides, using one-pot transglycosylation reactions. Over a period of 55 days, 2'-deoxyadenosine was produced continuously, with a product yield >90%. The dihalogenated nucleoside analogues 2,6-dichloropurine-2'-deoxyribonucleoside and 6-chloro-2-fluoro-2'-deoxyribonucleoside were also produced, with high conversion, but for shorter operation times, of 14 and 5.5 days, respectively. The EMR performed with specific productivities comparable to batch reactions. However, in the EMR, 220, 40, and 9 times more product per enzymatic unit was produced, for 2'-deoxyadenosine, 2,6-dichloropurine-2'-deoxyribonucleoside, and 6-chloro-2-fluoro-2'-deoxyribonucleoside, respectively. The application of the EMR using freely dissolved enzymes, facilitates a continuous process with integrated biocatalyst separation, which reduces the overall cost of the biocatalyst and enhances the downstream processing of nucleoside production.

FDA approved drugs with antiviral activity against SARS-CoV-2: From structure-based repurposing to host-specific mechanisms.

The continuing heavy toll of the COVID-19 pandemic necessitates development of therapeutic options. We adopted structure-based drug repurposing to screen FDA-approved drugs for inhibitory effects against main protease enzyme (Mpro) substrate-binding pocket of SARS-CoV-2 for non-covalent and covalent binding. Top candidates were screened against infectious SARS-CoV-2 in a cell-based viral replication assay. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. Additionally, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 µM), mebendazole (IC50 19 µM) and entacapone (IC50 9 µM). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential mechanisms. Although atovaquone is Dihydroorotate dehydrogenase (DHODH) inhibitor, we did not observe inhibition of DHODH at the respective SARS-CoV-2 IC50. Metabolomic profiling of atovaquone treated cells showed dysregulation of purine metabolism pathway metabolite, where ecto-5'-nucleotidase (NT5E) was downregulated by atovaquone at concentrations equivalent to its antiviral IC50. Atovaquone and mebendazole are promising candidates with SARS-CoV-2 antiviral activity. While mebendazole does appear to target Mpro, atovaquone may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.

Severe Prps1 Deficiency and Response to S-Adenosylmethionine and Nicotinamide Riboside Supplementation

Background: PRPS1 deficiency spectrum is an X-linked condition caused by pathogenic variants in PRPS1, which encodes for the PRPP enzyme involved in the purine synthesis pathway, among other metabolic pathways. Severely affected individuals, also known as Arts syndrome, have congenital sensorineural hearing loss, optic atrophy, developmental delays, ataxia, hypotonia, and recurrent infections. Infections often precipitate worsening of symptoms and many individuals pass away in childhood. Mildly to moderately affected individuals can have isolated hearing loss, also known as DFNX1, or hearing loss with later onset ataxia and optic neuropathy concerns, also known as CMTX5. Given the importance of PRPP in the role of purine synthesis as well as other cellular processes, including formation of NAD(P), supplementation of these pathways is a logical approach for these patients. 2 Arts syndrome patients were previously supplemented with S-adenosylmethionine, starting in mid-childhood, with improvement in infection severity and frequency, as well as stabilization of other symptoms. Recently another Arts syndrome patient was supplemented with S-adenosylmethionine and nicotinamide riboside, starting in early childhood, with improvement in infection frequency and developmental gains. Here we present a now 23 month old male patient with severe PRPS1 deficiency spectrum symptoms, who was started on S-adenosylmethionine and nicotinamide riboside supplementation. Result(s): This is a 23 month old male with developmental delay, retinal dystrophy, congenital bilateral sensorineural hearing loss, and hypotonia with a PRPS1 c.383A > T / p.Asp128Val likely pathogenic variant. He does not have a history of recurrent infections, however family reports relative isolation due to the Covid-19 pandemic. He sat unsupported at 10 months, crawled at 14 months, pulled to stand at 18 months, and is nonverbal. His uric acid testing was in the low range of normal. He had normal purine testing with low normal xanthine and hypoxanthine levels. At 19 months the patient started 20 mg/kg/d S-adenosylmethionine supplementation. At 20 months this was increased to 40 mg/kg/d S-adenosylmethionine and he started on 30 mg/kg/d nicotinamide riboside supplementation. Parents reported subjective improvement in strength and endurance with supplementation. He made significant developmental gains including walking with a walker. He had done well with occasional upper respiratory infections without regression in skills, worsening hypotonia, or increased respiratory needs. Unfortunately, very recently he had a cardiac arrest secondary to respiratory failure from rhinovirus/enterovirus and H. influzenzae pneumonia, for which he remains hospitalized at this time. Conclusion(s): This is the 4th reported patient with severe PRPS1 deficiency treated with S-adenosylmethionine supplementation and the 2nd reported patient treated with nicotinamide riboside supplementation. Both supplements have a limited side effect profile and have a biochemical basis for consideration in PRPS1 deficiency. He initially did well on supplementation with improvements in strength and endurance, as well as developmental gains, however his current trajectory remains to be seen. Unfortunately, NAD/NADP, ADP/ATP, and similar markers were unavailable to us and we plan to continue clinical monitoring on supplementation. Further studies are needed to evaluate the effectiveness of S-adenosylmethionine and nicotinamide riboside supplementation in these patients.Copyright © 2023

