Raman and NIR spectroscopy-based real-time monitoring of the membrane filtration process of a recombinant protein for the diagnosis of SARS-CoV-2.

This research shows the detailed comparison of Raman and near-infrared (NIR) spectroscopy as Process Analytical Technology tools for the real-time monitoring of a protein purification process. A comprehensive investigation of the application and model development of Raman and NIR spectroscopy was carried out for the real-time monitoring of a process-related impurity, imidazole, during the tangential flow filtration of Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike protein. The fast development of Raman and NIR spectroscopy-based calibration models was achieved using offline calibration data, resulting in low calibration and cross-validation errors. Raman model had an RMSEC of 1.53 mM, and an RMSECV of 1.78 mM, and the NIR model had an RMSEC of 1.87 mM and an RMSECV of 2.97 mM. Furthermore, Raman models had good robustness when applied in an inline measurement system, but on the contrary NIR spectroscopy was sensitive to the changes in the measurement environment. By utilizing the developed models, inline Raman and NIR spectroscopy were successfully applied for the real-time monitoring of a process-related impurity during the membrane filtration of a recombinant protein. The results enhance the importance of implementing real-time monitoring approaches for the broader field of diagnostic and therapeutic protein purification and underscore its potential to revolutionize the rapid development of biological products.

Application of a Receptor-Binding-Domain-Based Simple Immunoassay for Assessing Humoral Immunity against Emerging SARS-CoV-2 Virus Variants.

We have developed a simple, rapid, high-throughput RBD-based ELISA to assess the humoral immunity against emerging SARS-CoV-2 virus variants. The cDNAs of the His-tagged RBD proteins of the virus variants were stably engineered into HEK cells secreting the protein into the supernatant, and RBD purification was performed by Ni-chromatography and buffer exchange by membrane filtration. The simplified assay uses single dilutions of sera from finger-pricked native blood samples, purified RBD in 96-well plates, and a chromogenic dye for development. The results of this RBD-ELISA were confirmed to correlate with those of a commercial immunoassay measuring antibodies against the Wuhan strain, as well as direct virus neutralization assays assessing the cellular effects of the Wuhan and the Omicron (BA.5) variants. Here, we document the applicability of this ELISA to assess the variant-specific humoral immunity in vaccinated and convalescent patients, as well as to follow the time course of selective vaccination response. This simple and rapid assay, easily modified to detect humoral immunity against emerging SARS-CoV-2 virus variants, may help to assess the level of antiviral protection after vaccination or infection.

Expression, Function and Trafficking of the Human ABCG2 Multidrug Transporter Containing Mutations in an Unstructured Cytoplasmic Loop.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354-367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357-360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter.

Deciphering neuronal deficit and protein profile changes in human brain organoids from patients with creatine transporter deficiency.

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 GSK3ß, 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, GSK3ß, 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.

Interactions of the Anti-SARS-CoV-2 Agents Molnupiravir and Nirmatrelvir/Paxlovid with Human Drug Transporters.

Orally administered small molecules may have important therapeutic potential in treating COVID-19 disease. The recently developed antiviral agents, Molnupiravir and Nirmatrelvir, have been reported to be efficient treatments, with only moderate side effects, especially when applied in the early phases of this disease. However, drug-drug and drug-transporter interactions have already been noted by the drug development companies and in the application notes. In the present work, we have studied some of the key human transporters interacting with these agents. The nucleoside analog Molnupiravir (EIDD-2801) and its main metabolite (EIDD-1931) were found to inhibit CNT1,2 in addition to the ENT1,2 nucleoside transporters; however, it did not significantly influence the relevant OATP transporters or the ABCC4 nucleoside efflux transporter. The active component of Paxlovid (PF-07321332, Nirmatrelvir) inhibited the function of several OATPs and of ABCB1 but did not affect ABCG2. However, significant inhibition was observed only at high concentrations of Nirmatrelvir and probably did not occur in vivo. Paxlovid, as used in the clinic, is a combination of Nirmatrelvir (viral protease inhibitor) and Ritonavir (a "booster" inhibitor of Nirmatrelvir metabolism). Ritonavir is known to inhibit several drug transporters; therefore, we have examined these compounds together, in relevant concentrations and ratios. No additional inhibitory effect of Nirmatrelvir was observed compared to the strong transporter inhibition caused by Ritonavir. Our current in vitro results should help to estimate the potential drug-drug interactions of these newly developed agents during COVID-19 treatment.

