Dual computational and biological assessment of some promising nucleoside analogs against the COVID-19-Omicron variant.

Nucleoside analogs/derivatives (NAs/NDs) with potent antiviral activities are now deemed very convenient choices for the treatment of coronavirus disease 2019 (COVID-19) arisen by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. At the same time, the appearance of a new strain of SARS-CoV-2, the Omicron variant, necessitates multiplied efforts in fighting COVID-19. Counteracting the crucial SARS-CoV-2 enzymes RNA-dependent RNA polymerase (RdRp) and 3'-to-5' exoribonuclease (ExoN) jointly altogether using the same inhibitor is a quite successful new plan to demultiplicate SARS-CoV-2 particles and eliminate COVID-19 whatever the SARS-CoV-2 subtype is (due to the significant conservation nature of RdRps and ExoNs in the different SARS-CoV-2 strains). Successive in silico screening of known NAs finally disclosed six different promising NAs, which are riboprine/forodesine/tecadenoson/nelarabine/vidarabine/maribavir, respectively, that predictably can act through the planned dual-action mode. Further in vitro evaluations affirmed the anti-SARS-CoV-2/anti-COVID-19 potentials of these NAs, with riboprine and forodesine being at the top. The two NAs are able to effectively antagonize the replication of the new virulent SARS-CoV-2 strains with considerably minute in vitro anti-RdRp and anti-SARS-CoV-2 EC50 values of 189 and 408 nM for riboprine and 207 and 657 nM for forodesine, respectively, surpassing both remdesivir and the new anti-COVID-19 drug molnupiravir. Furthermore, the favorable structural characteristics of the two molecules qualify them for varied types of isosteric and analogistic chemical derivatization. In one word, the present important outcomes of this comprehensive dual study revealed the anticipating repurposing potentials of some known nucleosides, led by the two NAs riboprine and forodesine, to successfully discontinue the coronaviral-2 polymerase/exoribonuclease interactions with RNA nucleotides in the SARS-CoV-2 Omicron variant (BA.5 sublineage) and accordingly alleviate COVID-19 infections, motivating us to initiate the two drugs' diverse anti-COVID-19 pharmacological evaluations to add both of them betimes in the COVID-19 therapeutic protocols.

Association of Angiotensin Converting Enzyme 2 (ACE2) Gene Polymorphisms with Disease Severity in Indian Covid-19 Patients: A Next-gen Sequencing Approach

The clinical picture of COVID-19 ranges from asymptomatic to mild, moderate or severe disease sometimes leading to death. Differences in the interaction between SARS-CoV-2 Spike (S) protein and angiotensin converting enzyme 2 (ACE2) protein may lead to differences in disease severity. We studied whether ACE2 polymorphisms are associated with disease severity and outcome. We recruited 114 patients between July 2020 - March 2022 confirmed positive by RTPCR for COVID-19 with different degrees of severity (21 mild, 29 moderate, 34 severe, 30 death) and 30 controls (10 non-vaccinated+ 20 vaccinated) who were RT-PCR negative inspite of high-risk contact. Next-gen sequencing was done on MiSeq (Illumina) using amplicon-based targeted sequencing approach using a custom-designed panel to sequence all the exons of ACE2 gene. SPSS ver.26 was used for analysis. The following ACE2 variants were identified on the Local Run Manager (LRM) software from Illumina: (i) rs2285666 (c.439+4G>A) splice region variant, in controls (60%) and Patients (45.8%), (ii) rs4646140 (c.802+24G>A) intronic variant in 4/114 patients and 1/30 controls, (iii) rs41303171 ( c.2158A>G) missense variant in 2/114 patients, (iv) rs536749578 (c.2114+9T>C) intron variant, (v) rs763994205 (c.868A>C) missense variant and (vi) rs7595907 (c.656G>A) missense variant in 1/114 patient each only. rs2285666 was observed in equal frequency ( 60%) in vaccinated and non-vaccinated controls. rs2285666 was observed amongst different severity groups: Mild (80.95%), Moderate (37.93%), Severe (44.11%), and Death (56.67%) revealing association with disease severity, probably having a protective effect. However, these results need to be confirmed on larger sample sizes.

