COVID-19: potential therapeutics for pediatric patients

The global spread of COVID-19 has imparted significant economic, medical, and social burdens. Like adults, children are affected by this pandemic. However, milder clinical symptoms are often experienced by them. Only a minimal proportion of the affected patients may develop severe and complicated COVID-19. Supportive treatment is recommended in all patients. Antiviral and immunomodulatory medications are spared for hospitalized children with respiratory distress or severe to critical disease. Up till now, remdesivir is the only USFDA-approved anti-COVID-19 medication indicated in the majority of symptomatic patients with moderate to severe disease. Dexamethasone is solely recommended in patients with respiratory distress maintained on oxygen or ventilatory support. The use of these medications in pediatric patients is founded on evidence deriving from adult studies. No randomized controlled trials (RCTs) involving pediatric COVID-19 patients have assessed these medications’ efficacy and safety, among others. Similarly, three novel monoclonal anti-SARS-CoV-2 spike protein antibodies, bamlanivimab, casirivimab and imdevimab, have been recently authorized by the USFDA. Nonetheless, their efficacy has not been demonstrated by multiple RCTs. In this review, we aim to dissect the various potential therapeutics used in children with COVID-19. We aspire to provide a comprehensive review of the available evidence and display the mechanisms of action and the pharmacokinetic properties of the studied therapeutics. Our review offers an efficient and practical guide for treating children with COVID-19.Other Information Published in: Pharmacological Reports License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1007/s43440-021-00316-1</p

Frequency of the NFATC1 mutations according to the Exome Sequencing Project (ESP).

<p> <b>rs ID</b></p><p>dbSNP reference SNP identifier.</p><p> <b>EA Allele Count</b></p><p>The observed allele counts for the listed alleles in European American population. (delimited by /).</p><p> <b>AA Allele Count</b></p><p>The observed allele counts for the listed alleles in African American population. (delimited by /).</p><p> <b>Allele Count</b></p><p>The observed allele counts for the listed alleles in all populations. (delimited by /).</p><p> <b>MAF (%) (EA/AA/All):</b></p><p>the minor-allele frequency in percent listed in the order of European American (EA), African American(AA) and all populations (All). (delimited by /).</p

Hypothetical pathway involving NFATC1 in endocardial cushion proliferation and valve maturation.

<p>Hypothetical pathway involving NFATC1 in endocardial cushion proliferation and valve maturation.</p

NFATC1 mutations impair functional interactions with GATA5 and HAND2.

<p>A- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were transfected with/without HAND2 and the DEGS1 promoter coupled to luciferase reporter construct in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Wt NFATC1 and HAND2 synergistically activate DEGS1 promoter. This synergy was abrogated in all NFATC1 mutants. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05) B- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were transfected with/without PPP3CA and with/without GATA5 to assess their combinatorial regulation of the DEGS1 promoter in HeLa cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Wt NFATC1 cotransfected with GATA5 caused a synergistic activation of 35 fold, while transfection of Wt NFATC1 with PPP3CA and GATA5 caused even a stronger synergy reaching 68 fold. The synergestic activation was maintained in all mutants except for P66L/I701L double mutant where the synergy was totally lost. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05).</p

Effect of the NFATC1 missense SNPs on the cellular localization of the protein.

<p>A- Immunofluorescence of HeLa cells transfected with plasmids encoding for the Wt NFATC1 and NFATC1 Mutants (P66L, I701L, P66L/I701L). The localization of NFATC1 was visualized using an anti-Flag antibody. Nuclei of cells were visualized using the Hoechst dye (blue color). Wt and NFATC1 mutants localized to the cytoplasm in the absence of PPP3CA (red color). (Magnification ×40). B- Immunofluorescence of HeLa cells transfected with plasmids encoding for the Wt NFATC1 and NFATC1 Mutants (P66L, I701L, P66L/I701L) co-transfected with PP3CA. The localization of NFATC1 was visualized using an anti-Flag antibody (red color) while PP3CA was visualized using anti-HA antibody (green color). Nuclei of cells were visualized using Hoechst dye (blue color). Most of the cells co-transfected with the double NFATC1 mutant were retained in the cytoplasm around the nuclear membrane, whereas in the other cases, the protein was totally translocated to the nucleus. (Magnification ×40). Yellow arrows indicate cytoplasmic (peri-nuclear) staining, while red arrows indicate nuclear staining.</p

DNA binding affinity of the mutated NFATC1 proteins.

<p>A- NFATC1 extracts from HEK 293 cells transfected with Wt NFATC1 and Mutants (P66L – I701L – P66L/I701L) were resolved on an SDS-PAGE prior to gel shift assays. Western blots showed equal amounts of expressed proteins as depicted by the anti-Flag antibody. (Ctrl refers to nuclear extracts from mock-transfected cells). B- EMSA was performed using equal amounts of the overexpressed NFATC1 proteins from HEK 293 cells transfected with Wt NFATC1 and NFATC1 mutants (P66L, I701L, P66L/I701L) and NFAT-consensus binding site as a probe. – ve sign indicates absence of nuclear extracts/* indicates NFATC1 monomer/** indicates NFATC1 Dimer/→ refers to the ³²P labeled free DNA probe. C- Quantification of the NFATC1 dimers in the EMSA using the TotalLab2010 software from Amersham shows a 30% decrease in DNA binding affinity of the single and double mutant as compared to the wild type NFATC1 protein.</p

Mendelian inheritance of the different NFATC1 SNPs.

<p>Genotype-phenotype correlations showed that in addition to the indexed patient with tricuspid atresia who died at 17 years of age, his “healthy” father carried the four different SNPs. None of the siblings, nor the mother who all are healthy have any of these SNPs.</p

Sequencing results showing the different NFATC1 SNPs.

<p>Representative chromatograms of the different missense SNPs in exons 2 and 8 (A and B respectively) and synonymous SNPs in exon 2 and 3 (C and D respectively).The boxed region indicates the place of the polymorphisms in the patient as compared to a normal sequence. In all cases, the SNPs occur on one allele as visualized by overlapping peaks at the indicated position inside the box. In (A) a cytosine is substituted by a thymine, in (B) an adenine is substituted by a cysteine, in (C) a guanine is substituted by a thymine, and in (D) a cytosine is substituted by a thymine.</p

Transcriptional activity of the mutated NFATC1 proteins.

<p>A- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were cotransfected with the human CCND1 promoter coupled luciferase reporter construct in the presence or absence of activated clacineurin (PPP3CA) in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05) B- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were cotransfected with the human DEGS1 promoter coupled luciferase reporter construct in the presence or absence of activated clacineurin (PPP3CA) in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05).</p