Genetic polymorphisms and decreased protein expression of ABCG2 urate transporters are associated with susceptibility to gout, disease severity and renal-overload hyperuricemia.

Gout is a common crystal induced disease of high personal and social burden, characterised by severe arthritis and comorbidity if untreated. Impaired function of ABCG2 transporter is causative in gout and may be responsible for renal-overload type hyperuricemia. Despite its importance, there is limited information on how clinical parameters correlate with protein expression and that with genetic changes. Urate and clinical parameters of 78 gouty patients and healthy controls were measured among standardised circumstances from a Hungarian population. ABCG2 membrane expression of red blood cells was determined by flow cytometry-based method and SNPs of this protein were analysed by TaqMan-based qPCR. The prevalence of ABCG2 functional polymorphisms in gouty and control patients were 32.1 and 13.7%, respectively. Most common SNP was Q141K while one sample with R236X, R383C and the lately described M71V were found in the gouty population. These polymorphisms showed strong linkage with decreased protein expression while the latter was also associated with higher fractional urate excretion (FUE) and urinary urate excretion (UUE). This study firstly evaluated ABCG2 protein expression in a clinically defined gouty population while also proving its associations between ABCG2 genetic changes and renal-overload hyperuricemia. The paper also highlighted relations between ABCG2 SNPs, gout susceptibility and disease severity characterised by an early onset disease with frequent flares and tophi formation.

Genetic Modulation of the GLUT1 Transporter Expression-Potential Relevance in Complex Diseases.

The human GLUT1 (SLC2A1) membrane protein is the key glucose transporter in numerous cell types, including red cells, kidney, and blood-brain barrier cells. The expression level of this protein has a role in several diseases, including cancer and Alzheimer's disease. In this work, to investigate a potential genetic modulation of the GLUT1 expression level, the protein level was measured in red cell membranes by flow cytometry, and the genetic background was analyzed by qPCR and luciferase assays. We found significant associations between red cell GLUT1 levels and four single nucleotide polymorphisms (SNP) in the coding SLC2A1 gene, that in individuals with the minor alleles of rs841848, rs1385129, and rs11537641 had increased, while those having the variant rs841847 had decreased erythrocyte GLUT1 levels. In the luciferase reporter studies performed in HEK-293T and HepG2 cells, a similar SNP-dependent modulation was observed, and lower glucose, serum, and hypoxic condition had variable, cell- and SNP-specific effects on luciferase expression. These results should contribute to a more detailed understanding of the genetic background of membrane GLUT1 expression and its potential role in associated diseases.

Selective Fluorescent Probes for High-Throughput Functional Diagnostics of the Human Multidrug Transporter P-Glycoprotein (ABCB1).

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells.

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.

Genetics of Pheochromocytomas and Paragangliomas Determine the Therapeutical Approach.

Pheochromocytomas and paragangliomas are the most heritable endocrine tumors. In addition to the inherited mutation other driver mutations have also been identified in tumor tissues. All these genetic alterations are clustered in distinct groups which determine the pathomechanisms. Most of these tumors are benign and their surgical removal will resolve patient management. However, 5-15% of them are malignant and therapeutical possibilities for them are limited. This review provides a brief insight about the tumorigenesis associated with pheochromocytomas/paragangliomas in order to present them as potential therapeutical targets.

RBD-specific antibody responses after two doses of BBIBP-CorV (Sinopharm, Beijing CNBG) vaccine.

BACKGROUND: Limited information is available on the effectiveness of the BBIBP-CorV (Sinopharm, Beijing CNBG) vaccine, especially in the elderly, despite the fact that it is approved in more than 50 countries. METHODS: RBD-specific antibody titres, as a rapidly available and highly predictive surrogate marker, were measured after two doses of the BBIBP-CorV vaccine in 450 subjects. Results were analyzed in a multivariable model accounting for age, sex and time since the administration of the second dose of the vaccine. RESULTS: Sex and time since the second dose had little association with the antibody titres. Age, however, was highly relevant: measurable antibody levels were present in about 90% of individuals below the age of 50, but antibody production after BBIBP-CorV vaccination was strongly reduced with increasing age. A large number of elderly subjects, reaching 25% at 60 years, and up to 50% at ages over 80, were found not to produce any protective antibody. CONCLUSIONS: RBD-specific antibody titre, as a correlate of protection for COVID-19 disease susceptibility, should help to evaluate the effectiveness of the BBIBP-CorV vaccine. Results suggest that proper measures should be undertaken to prevent a potential outbreak of COVID-19 in BBIBP-CorV vaccinated but eventually unprotected elderly individuals.