Plant-Derived Natural Non-Nucleoside Analog Inhibitors (NNAIs) against RNA-Dependent RNA Polymerase Complex (nsp7/nsp8/nsp12) of SARS-CoV-2.

The emergence of fast-spreading SARS-CoV-2 mutants has sparked a new phase of COVID-19 pandemic. There is a dire necessity for antivirals targeting highly conserved genomic domains on SARS-CoV-2 that are less prone to mutation. The nsp12, also known as the RNA-dependent RNA-polymerase (RdRp), the core component of 'SARS-CoV-2 replication-transcription complex', is a potential well-conserved druggable antiviral target. Several FDA-approved RdRp 'nucleotide analog inhibitors (NAIs)' such as remdesivir, have been repurposed to treat COVID-19 infections. The NAIs target RdRp protein translation and competitively block the nucleotide insertion into the RNA chain, resulting in the inhibition of viral replication. However, the replication proofreading function of nsp14-ExoN could provide resistance to SARS-CoV-2 against many NAIs. Conversely, the 'non-nucleoside analog inhibitors (NNAIs)' bind to allosteric sites on viral polymerase surface, change the redox state; thereby, exert antiviral activity by altering interactions between the enzyme substrate and active core catalytic site of the RdRp. NNAIs neither require metabolic activation (unlike NAIs) nor compete with intracellular pool of nucleotide triphosphates (NTPs) for anti-RdRp activity. The NNAIs from phytonutrient origin are potential antiviral candidates compared to their synthetic counterparts. Several in-silico studies reported the antiviral spectrum of natural phytonutrient-NNAIs such as Suramin, Silibinin (flavonolignan), Theaflavin (tea polyphenol), Baicalein (5,6,7-trihydroxyflavone), Corilagin (gallotannin), Hesperidin (citrus bioflavonoid), Lycorine (pyrrolidine alkaloid), with superior redox characteristics (free binding energy, hydrogen-bonds, etc.) than antiviral drugs (i.e. remdesivir, favipiravir). These phytonutrient-NNAIs also exert anti-inflammatory, antioxidant, immunomodulatory and cardioprotective functions, with multifunctional therapeutic benefits in the clinical management of COVID-19.

Oligonucleotide usage in coronavirus genomes mimics that in exon regions in host genomes.

BACKGROUND: Viruses use various host factors for their growth, and efficient growth requires efficient use of these factors. Our previous study revealed that the occurrence frequency of oligonucleotides in the influenza virus genome is distinctly different among derived hosts, and the frequency tends to adapt to the host cells in which they grow. We aimed to study the adaptation mechanisms of a zoonotic virus to host cells. METHODS: Herein, we compared the frequency of oligonucleotides in the genome of alpha- and betacoronavirus with those in the genomes of humans and bats, which are typical hosts of the viruses. RESULTS: By comparing the oligonucleotide frequency in coronaviruses and their host genomes, we found a statistically tested positive correlation between the frequency of coronaviruses and that of the exon regions of the host from which the virus is derived. To examine the characteristics of early-stage changes in the viral genome, which are assumed to accompany the host change from non-humans to humans, we compared the oligonucleotide frequency between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the beginning of the pandemic and the prevalent variants thereafter, and found changes towards the frequency of the host exon regions. CONCLUSIONS: In alpha- and betacoronaviruses, the genome oligonucleotide frequency is thought to change in response to the cellular environment in which the virus is replicating, and actually the frequency has approached the frequency in exon regions in the host.

Growth hormone deficiency and SHOX variants present in 2 siblings short for the family target