Humoral Responses Following SARSCoV- 2 Vaccination in Patients with Commonly Used Immunosuppressants in Neuromuscular Disorders

Background: Disease-specific studies have reported impaired humoral responses after SARS-CoV-2 vaccination in patients with immune-mediated inflammatory disorders (IMIDs) treated with specific immunosuppressants and immunomodulating agents. The objective of this study is to investigate the humoral immune response after SARS-CoV-2 vaccination in patients using immunosuppressive and immunomodulating mono- and combination therapies, focussing on frequently prescribed therapies for inflammatory neuromuscular diseases. Methods: National prospective observational cohort study in selected patients with prevalent IMIDs including neuromuscular disease, and immunosuppressive or immunomodulating monotherapy (n=1273), combination therapies (n=419), patients without immunosuppressants (n=473), and healthy controls (n=174). Anti-RBD IgG responses and neutralisation capacity were monitored following standard vaccination regimens and a three-vaccination regimen in subgroups. Hybrid immune responses, i.e. vaccination after previous SARS-CoV-2 infection, were studied as a proxy for recall responses. Findings: Sera from 1869 participants without and 470 participants with previous SARS-CoV-2 infection were analysed. We included 168 (7 2%) patients with inflammatory neuropathies and myopathies, and 127 (5 4%) patients with myasthenia gravis. Humoral responses did not differ between disorders. Anti-CD20 therapy and mycophenolate mofetil combined with corticosteroids were associated with lower relative risks (RR) for reaching seroconversion following standard vaccination (RR: 0 32 and 0 61 respectively). The monotherapies corticosteroids, purine antagonists, methotrexate, mycophenolate mofetil and IVIg were not associated with a lower RR for reaching seroconversion (RR: 0 97, 0 98, 1 01, 0 86, and 0 99, respectively). Similarly, corticosteroids combined with either methotrexate or purine antagonists was not associated with a lower RR for reaching seroconversion (RR 0 89). A third vaccination increased seroconversion for mycophenolate mofetil combination treatments but not for anti-CD20 therapies. Most immunosuppressant groups showed moderately reduced antibody titres after standard vaccination that, in subgroups, did not increase after a third vaccination, although seroconversion rates and neutralisation capacity were unaffected. In participants with previous SARS-CoV-2 infection, SARS-CoV-2 antibodies were boosted after vaccination, regardless of immunosuppressive treatment. Interpretation: Humoral responses following vaccination are impaired by specific immunosuppressants, most relevant for neuromuscular diseases being anti-CD20 and mycophenolate mofetil combination treatments. After standard vaccination regimens most immunosuppressants show equal seroconversion to controls although antibody titres may be moderately reduced. As neutralisation capacity and recall responses are also preserved in these patients, this is not likely to translate in loss of (short term) protection. Alternatively, in immunosuppressants showing poor humoral responses after standard vaccination regimens such as, a third vaccination resulted in additional seroconversion in mycophenolate mofetil combination treatments whereas the effect for anti-CD20 therapy was limited.