Potential role of the ABCG2-Q141K polymorphism in type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease and variations in multispecific membrane transporter functions may affect T2DM development, complications or treatment. In this work we have analyzed the potential effects of a major polymorphism, the Q141K variant of the ABCG2 transporter in T2DM. The ABCG2 protein is a multispecific xeno- and endobiotic transporter, affecting drug metabolism and playing a key role in uric acid extrusion. The ABCG2-Q141K variant, with reduced expression level and function, is present in 15-35% of individuals, depending on the genetic background of the population, and has been shown to significantly affect gout development. Several other diseases, including hypertension, chronic renal failure, and T2DM have also been reported to be associated with high serum uric acid levels, suggesting that ABCG2 may also play a role in these conditions. In this work we have compared relatively small cohorts (n = 203) of T2DM patients (n = 99) and healthy (n = 104) individuals regarding the major laboratory indicators of T2DM and determined the presence of the SNP rs2231142 (C421A), resulting the ABCG2-Q141K protein variant. We found significantly higher blood glucose and HbA1c levels in the T2DM patients carrying the ABCG2-Q141K variant. These findings may emphasize the potential metabolic role of ABCG2 in T2DM and indicate that further research should explore how prevention and treatment of this disease may be affected by the frequent polymorphism of ABCG2.

Interactions of anti-COVID-19 drug candidates with hepatic transporters may cause liver toxicity and affect pharmacokinetics.

Transporters in the human liver play a major role in the clearance of endo- and xenobiotics. Apical (canalicular) transporters extrude compounds to the bile, while basolateral hepatocyte transporters promote the uptake of, or expel, various compounds from/into the venous blood stream. In the present work we have examined the in vitro interactions of some key repurposed drugs advocated to treat COVID-19 (lopinavir, ritonavir, ivermectin, remdesivir and favipiravir), with the key drug transporters of hepatocytes. These transporters included ABCB11/BSEP, ABCC2/MRP2, and SLC47A1/MATE1 in the canalicular membrane, as well as ABCC3/MRP3, ABCC4/MRP4, SLC22A1/OCT1, SLCO1B1/OATP1B1, SLCO1B3/OATP1B3, and SLC10A1/NTCP, residing in the basolateral membrane. Lopinavir and ritonavir in low micromolar concentrations inhibited BSEP and MATE1 exporters, as well as OATP1B1/1B3 uptake transporters. Ritonavir had a similar inhibitory pattern, also inhibiting OCT1. Remdesivir strongly inhibited MRP4, OATP1B1/1B3, MATE1 and OCT1. Favipiravir had no significant effect on any of these transporters. Since both general drug metabolism and drug-induced liver toxicity are strongly dependent on the functioning of these transporters, the various interactions reported here may have important clinical relevance in the drug treatment of this viral disease and the existing co-morbidities.

Modulation of the Human Erythroid Plasma Membrane Calcium Pump (PMCA4b) Expression by Polymorphic Genetic Variants.

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.

Analytical Performance of NGS-Based Molecular Genetic Tests Used in the Diagnostic Workflow of Pheochromocytoma/Paraganglioma.

Next Generation Sequencing (NGS)-based methods are high-throughput and cost-effective molecular genetic diagnostic tools. Targeted gene panel and whole exome sequencing (WES) are applied in clinical practice for assessing mutations of pheochromocytoma/paraganglioma (PPGL) associated genes, but the best strategy is debated. Germline mutations of at the least 18 PPGL genes are present in approximately 20-40% of patients, thus molecular genetic testing is recommended in all cases. We aimed to evaluate the analytical and clinical performances of NGS methods for mutation detection of PPGL-associated genes. WES (three different library preparation and bioinformatics workflows) and an in-house, hybridization based gene panel (endocrine-onco-gene-panel- ENDOGENE) was evaluated on 37 (20 WES and 17 ENDOGENE) samples with known variants. After optimization of the bioinformatic workflow, 61 additional samples were tested prospectively. All clinically relevant variants were validated with Sanger sequencing. Target capture of PPGL genes differed markedly between WES platforms and genes tested. All known variants were correctly identified by all methods, but methods of library preparations, sequencing platforms and bioinformatical settings significantly affected the diagnostic accuracy. The ENDOGENE panel identified several pathogenic mutations and unusual genotype-phenotype associations suggesting that the whole panel should be used for identification of genetic susceptibility of PPGL.