P1 was referred with short stature aged 2.4 yrs (HtSDS -2.1). Mother's height 165cm (+0.5 SD), father's 185cm (+1.55 SD). Growth hormone (GH) deficiency was diagnosed following two GH stimulation tests (peak GH 6.8 ng/ml at 3.2 yrs, 5.17 ng/ml at 3.7 yrs) IGF1 4.6 nmol/L (1.7-27.6) at 2.5 yrs. Pituitary MRI was normal. GH treatment was started at 5 years at HtSD -2.2. Once P1 demonstrated catchup growth (up one centile in 7 months), P1's older sister P2 was referred for investigation of short stature (HtSDS -1.5). She was not dysmorphic but her mother noted antenatal scans had short foetal femur lengths on the 5th centile. Birth weight was on the 10th centile at 2.65kg at 38 weeks gestation. Short stature was present from age 3 with height tracking along the 2nd centile. Her GH was 7.17 ng/ml at time 0 on a growth hormone stimulation test, IGF1 25.9 nmol/L (3.1 - 51.9). This was performed during the COVID pandemic and could not be repeated. It was felt that GH deficiency was unlikely so a skeletal survey was requested. This showed mild Madelung deformity and short 4th/5th metacarpals. Subsequent SHOX analysis revealed that she was heterozygous for 2 SHOX dosage abnormalities in cis. The first was a deletion located between 133 kb and 163 kb downstream of the SHOX gene. The second was a duplication that included the final exons of the SHOX gene (exons4-6a). Family testing revealed the same SHOX variants in P1's father, who was the smallest in class in early childhood and had grown late into adulthood, reaching a tall final height at aged 21. Mother and P1 did not have these SHOX variants. P1 has demonstrated excellent catch-up growth on GH treatment, HtSDS +0.4. IGF1 36.5 nmol/L (2.7-45.5) at 7.3 years. P2 has been on GH treatment for over a year, latest HtSDS - 1. Conclusion(s): Both siblings were short for the family target. Following a diagnosis of GH deficiency in one sibling, the investigative pathway for the other was assumed. Routine karyotype was performed after GH stimulation test with SHOX analysis requested after the skeletal survey report. The siblings' father has the same SHOX variants as P2 and is tall. Biological effects of SHOX deletion appears to be variable in this family and is present in P2 radiologically. The duplication is of uncertain significance.

Strong Dual Antipolymerase/Antiexonuclease Actions of Some Aminothiadiazole Antioxidants: A Promising In-Silico/In-Vitro Repurposing Research Study against the COVID-19 Omicron Virus (B.1.1.529.3 Lineage).

Currently, nitrogen-containing heterocyclic virucides take the lead as top options for treating the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and their escorting disease, the coronavirus disease 2019 (COVID-19). But unfortunately, the sudden emergence of a new strain of SARS-CoV-2, the Omicron variant and its lineages, complicated matters in the incessant COVID-19 battle. Goaling the two paramount coronaviral-2 multiplication enzymes RNA-dependent RNA polymerase (RdRp) and 3'-to-5' exoribonuclease (ExoN) at synchronous times using single ligand is a quite effective new binary avenue to restrain SARS-CoV-2 reproduction and cease COVID-19 progression irrespective of the SARS-CoV-2 strain type, as RdRps and ExoNs are vastly conserved in all SARS-CoV-2 strains. The presented in-silico/in-vitro research winnowed our own small libraries of antioxidant nitrogenous heterocyclic compounds, inspecting for the utmost convenient drug candidates expectedly capable of effectively working through this dual tactic. Computational screening afforded three promising compounds of the antioxidant 1,3,4-thiadiazole class, which were named ChloViD2022, Taroxaz-26, and CoViTris2022. Subsequent biological examination, employing the in-vitro anti-RdRp/anti-ExoN and anti-SARS-CoV-2 assays, exclusively demonstrated that ChloViD2022, CoViTris2022, and Taroxaz-26 could efficiently block the replication of the new lineages of SARS-CoV-2 with considerably minute anti-RdRp and anti-SARS-CoV-2 EC50 values of about 0.18 and 0.44 µM for ChloViD2022, 0.22 and 0.72 µM for CoViTris2022, and 0.25 and 0.78 µM for Taroxaz-26, in the order, overtaking the standard anti-SARS-CoV-2 drug molnupiravir. These biochemical findings were optimally presupported by the results of the prior in-silico screening, suggesting that the three compounds might potently hit the catalytic active sites of the virus's RdRp and ExoN enzymes. Furthermore, the perfect pharmacophoric features of ChloViD2022, Taroxaz-26, and CoViTris2022 molecules make them typical dual inhibitors of SARS-CoV-2 replication and proofreading, with their relatively flexible structures eligible for diverse forms of chemical modification. In sum, the current important results of this thorough research work exposed the interesting repurposing potential of the three 2-amino-1,3,4-thiadiazole ligands, ChloViD2022, Taroxaz-26, and CoViTris2022, to effectively conflict with the vital biointeractions between the coronavirus's polymerase/exoribonuclease and the four essential RNA nucleotides, and, accordingly, arrest COVID-19 disease, persuading the relevant investigators to quickly begin the three agents' comprehensive preclinical and clinical anti-COVID-19 assessments.

