Interaction of crown ethers with the ABCG2 transporter and their implication for multidrug resistance reversal.

Overexpression of ABC transporters, such as ABCB1 and ABCG2, plays an important role in mediating multidrug resistance (MDR) in cancer. This feature is also attributed to a subpopulation of cancer stem cells (CSCs), having enhanced tumourigenic potential. ABCG2 is specifically associated with the CSC phenotype, making it a valuable target for eliminating aggressive and resistant cells. Several natural and synthetic ionophores have been discovered as CSC-selective drugs that may also have MDR-reversing ability, whereas their interaction with ABCG2 has not yet been explored. We previously reported the biological activities, including ABCB1 inhibition, of a group of adamantane-substituted diaza-18-crown-6 (DAC) compounds that possess ionophore capabilities. In this study, we investigated the mechanism of ABCG2-inhibitory activity of DAC compounds and the natural ionophores salinomycin, monensin and nigericin. We used a series of functional assays, including real-time microscopic analysis of ABCG2-mediated fluorescent substrate transport in cells, and docking studies to provide comparative aspects for the transporter-compound interactions and their role in restoring chemosensitivity. We found that natural ionophores did not inhibit ABCG2, suggesting that their CSC selectivity is likely mediated by other mechanisms. In contrast, DACs with amide linkage in the side arms demonstrated noteworthy ABCG2-inhibitory activity, with DAC-3Amide proving to be the most potent. This compound induced conformational changes of the transporter and likely binds to both Cavity 1 and the NBD-TMD interface. DAC-3Amide reversed ABCG2-mediated MDR in model cells, without affecting ABCG2 expression or localization. These results pave the way for the development of new crown ether compounds with improved ABCG2-inhibitory properties.

Typical achondroplasia secondary to a unique insertional variant of FGFR3 with in vitro demonstration of its effect on FGFR3 function.

We describe an individual in whom clinical and radiographic features are typical for achondroplasia, but in whom the common variants of FGFR3 that result in achondroplasia are absent. Whole exome sequencing demonstrated a novel, de novo 6 base pair tandem duplication in FGFR3 that results in the insertion of Ser-Phe after position Leu324. in vitro studies showed that this variant results in aberrant dimerization, excessive spontaneous phosphorylation of FGFR3 dimers and excessive, ligand-independent tyrosine kinase activity. Together, these data suggest that this variant leads to constitutive disulfide bond-mediated dimerization, and that this, surprisingly, occurs to an extent similar to the neonatal lethal thanatophoric dysplasia type I Ser249Cys variant.

Phenotypic Variability of Patients With PAX8 Variants Presenting With Congenital Hypothyroidism and Eutopic Thyroid.

PURPOSE: Thyroid dyshormonogenesis is a heterogeneous group of hereditary diseases produced by a total/partial blockage of the biochemical processes of thyroid-hormone synthesis and secretion. Paired box 8 (PAX8) is essential for thyroid morphogenesis and thyroid hormone synthesis. We aimed to identify PAX8 variants in patients with thyroid dyshormonogenesis and to analyze them with in vitro functional studies. PATIENTS AND METHODS: Nine pediatric patients with a eutopic thyroid gland were analyzed by the Catalan screening program for congenital hypothyroidism. Scintigraphies showed absent, low, or normal uptake. Only one patient had a hypoplastic gland. On reevaluation, perchlorate discharge test was negative or compatible with partial iodine-organization deficit. After evaluation, 8 patients showed permanent mild or severe hypothyroidism. Massive-sequencing techniques were used to detect variants in congenital hypothyroidism-related genes. In vitro functional studies were based on transactivating activity of mutant PAX8 on a TG-gene promoter and analyzed by a dual-luciferase assays. RESULTS: We identified 7 heterozygous PAX8 exonic variants and 1 homozygous PAX8 splicing variant in 9 patients with variable phenotypes of thyroid dyshormonogenesis. Five were novel and 5 variants showed a statistically significant impaired transcriptional activity of TG promoter: 51% to 78% vs the wild type. CONCLUSIONS: Nine patients presented with PAX8 candidate variants. All presented with a eutopic thyroid gland and 7 had deleterious variants. The phenotype of affected patients varies considerably, even within the same family; but, all except the homozygous patient presented with a normal eutopic thyroid gland and thyroid dyshormonogenesis. PAX8 functional studies have shown that 6 PAX8 variants are deleterious. Our studies have proven effective in evaluating these variants.

Molecular and clinical genetics of the transcription factor GLIS3 in Chinese congenital hypothyroidism.

The transcription factor GLIS3 is an important factor in hormone biosynthesis and thyroid development, and mutations in GLIS3 are relatively rare. Deletions of more than one of the 11 exons of GLIS3 occur in most patients with various extrathyroidal abnormalities and congenital hypothyroidism (CH), and only 18 missense variants of GLIS3 related to thyroid disease have been reported. The aim of this study was to report the family history and molecular basis of patients with CH who carry GLIS3 variants. Three hundred and fifty-three non-consanguineous infants with CH were recruited and subjected to targeted exome sequencing of CH-related genes. The transcriptional activity and cellular localization of the variants in GLIS3 were investigated in vitro. We identified 20 heterozygous GLIS3 exonic missense variants, including eight novel sites, in 19 patients with CH. One patient carried compound heterozygous GLIS3 variants (p.His34Arg and p.Pro835Leu). None of the variants affected the nuclear localization. However, three variants (p.His34Arg, p.Pro835Leu, and p.Ser893Phe) located in the N-terminal and C-terminal regions of the GLIS3 protein downregulated the transcriptional activation of several genes required for thyroid hormone (TH) biosynthesis. This study of patients with CH extends the current knowledge surrounding the spectrum of GLIS3 variants and the mechanisms by which they cause TH biosynthesis defects.