Three Dimensional Cell Culturing for Modeling Adrenal and Pituitary Tumors.

In vitro monolayer conditions are not able to reproduce the complexity of solid tumors, still, there is scarce information about the 3D cell culture models of endocrine tumor types. Therefore, our aim was to develop in vitro 3D tumor models by different methodologies for adrenocortical carcinoma (H295R), pituitary neuroendocrine tumor (RC-4B/C and GH3) and pheochromocytoma (PC-12). Various methodologies were tested. Cell biological assays (cell viability, proliferation and live cell ratio) and steroid hormone production by HPLC-MS/MS method were applied to monitor cellular well-being. Cells in hanging drops and embedded in matrigel formed multicellular aggregates but they were difficult to handle and propagate for further experiments. The most widely used methods: ultra-low attachment plate (ULA) and spheroid inducing media (SFDM) were not the most viable 3D model of RC-4B/C and GH3 cells that would be suitable for further experiments. Combining spheroid generation with matrigel scaffold H295R 3D models were viable for 7 days, RC-4B/C and GH3 3D models for 7-10 days. ULA and SFDM 3D models of PC-12 cells could be used for further experiments up to 4 days. Higher steroid production in 3D models compared to conventional monolayer culture was detected. Endocrine tumor cells require extracellular matrix as scaffold for viable 3D models that can be one reason behind the lack of the usage of endocrine 3D cultures. Our models help understanding the pathogenesis of endocrine tumors and revealing potential biomarkers and therapeutic targets. They could also serve as an excellent platform for preclinical drug test screening.

Alterations in erythrocyte membrane transporter expression levels in type 2 diabetic patients.

Type 2 diabetes mellitus (T2DM) is one of the most common multifactorial diseases and several membrane transporters are involved in its development, complications and treatment. We have recently developed a flow-cytometry assay panel for the quantitative determination of red cell membrane protein levels with potential relevance in diseases. Here we report a detailed phenotypic analysis of a medium scale, clinically based study on the expression of T2DM-related membrane proteins, the GLUT1, GLUT3, MCT1, URAT1, ABCA1, ABCG2 and the PMCA4 transporters in erythrocytes. By comparing age-matched control subjects and three groups of T2DM patients (recently diagnosed, successfully managed, and patients with disease-related complications), we found significant differences in the membrane expression levels of the transporters in these groups. This is a first detailed analysis of T2DM related alterations in erythrocyte membrane transporter protein levels, and the results suggest significant changes in some of the transporter expression levels in various patient groups. By performing a further, more detailed analysis of the clinical and molecular biology parameters, these data may serve as a basis of establishing new, personalized diagnostic markers helping the prevention and treatment of type 2 diabetes.

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.

The transport pathway in the ABCG2 protein and its regulation revealed by molecular dynamics simulations.

Atomic-level structural insight on the human ABCG2 membrane protein, a pharmacologically important transporter, has been recently revealed by several key papers. In spite of the wealth of structural data, the pathway of transmembrane movement for the large variety of structurally different ABCG2 substrates and the physiological lipid regulation of the transporter has not been elucidated. The complex molecular dynamics simulations presented here may provide a breakthrough in understanding the steps of the substrate transport process and its regulation by cholesterol. Our analysis revealed drug binding cavities other than the central binding site and delineated a putative dynamic transport pathway for substrates with variable structures. We found that membrane cholesterol accelerated drug transport by promoting the closure of cytoplasmic protein regions. Since ABCG2 is present in all major biological barriers and drug-metabolizing organs, influences the pharmacokinetics of numerous clinically applied drugs, and plays a key role in uric acid extrusion, this information may significantly promote a reliable prediction of clinically important substrate characteristics and drug-drug interactions.