Potent Dual Polymerase/Exonuclease Inhibitory Activities of Antioxidant Aminothiadiazoles Against the COVID-19 Omicron Virus: A Promising In Silico/In Vitro Repositioning Research Study.

Recently, natural and synthetic nitrogenous heterocyclic antivirals topped the scene as first choices for the treatment of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and their accompanying disease, the coronavirus disease 2019 (COVID-19). Meanwhile, the mysterious evolution of a new strain of SARS-CoV-2, the Omicron variant and its sublineages, caused a new defiance in the continual COVID-19 battle. Hitting the two principal coronaviral-2 multiplication enzymes RNA-dependent RNA polymerase (RdRp) and 3'-to-5' exoribonuclease (ExoN) synchronously using the same ligand is a highly effective novel dual pathway to hinder SARS-CoV-2 reproduction and stop COVID-19 progression irrespective of the SARS-CoV-2 variant type since RdRps and ExoNs are widely conserved among all SARS-CoV-2 strains. Herein, the present computational/biological study screened our previous small libraries of nitrogenous heterocyclic compounds, searching for the most ideal drug candidates predictably able to efficiently act through this double approach. Theoretical filtration gave rise to three promising antioxidant nitrogenous heterocyclic compounds of the 1,3,4-thiadiazole type, which are CoViTris2022, Taroxaz-26, and ChloViD2022. Further experimental evaluation proved for the first time, utilizing the in vitro anti-RdRp/ExoN and anti-SARS-CoV-2 bioassays, that ChloViD2022, CoViTris2022, and Taroxaz-26 could effectively inhibit the replication of the new virulent strains of SARS-CoV-2 with extremely minute in vitro anti-RdRp and anti-SARS-CoV-2 EC50 values of 0.17 and 0.41 µM for ChloViD2022, 0.21 and 0.69 µM for CoViTris2022, and 0.23 and 0.73 µM for Taroxaz-26, respectively, transcending the anti-COVID-19 drug molnupiravir. The preliminary in silico outcomes greatly supported these biochemical results, proposing that the three molecules potently strike the key catalytic pockets of the SARS-CoV-2 (Omicron variant) RdRp's and ExoN's vital active sites. Moreover, the idealistic pharmacophoric hallmarks of CoViTris2022, Taroxaz-26, and ChloViD2022 molecules relatively make them typical dual-action inhibitors of SARS-CoV-2 replication and proofreading, with their highly flexible structures open for various kinds of chemical derivatization. To cut it short, the present pivotal findings of this comprehensive work disclosed the promising repositioning potentials of the three 2-aminothiadiazoles, CoViTris2022, Taroxaz-26, and ChloViD2022, to successfully interfere with the crucial biological interactions of the coronaviral-2 polymerase/exoribonuclease with the four principal RNA nucleotides, and, as a result, cure COVID-19 infection, encouraging us to rapidly start the three drugs' broad preclinical/clinical anti-COVID-19 evaluations. Dual SARS-CoV-2 polymerase (RdRp) and exoribonuclease (ExoN) inhibition via nucleoside mimicry is a very effective novel approach for COVID-19 infection therapy. Hydroxylated nitrogenous heterocyclic compounds are currently considered first choices in COVID-19 therapy. Extensive computational investigations disclosed three synthetic 5-substituted-2-amino-1,3,4-thiadiazoles, CoViTris2022, Taroxaz-26, and ChloViD2022, with ideal anti-RdRp/ExoN features. ChloViD2022 was ranked the top among the three NAs, with biochemical anti-RdRp EC50 value of 0.17 µM. ChloViD2022 accordingly displayed excellent anti-SARS-CoV-2 EC50 value of 0.41 µM against the Omicron variant.