EXPERIENCE IN THE TREATMENT OF HAIRY CELL LEUKEMIA IN KAZAKHSTAN

Background: Hairy cell leukemia is a rare oncohematological disease that manifests itself in damage to the bone marrow and spleen. One of the characteristic changes in the bone marrow are large cells, a rounded nucleus and thin outgrowths - 'hairs' of the cytoplasm. The standard of care are drugs: cladribine and purine analogues-pentostatin. The complexity of therapy lies in the high risk of infections due to prolonged cytopenia during therapy. Aims: The aim of the study was to analyze the detection of hairy cell leukemia in Kazakhstan and the effectiveness of the cladribine regimen. Methods: In Kazakhstan 30 patients were diagnosed over the last 5-year period. All patients underwent cytological, histological examination of the bone marrow, bone marrow immunophenotyping. Thirty patient s were treated with cladribine intravenous infusion 0.1 mg/kg per day for 7 days. The average age was 54.6years (from 29 to 80 years), men-10, women-20. With a median follow-up of 4 years, the complete response (CR) was -25 patients, progression of disease - 3 patients, two patients die from infection. Results: Median duration of response was 38 months (range, 0.0+ to 52.0+). Median overall survival was not reached in all patients with CR. All patients had hematological complications in the form of grade 4 leukopenia - 30 patients, grade 4 thrombocytopenia - 28 patients, grade 3 thrombocytopenia - 2, patients grade 2 anemia - 9 patients. Emetic syndrome in the form of vomiting of the 3rd degree was in one patient, diarrhea of the 2nd degree in one patient. Eleven patients had febrile neutropenia. Two patients had a fatal outcome due to the development of pneumonia and subsequent sepsis. Summary/Conclusion: Treatment of patients with hairy cell leukemia with a purine analogue showed high efficiency, but was accompanied by prolonged cytopenia and frequent secondary infection. It is necessary to consider the issue of vaccination of this category of patients with anti-pneumococcal vaccine and also to conduct continuous monitoring. Ten patients with CR were vaccinated against coronavirus infection.

Post-COVID syndrome is associated with increased extracellular purine bases and neutrophil extracellular traps in the blood plasma

Post-COVID syndrome is characterized by fatigue, reduced exercise tolerance, muscle and joint pain, and psycho-emotional disorders. In the development of a generalized body response in a viral infection, abnormal defense responses are of great importance. We studied neutrophils, neutrophil extracellular traps (NETs), DNA degradation products (purine nitrogenous bases, PNBs), and traditional biochemical parameters. Aim. To determine biochemical parameters and the number of NETs and PNBs in the peripheral blood of patients with post-COVID syndrome. Materials and methods. The study included outpatients (n = 21) aged 18–59 years (36 [27 ÷ 50]). The control group consisted of 20 individuals aged 18–59 years (38.5 [29 ÷ 51.5]) without a past medical history of the coronavirus infection. All patients underwent a physical examination, their medical history was assessed, and the level of NETs and PNBs in the venous blood was determined. Results. 11 patients had a mild form of the disease in their past medical history, 7 – moderate, and 3 – severe. The most common symptoms in the patients were fatigue, headache, epigastric pain, dizziness, and joint pain. Hair loss and dyspnea were less common. The concentration of NETs and PNBs was higher in the patients with post-COVID syndrome than in the control group (p < 0.05). We detected NETs in the patients with post-COVID syndrome only in the form of filamentous structures. The concentration of extracellular purine bases in the blood of the patients with post-COVID syndrome was the highest in patients with moderate and severe acute periods. In patients with a mild acute period, the concentration of PNBs was 7.38 [0.0 ÷ 60.7] mg / ml, and in patients with moderate and severe acute periods – 19.15 [0.0 ÷ 33.5] and 34.19 [3.35 ÷ 70.0] mg / ml, respectively. Conclusion. Extracellular purine bases in concentrations capable of causing secondary alteration of cells are found in the peripheral blood of patients with post-COVID syndrome. Post-COVID syndrome is accompanied by the formation of filamentous NETs in the blood of patients. © 2022 Siberian State Medical University. All rights reserved.