Interactions of Potential Anti-COVID-19 Compounds with Multispecific ABC and OATP Drug Transporters.

During the COVID-19 pandemic, several repurposed drugs have been proposed to alleviate the major health effects of the disease. These drugs are often applied with analgesics or non-steroid anti-inflammatory compounds, and co-morbid patients may also be treated with anticancer, cholesterol-lowering, or antidiabetic agents. Since drug ADME-tox properties may be significantly affected by multispecific transporters, in this study, we examined the interactions of the repurposed drugs with the key human multidrug transporters present in the major tissue barriers and strongly affecting the pharmacokinetics. Our in vitro studies, using a variety of model systems, explored the interactions of the antimalarial agents chloroquine and hydroxychloroquine; the antihelmintic ivermectin; and the proposed antiviral compounds ritonavir, lopinavir, favipiravir, and remdesivir with the ABCB1/Pgp, ABCG2/BCRP, and ABCC1/MRP1 exporters, as well as the organic anion-transporting polypeptide (OATP)2B1 and OATP1A2 uptake transporters. The results presented here show numerous pharmacologically relevant transporter interactions and may provide a warning on the potential toxicities of these repurposed drugs, especially in drug combinations at the clinic.

Functional analysis of six uncharacterised mutations in LDLR gene.

BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is a primary hyperlipemia. It is an autosomal dominant genetic disorder of lipoproteins metabolism mainly caused by mutations in the low density lipoprotein receptor gene (LDLR). We aimed to investigate the functional impact on the low density lipoprotein receptor (LDLR) activity of six uncharacterised variants located in the coding region of the LDLR gene, namely c.428G > T, c.640T > C, c.1708C > T, c.1736A > T, c.1981C > G and c.2114C > G (NM_000527.4) and to attempt to define their clinical status. METHODS: Functional studies were carried out using site-directed mutagenesis techniques and expression of LDLR protein in vitro. Results were correlated with clinical data and in silico analyses in order to assess the physiopathological role of these variants. RESULTS: This work provides functional information about 6 uncharacterised mutations in LDLR. CONCLUSIONS: The six variants studied here appeared to affect the LDLR function in vitro to different degrees, ranging from receptors with normal to slightly reduced activity to receptors exhibiting less than 10% of the wild-type activity. According to these studies and The American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines, two variants could be classified as "Likely Benign" (p.(Ala705Gly) and p.(Leu570Phe)), three variants as "Pathogenic" (p.(Asp579Val), p.(Cys143Phe) and p.(Trp214Arg)) and one variant as "Likely Pathogenic" (p.(Pro661Ala)).

Role of the 5-HTTLPR and SNP Promoter Polymorphisms on Serotonin Transporter Gene Expression: a Closer Look at Genetic Architecture and In Vitro Functional Studies of Common and Uncommon Allelic Variants.

The serotonin (5-hydroxytriptamine (5-HT)) transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) is a variable number tandem repeats (VNTR) located in the promoter region of the human 5-HTT-encoding gene SLC6A4. This length polymorphism gives rise to different promoter variants, variously influencing SLC6A4 expression. Over the years, an extensive literature has investigated the relationships between these promoter variants and SLC6A4 gene expression, since these variants have been variously associated to complex neuropsychiatric conditions and traits. In this review, we detail the genetic architecture of the 5-HTTLPR allelic variants reported so far, with a closer look at the two single nucleotide polymorphisms (SNPs) rs25531 and rs25532 that lies in the VNTR and thus increase genetic variability of the SLC6A4 promoter. We summarize the hypothesized molecular mechanisms underlying this variation. We also provide an update on common and uncommon 5-HTTLPR allelic variants reviewing the available data on functional in vitro analysis of their regulatory effect on SLC6A4 gene transcription. Controversial findings are highlighted and critically discussed. A deeper knowledge of the "5-HTTLPR universe" will be useful to better understand the molecular basis of serotonin homeostasis and the pathological basis underlying serotonin-related neuropsychiatric conditions and traits.

Biochemical Characterization of APOBEC3H Variants: Implications for Their HIV-1 Restriction Activity and mC Modification.

APOBEC3H (A3H) is the most polymorphic member of the APOBEC3 family. Seven haplotypes (hap I-VII) and four mRNA splicing variants (SV) of A3H have been identified. The various haplotypes differ in anti-HIV activity, which is attributed to differences in protein stability, subcellular distribution, and/or RNA binding and virion packaging. Here, we report the first comparative biochemical studies of all the A3H variants using highly purified proteins. We show that all haplotypes were stably expressed and could be purified to homogeneity by Escherichia coli expression. Surprisingly, four out of the seven haplotypes showed high cytosine (C) deaminase activity, with hap V displaying extremely high activity that was comparable to the highly active A3A. Furthermore, all four haplotypes that were active in C deamination were also highly active on methylated C (mC), with hap II displaying almost equal deamination efficiency on both. The deamination activity of these A3H variants correlates well with their reported anti-HIV activity for the different haplotypes, suggesting that deaminase activity may be an important factor in determining their respective anti-HIV activities. Moreover, mC deamination of A3H displayed a strong preference for the sequence motif of T-mCpG-C/G, which may suggest a potential role in genomic mC modification at the characteristic "CpG" island motif.