A Series of Adenosine Analogs as the First Efficacious Anti-SARS-CoV-2 Drugs against the B.1.1.529.4 Lineage: A Preclinical Repurposing Research Study.

Given the rapid progression of the coronavirus disease 2019 (COVID-19) pandemic, an ultrafast response was urgently required to handle this major public crisis. To contain the pandemic, investments are required to develop diagnostic tests, prophylactic vaccines, and novel therapies. Lately, nucleoside analog (NA) antivirals topped the scene as top options for the treatment of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Meanwhile, the continuous generation of new lineages of the SARS-CoV-2 Omicron variant caused a new challenge in the persistent COVID-19 battle. Hitting the two crucial SARS-CoV-2 enzymes RNA-dependent RNA polymerase (RdRp) and 3'-to-5' exoribonuclease (ExoN) collectively together using only one single ligand is a very successful new approach to stop SARS-CoV-2 multiplication and combat COVID-19 irrespective of the SARS-CoV-2 variant type because RdRps and ExoNs are broadly conserved among all SARS-CoV-2 strains. Herein, the current comprehensive study investigated most NAs libraries, searching for the most ideal drug candidates expectedly able to perfectly act through this double tactic. Gradual computational filtration gave rise to six different promising NAs, which are riboprine, forodesine, tecadenoson, nelarabine, vidarabine, and maribavir, respectively. Further biological assessment proved for the first time, using the in vitro anti-RdRp/ExoN and anti-SARS-CoV-2 bioassays, that riboprine and forodesine, among all the six tested NAs, are able to powerfully inhibit the replication of the new virulent strains of SARS-CoV-2 with extremely minute in vitro anti-RdRp and anti-SARS-CoV-2 EC50 values of about 0.22 and 0.49 µM for riboprine and about 0.25 and 0.73 µM for forodesine, respectively, surpassing both remdesivir and the new anti-COVID-19 drug molnupiravir. The prior in silico data supported these biochemical findings, suggesting that riboprine and forodesine molecules strongly hit the key catalytic pockets of the SARS-CoV-2 (Omicron variant) RdRp's and ExoN's main active sites. Additionally, the ideal pharmacophoric features of riboprine and forodesine molecules render them typical dual-action inhibitors of SARS-CoV-2 replication and proofreading, with their relatively flexible structures open for diverse types of chemical derivatization. In Brief, the current important results of this comprehensive study revealed the interesting repurposing potentials of, mainly, the two nucleosides riboprine and forodesine to effectively shut down the polymerase/exoribonuclease-RNA nucleotides interactions of the SARS-CoV-2 Omicron variant and consequently treat COVID-19 infections, motivating us to rapidly begin the two drugs' broad preclinical/clinical anti-COVID-19 bioevaluations, hoping to combine both drugs soon in the COVID-19 treatment protocols.

Unusual Clinical Experience in BRAF Exon 15 p.K601E-Mutated Lung Cancer: A Case Report and Brief Review of the Literature

Molecular profiling has revolutionized the treatment of metastatic NSCLC. Uncommon mutations have been reported primarily in EGFR and BRAF genes and are frequently associated with atypical clinical presentations. Here, we present a rare case of a patient affected by BRAF exon 15 p.K601E-mutated lung cancer with synchronous peritoneal carcinomatosis. First line treatment with chemo-immunotherapy combinations provided a PFS of 8–9 months, whereas a second line treatment with BRAF and MEK inhibitors elicited a dissociated response. The latter clinical outcome suggests that these inhibitors have only partial activity against this rare mutation.

Viral Nucleases from Herpesviruses and Coronavirus in Recombination and Proofreading: Potential Targets for Antiviral Drug Discovery.