AND KIDNEY OF MALE ALBINO RATS

As a result of the scale of the Covid 19 pandemic that launched from China in December 2019 and to limit its spread, several treatments were used to control this virus, including, hydroxychloroquine, favipiravir, lopinavir, remidesivir, tocilizumab, and anakinra. Favipiravir is an antiviral drug that works by inhibiting RNA-dependent RNA polymerase, favipiravir inhibited viral genome replication, which was most noticeable in the middle of the viral proliferation period. Favipiravir was found to have antiviral activity, Purine nucleosides or purine bases inhibit favipiravir, meaning that it competes with purine nucleosides rather than pyrimidine nucleosides, In a time-of-drugaddition test, to treat a variety of RNA viruses (influenza, West Nile, yellow fever, flaviviruses, arenaviruses, bunyaviruses and alphaviruses). Here, we show for the first time the histologycal effect of favipiravir on the liver and kidneys using albino rats, using light microscopy, where the optical microscopic revealed that normal doses in liver showed hepatic cords arranged, normal central vein and mild sinusoildal infiltration of mono nuclear leukocytes mainly lymphocytes, the hepatocytes showed mild granular cytoplasm while double doses showed little hemorrhagic foci and disarrangement of hepatic cords. The magnified sections revealed few of hepatocytes showed mild cloudy swelling associated with little figures of cellular necrosis. As for kidneys, the optical microscopic observations showed multiple foci of hemorrhage, the magnified section revealed congestion of glomerular capillary tuft and few of renal tubules showed mild granular or vacular degeneration. On the other hand, sections of renal medulla revealed normal appearance for normal doses while renal cortex and medulla were showed marked interstitial nephritis, which characterized by interstitial thickening due to infiltration of mono nuclear leukocytes and the renal tubules showed sever vacular degeneration and necrosis for double doses. These results can guide the safe use of favipiravir and reduce the risks to tissue the liver and kidney by using double doses.

Synthesis of Pyrimidine Conjugates with 4-(6-Amino-hexanoyl)-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine and Evaluation of Their Antiviral Activity.

A series of pyrimidine conjugates containing a fragment of racemic 7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine and its (S)-enantiomer attached via a 6-aminohexanoyl fragment were synthesized by the reaction of nucleophilic substitution of chlorine in various chloropyrimidines. The structures of the synthesized compounds were confirmed by 1H, 19F, and 13C NMR spectral data. Enantiomeric purity of optically active derivatives was confirmed by chiral HPLC. Antiviral evaluation of the synthesized compounds has shown that the replacement of purine with a pyrimidine fragment leads to a decrease in the anti-herpesvirus activity compared to the lead compound, purine conjugate. The studied compounds did not exhibit significant activity against influenza A (H1N1) virus.

Enzyme-mediated synthesis of Molnupiravir: paving the way for the application of biocatalysis in pharmaceutical industry

Molnupiravir (Lagevrio (R)) is an orally-administered anti-COVID-19 agent. Due to the urgency to meet the worldwide demand and the growing environmental concern, there is a need for speed in the industrial implementation of novel and efficient bioprocesses for Molnupiravir synthesis. This concept paper aims to review the most relevant milestones that have guided important developments in the enzyme-mediated synthesis of Molnupiravir, including detailed comments on the advantages and drawbacks of the different synthetic routes. Finally, based on a personal perspective, new greener processes for Molnupiravir manufacturing are proposed and discussed.

A REVIEW ON THE DEVELOPMENT OF FAVIPRAVIR AGAINST SARS COV 2 INFECTION

Covid 19, the disease first identified in the Chine city of Wuhan in December 2019 had been declared as a pandemic by WHO. This pandemic caused by Sars Cov 2 has resulted in 165.5 million infections and 3.5 million deaths globally, as of now. Till now no drug isavailable to fight against this deadly disease. The strategy adopted by drug discovery groups is drug repurposing which has not met much success with chloroquine as well as remdesivir. A relatively new candidate in the fray is favipravir which was originally developed by Toyama chemical company against influenza strains. Few synthetic routes are developed for this compound and the safety concerns are relatively few. If favourable results from the ongoing clinicaltrials arise, that may provide the therapeutic community a lethalweapon against the virus.

COVID-19 and One-Carbon Metabolism.

