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.

The human ABCG2 is an important plasma membrane multidrug transporter, involved in uric acid secretion, modulation of absorption of drugs, and in drug resistance of cancer cells. Variants of the ABCG2 transporter, affecting cellular processing and trafficking, have been shown to cause gout and increased drug toxicity. In this paper, we overview the key cellular pathways involved in the processing and trafficking of large membrane proteins, focusing on ABC transporters. We discuss the information available for disease-causing polymorphic variants and selected mutations of ABCG2, causing increased degradation and impaired travelling of the transporter to the plasma membrane. In addition, we provide a detailed in silico analysis of an as yet unrecognized loop region of the ABCG2 protein, in which a recently discovered mutation may actually promote ABCG2 membrane expression. We suggest that post-translational modifications in this unstructured loop at the cytoplasmic surface of the protein may have special influence on ABCG2 processing and trafficking.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein.

The ABCG2 membrane protein is a key xeno- and endobiotic transporter, modulating the absorption and metabolism of pharmacological agents and causing multidrug resistance in cancer. ABCG2 is also involved in uric acid elimination and its impaired function is causative in gout. Analysis of ABCG2 expression in the erythrocyte membranes of healthy volunteers and gout patients showed an enrichment of lower expression levels in the patients. By genetic screening based on protein expression, we found a relatively frequent, novel ABCG2 mutation (ABCG2-M71V), which, according to cellular expression studies, causes reduced protein expression, although with preserved transporter capability. Molecular dynamics simulations indicated a stumbled dynamics of the mutant protein, while ABCG2-M71V expression in vitro could be corrected by therapeutically relevant small molecules. These results suggest that personalized medicine should consider this newly discovered ABCG2 mutation, and genetic analysis linked to protein expression provides a new tool to uncover clinically important mutations in membrane proteins.

In the human ATP2B4 gene, coding for the plasma membrane calcium pump PMCA4b, a minor haplotype results in the decreased expression of this membrane protein in erythroid cells. The presence of this haplotype and the consequently reduced PMCA4b expression have been suggested to affect red blood cell hydration and malaria susceptibility. By using dual-luciferase reporter assays, we have localized the erythroid-specific regulatory region within the haplotype of the ATP2B4 gene, containing predicted GATA1 binding sites that are affected by SNPs in the minor haplotype. Our results show that, in human erythroid cells, the regulation of ATP2B4 gene expression is significantly affected by GATA1 expression, and we document the role of specific SNPs involved in predicted GATA1 binding. Our findings provide a mechanistic explanation at the molecular level for the reduced erythroid-specific PMCA4b expression in carriers of ATP2B4 gene polymorphic variants.

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3β, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3β, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.