In this review, we explore recombination in two very different virus families that have become major threats to human health. The Herpesviridae are a large family of pathogenic double-stranded DNA viruses involved in a range of diseases affecting both people and animals. Coronaviridae are positive-strand RNA viruses (CoVs) that have also become major threats to global health and economic stability, especially in the last two decades. Despite many differences, such as the make-up of their genetic material (DNA vs. RNA) and overall mechanisms of genome replication, both human herpes viruses (HHVs) and CoVs have evolved to rely heavily on recombination for viral genome replication, adaptation to new hosts and evasion of host immune regulation. In this review, we will focus on the roles of three viral exonucleases: two HHV exonucleases (alkaline nuclease and PolExo) and one CoV exonuclease (ExoN). We will review the roles of these three nucleases in their respective life cycles and discuss the state of drug discovery efforts against these targets.

Opportunities and Challenges in Targeting the Proofreading Activity of SARS-CoV-2 Polymerase Complex.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. While the development of vaccines and the emergence of antiviral therapeutics is promising, alternative strategies to combat COVID-19 (and potential future pandemics) remain an unmet need. Coronaviruses feature a unique mechanism that may present opportunities for therapeutic intervention: the RNA polymerase complex of coronaviruses is distinct in its ability to proofread and remove mismatched nucleotides during genome replication and transcription. The proofreading activity has been linked to the exonuclease (ExoN) activity of non-structural protein 14 (NSP14). Here, we review the role of NSP14, and other NSPs, in SARS-CoV-2 replication and describe the assays that have been developed to assess the ExoN function. We also review the nucleoside analogs and non-nucleoside inhibitors known to interfere with the proofreading activity of NSP14. Although not yet validated, the potential use of non-nucleoside proofreading inhibitors in combination with chain-terminating nucleosides may be a promising avenue for the development of anti-CoV agents.

Identification of Complement-Related Missense Variants in Pediatric Patients with Acute and Post COVID-19 Syndromes

Background: Complement dysregulation has been documented in the molecular pathophysiology of COVID-19 and recently implicated in the relevant pediatric patient inflammatory responses. Aims: Based on our previous data in adults, we hypothesized that signatures of complement genetic variants would also be detectable in pediatric patients exhibiting COVID-19 signs and symptoms. Methods: We prospectively studied consecutive pediatric patients from our COVID-19 Units (November 2020-March 2021). COVID-19 was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Patient data were recorded by treating physicians that followed patients up to discharge. DNA was obtained from peripheral blood samples. Probes were designed using the Design studio (Illumina). Amplicons cover exons of complement-associated genes (C3, C5, CFB, CFD, CFH, CFHR1, CFI, CD46, CD55, MBL2, MASP1, MASP2, COLEC11, FCN1, FCN3 as well as ADAMTS13 and ΤHBD) spanning 15 bases into introns. We used 10ng of initial DNA material. Libraries were quantified using Qubit and sequenced on a MiniSeq System in a 2x150 bp run. Analysis was performed using the TruSeq Amplicon application (BaseSpace). Alignment was based on the banded Smith-Waterman algorithm in the targeted regions (specified in a manifest file). We performed variant calling with the Illumina-developed Somatic Variant Caller in germline mode and variant allele frequency higher than 20%. Both Ensembl and Refseq were used for annotation of the output files. A preliminary analysis (A) for the identification of variants of clinical significance was based on annotated ClinVar data, while a further and more selective analysis (B) was performed to identify missense complement coding variants that may biochemically contribute to the deregulation of innate responses during infection. This analysis was mainly based on the dbSNP and UniProt databases and available literature. Results: We studied 80 children and adolescents, 8 of whom developed inflammatory syndromes (MIS-C group). Among them, 41 were hospitalized and eventually all survived. 1. In our preliminary analysis, patients exhibited heterogeneous variant profiles including pathogenic, benign, likely benign, and variants of unknown significance (median number of variants: 97, range: 61-103). We found a variant of ADAMTS13 (rs2301612, missense) in 39 patients. We also detected two missense risk factor variants, previously detected in complement-related diseases: rs2230199 in C3 (33 patients);and rs800292 in CFH (36 patients). Among them, 40 patients had a combination of these characterized variants. This combination was significantly associated with the presence of dyspnea (p=0.031) and cough (p=0.042). Furthermore, 27 patients had a pathogenic variant in MBL2 (rs1800450), and 7 a pathogenic deletion in FCN3 that have been previously associated with inflammatory syndromes. 2. The results of our further analysis are summarized in Figure. We identified common variants, some well represented by relatively high frequencies (>70%) (rs11098044 in CFI, rs1061170 in CFH and rs12711521 in MASP2) and others less abundant, but varying considerably between the hospitalized group, the non-admitted group and the MIS-C individuals (rs2230199 in C3, rs1065489 in CFH, rs12614 and rs641153 in CFB, rs1800450 in MBL2, rs2273346 and rs72550870 in MASP2, rs72549154 in MASP3 and rs7567833 in COLEC11, all highlighted in Figure in red).). Structurally, the majority of these common variants of interest encode charge reversal mutations. These may influence protein-protein interactions in complex formations that are important for complement activation and/or regulation. Conclusion: In pediatric COVID-19 we have detected a novel set of complement missense coding variants some of which have been implicated earlier in inflammatory syndromes and endothelial stress responses. Certain combinations of mutations of alternative and/or lectin pathway components may increase the threshold dynamics of complement consumption and therefore alter COVID-19 phenotypes. [Formula prese ted] Disclosures: Gavriilaki: Alexion, Omeros, Sanofi Corporation: Consultancy;Gilead Corporation: Honoraria;Pfizer Corporation: Research Funding. Anagnostopoulos: Abbvie: Other: clinical trials;Sanofi: Other: clinical trials;Ocopeptides: Other: clinical trials;GSK: Other: clinical trials;Incyte: Other: clinical trials;Takeda: Other: clinical trials;Amgen: Other: clinical trials;Janssen: Other: clinical trials;novartis: Other: clinical trials;Celgene: Other: clinical trials;Roche: Other: clinical trials;Astellas: Other: clinical trials.