Dysregulation of one-carbon metabolism affects a wide range of biological processes and is associated with a number of diseases, including cardiovascular disease, dementia, neural tube defects, and cancer. Accumulating evidence suggests that one-carbon metabolism plays an important role in COVID-19. The symptoms of long COVID-19 are similar to those presented by subjects suffering from vitamin B12 deficiency (pernicious anemia). The metabolism of a cell infected by the SARS-CoV-2 virus is reshaped to fulfill the need for massive viral RNA synthesis, which requires de novo purine biosynthesis involving folate and one-carbon metabolism. Many aspects of host sulfur amino acid metabolism, particularly glutathione metabolism underlying antioxidant defenses, are also taken over by the SARS-CoV-2 virus. The purpose of this review is to summarize recent findings related to one-carbon metabolism and sulfur metabolites in COVID-19 and discuss how they inform strategies to combat the disease.

Diagnostic and therapeutic recommendations of the Polish Society of Haematologists and Transfusiologists and Polish Adult Leukemia Group-CLL for chronic lymphocytic leukemia in 2021

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, with a median age at diagnosis of approximately 70 years. The natural course of the disease varies greatly, and patients with non-progressive and asymptomatic leukemia do not require treatment. The results of CLL treatment have improved significantly in recent years, mainly due to the introduction of new, more effective drugs, including BCR inhibitors and BCL2 inhibitors. The new drugs are used continuously, while venetoclax in combination with anti-CD20 antibodies is used for 24 (rituximab) or 12 (obinutuzumab) months, depending on the type of antibody and line of therapy. The choice of treatment protocol should largely depend on the assessment of 17p deletion/TP53 mutation and immunoglobulin variable heavy chain (IGVH) mutation status, which correlate with a worse response to immunochemotherapy. The role of immunochemotherapy, which until recently was the mainstay of CLL treatment, has now significantly decreased. In the first-line, it is recommended only in patients without 17p deletion/TP53 mutation, with mutated IGVH. Other patients should receive novel targeted therapies. However, at the time of the preparation of these recommendations, these therapies are not available in the firs-line of treatment in Poland. Novel targeted therapies play a major role in the treatment of refractory/relapsed CLL, and immunochemotherapy is recommended primarily in patients with a long-term response to first-line therapy. In this article, we present an update of the guidelines for the diagnosis and treatment of CLL, including the treatment of autoimmune complications, as well as the prophylaxis and treatment of infections, developed by the Polish Society of Haematologists and Transfusiologists and PALG-CLL Working Group.

Can Probiotics and Diet Promote Beneficial Immune Modulation and Purine Control in Coronavirus Infection?

Infection caused by the SARS-CoV-2 coronavirus worldwide has led the World Health Organization to declare a COVID-19 pandemic. Because there is no cure or treatment for this virus, it is emergingly urgent to find effective and validated methods to prevent and treat COVID-19 infection. In this context, alternatives related to nutritional therapy might help to control the infection. This narrative review proposes the importance and role of probiotics and diet as adjunct alternatives among the therapies available for the treatment of this new coronavirus. This review discusses the relationship between intestinal purine metabolism and the use of Lactobacillus gasseri and low-purine diets, particularly in individuals with hyperuricemia, as adjuvant nutritional therapies to improve the immune system and weaken viral replication, assisting in the treatment of COVID-19. These might be promising alternatives, in addition to many others that involve adequate intake of vitamins, minerals and bioactive compounds from food.

Immune reconstitution in patients with classical hairy cell leukemia during BRAF inhibitor treatment

For patients with classical hairy cell leukemia (HCL) standard treatment options such as purine analogues (PA) achieve a durable response, but are associated with severe immunosuppression. In particular, PAs cause long-lasting depletion of CD4+-lymphocytes. The BRAF inhibitor vemu-rafenib is effective in HCL but its use in first line treatment is currently limited to select clinical situations such as active infection. There is a lack of clinical data on the impact of BRAF inhibitors on immune function or response to vaccines in HCL. Here, we report the use of vemurafenib in four patients with HCL during the coronavirus disease 2019 (COVID-19) pandemic with detailed immune monitoring during treatment. All patients responded to BRAFi with normalization of peripheral blood counts. None of the patients developed neutropenia or severe infection. We observed stable CD4+ and CD8+ T-lymphocyte counts while receiving vemurafenib (median treatment duration 131 days). Immunoglobulin levels were normal in all patients without decline. 3 out of 4 patients received the SARS-CoV-2 vaccination (Pfizer-BioNTech) during vemuraf-enib treatment. The IgG antibody levels against the spike-protein of SARS-CoV-2 were detectable in 3 out of 3 patients (2-12 weeks after the second vaccination). Our findings suggest that BRAF inhibitors have limited effect on cellular and humoral immune function. The findings may support the use of BRAF inhibitors during the current pandemic to avoid the potentially detrimental effects of PA and thus minimize COVID-19 related morbidity and mortality in patients without SARS-CoV-2 vaccination.