A Case of Severe Left-Ventricular Noncompaction Associated with Splicing Altering Variant in the FHOD3 Gene.

Left ventricular noncompaction (LVNC) is a highly heterogeneous primary disorder of the myocardium. Its clinical features and genetic spectrum strongly overlap with other types of primary cardiomyopathies, in particular, hypertrophic cardiomyopathy. Study and the accumulation of genotype-phenotype correlations are the way to improve the precision of our diagnostics. We present a familial case of LVNC with arrhythmic and thrombotic complications, myocardial fibrosis and heart failure, cosegregating with the splicing variant in the FHOD3 gene. This is the first description of FHOD3-dependent LVNC to our knowledge. We also revise the assumed mechanism of pathogenesis in the case of FHOD3 splicing alterations.

Effects of VDR polymorphisms on the COVID-19 Symptoms

Vitamin D [25 (OH)D] plays a role in many of biological processes, such as bone metabolism, immunomodulation, cell proliferation, differentiation, and regulation. Also, it has anti-inflammatory, antifibrotic, and antioxidant effects. Due to the immunomodulatory effects of 25 (OH)D, its deficiency is blamed for a higher risk for COVID-19 infection. Serum concentrations of 25 (OH)D were inversely associated with proinflammatory cytokines such as increased IL-6, CRP levels, and increased risk of pneumonia or ARDS. Lower 25 (OH)D concentrations are associated with a higher risk for infections, especially from the respiratory tract [1]. Chronic vitamin D deficiency can induce the renin-angiotensin system activation and leads to fibrotic changes that can cause lung injury by inducing proinflammatory cytokine production in human monocytes/macrophages (2). Increased frequency of COVID-19 infection at high latitudes and worse prognosis of these cases made clinicians to think that 25 (OH)D levels may affect the risk and prognosis of COVID-19 infection [3]. In previous reports, in the early pandemic, a higher prevalence of vitamin D deficiency has been reported to be related to high rates of COVID-19 infection, higher risk of invasive mechanical ventilation (IMV), and mortality [6]. Whilst, it is reported that 25 (OH)D may not protect against COVID-19 infection in recent studies. Moreover, it was not associated with disease severity or lethality [4-6]. The active form of vitamin D binds to its receptor (VDR) and modulates its responses. VDR is located on chromosome 12q13, consisting of 9 exons. Vitamin D-VDR signaling regulates the expression of a wide range of physiological functions. Herein, VDR polymorphisms cause a dysfunctional receptor that affects VDR activity. Both innate and adaptive immune responses can vary according to different polymorphisms of VDR. Also VDR polymorphisms have been previously found to be associated with bacterial infections such as tuberculosis [7] and severe Respiratory Syncytial Virus (RSV) bronchiolitis in respect to vitamin D deficiency [8]. Moreover, it was demonstrated that different VDR polymorphisms such as FokI, BsmI, ApaI, and TaqI could change the course of RSV infection in several studies, respectively [8-10]. This study aimed to evaluate if there is any association between the VDR gene polymorphism at FokI, TaqI, BsmI, and ApaI alleles and the prognosis of COVID-19 in respect to vitamin D deficiency. Two-hundred ninety-seven (n=297) patients with reverse-transcription polymerase chain reaction (RT-PCR)-confirmed COVID-19 who were admitted to Marmara University Education and Research Hospital between April and October 2020 were enrolled. The severity of COVID-19 patients was classified into 1-10 according to WHO criteria. The patients' requirement