The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models.

The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 µM, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) and ~100-fold reductions in Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 100 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus.

Genome-Scale Metabolic Model of Infection with SARS-CoV-2 Mutants Confirms Guanylate Kinase as Robust Potential Antiviral Target.

The current SARS-CoV-2 pandemic is still threatening humankind. Despite first successes in vaccine development and approval, no antiviral treatment is available for COVID-19 patients. The success is further tarnished by the emergence and spreading of mutation variants of SARS-CoV-2, for which some vaccines have lower efficacy. This highlights the urgent need for antiviral therapies even more. This article describes how the genome-scale metabolic model (GEM) of the host-virus interaction of human alveolar macrophages and SARS-CoV-2 was refined by incorporating the latest information about the virus's structural proteins and the mutant variants B.1.1.7, B.1.351, B.1.28, B.1.427/B.1.429, and B.1.617. We confirmed the initially identified guanylate kinase as a potential antiviral target with this refined model and identified further potential targets from the purine and pyrimidine metabolism. The model was further extended by incorporating the virus' lipid requirements. This opened new perspectives for potential antiviral targets in the altered lipid metabolism. Especially the phosphatidylcholine biosynthesis seems to play a pivotal role in viral replication. The guanylate kinase is even a robust target in all investigated mutation variants currently spreading worldwide. These new insights can guide laboratory experiments for the validation of identified potential antiviral targets. Only the combination of vaccines and antiviral therapies will effectively defeat this ongoing pandemic.

Methotrexate inhibits SARS-CoV-2 virus replication "in vitro".

In early 2020 the new respiratory syndrome COVID-19 (caused by the zoonotic SARS-CoV-2 virus) spread like a pandemic, starting from Wuhan, China, causing severe economic depression. Despite some advances in drug treatments of medical complications in the later stages of the disease, the pandemic's death toll is tragic, as no vaccine or specific antiviral treatment is currently available. By using a systems approach, we identify the host-encoded pathway, which provides ribonucleotides to viral RNA synthesis, as a possible target. We show that methotrexate, an FDA-approved inhibitor of purine biosynthesis, potently inhibits viral RNA replication, viral protein synthesis, and virus release. The effective antiviral methotrexate concentrations are similar to those used for established human therapies using the same drug. Methotrexate should be most effective in patients at the earliest appearance of symptoms to effectively prevent viral replication, diffusion of the infection, and possibly fatal complications.

An Expedient Synthesis of Flexible Nucleosides through Enzymatic Glycosylation of Proximal and Distal Fleximer Bases.

The structurally unique "fleximer" nucleosides were originally designed to investigate how flexibility in a nucleobase could potentially affect receptor-ligand recognition and function. Recently they have been shown to have low-to-sub-micromolar levels of activity against a number of viruses, including coronaviruses, filoviruses, and flaviviruses. However, the synthesis of distal fleximers in particular has thus far been quite tedious and low yielding. As a potential solution to this issue, a series of proximal fleximer bases (flex-bases) has been successfully coupled to both ribose and 2'-deoxyribose sugars by using the N-deoxyribosyltransferase II of Lactobacillus leichmannii (LlNDT) and Escherichia coli purine nucleoside phosphorylase (PNP). To explore the range of this facile approach, transglycosylation experiments on a thieno-expanded tricyclic heterocyclic base, as well as several distal and proximal flex-bases were performed to determine whether the corresponding fleximer nucleosides could be obtained in this fashion, thus potentially significantly shortening the route to these biologically significant compounds. The results of those studies are reported herein.