for noninvasive mechanical ventilation (NIMV) or reservoir mask, their requirement for admission to intensive care unit (ICU), mortality, and WHO clinical progression scales were reviewed. Four variant regions of vitamin D receptor (VDR);FokI, BsmI, ApaI, and TaqI were determined using the Restriction Fragment Length Polymorphism (RFLP) technique. To conclude;The effect of VDR polymorphisms on the receptor function causes intensive care unit treatment, disease severity and mortality differences among patients with covid-19 infection in the clinical set-up. VDR Ff genotype was related with disease severity, TT with disease severity and aa with mortality respectively. As a result we have detected that 25 (OH)D levels were not related to COVID-19 infection severity and mortality. Additionally, it indicated that VDR polymorphisms are independently associated with the severity of COVID-19 and the survival of patients. More extensive studies are needed to determine the impact of polymorphisms on COVID-19 and explain the underlying cause.

The Antiviral Effect of the Chemical Compounds Targeting DED/EDh Motifs of the Viral Proteins on Lymphocytic Choriomeningitis Virus and SARS-CoV-2.

Arenaviruses and coronaviruses include several human pathogenic viruses, such as Lassa virus, Lymphocytic choriomeningitis virus (LCMV), SARS-CoV, MERS-CoV, and SARS-CoV-2. Although these viruses belong to different virus families, they possess a common motif, the DED/EDh motif, known as an exonuclease (ExoN) motif. In this study, proof-of-concept studies, in which the DED/EDh motif in these viral proteins, NP for arenaviruses, and nsp14 for coronaviruses, could be a drug target, were performed. Docking simulation studies between two structurally different chemical compounds, ATA and PV6R, and the DED/EDh motifs in these viral proteins indicated that these compounds target DED/EDh motifs. The concentration which exhibited modest cell toxicity was used with these compounds to treat LCMV and SARS-CoV-2 infections in two different cell lines, A549 and Vero 76 cells. Both ATA and PV6R inhibited the post-entry step of LCMV and SARS-CoV-2 infection. These studies strongly suggest that DED/EDh motifs in these viral proteins could be a drug target to combat two distinct viral families, arenaviruses and coronaviruses.

Bisindolylmaleimide IX: A novel anti-SARS-CoV2 agent targeting viral main protease 3CLpro demonstrated by virtual screening pipeline and in-vitro validation assays.

SARS-CoV-2, the virus that causes COVID-19 consists of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs/compounds. We targeted seven proteins with enzymatic activities known to be essential at different stages of the viral cycle including PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2'-O-MT. For virtual screening, energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard (Schrodinger LLC 2020-1). Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs and investigational molecules (n = 5903). The screening was performed against viral targets using three sequential docking modes (i.e., HTVS, SP, and XP). Virtual screening identified ∼290 potential inhibitors based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. The top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. The resulting top eight hits were tested for their SARS-CoV-2 anti-viral activity in-vitro. Among these, a known inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), was found to be a potent inhibitor of SARS-CoV-2. Further, target validation through enzymatic assays confirmed 3CLpro to be the target. This is the first study that has showcased BIM IX as a COVID-19 inhibitor thereby validating our pipeline.