Novel drug transporter substrates identification: An innovative approach based on metabolomic profiling, in silico ligand screening and biological validation.

Solute carrier (SLC) transport proteins are fundamental for the translocation of endogenous compounds and drugs across membranes, thus playing a critical role in disease susceptibility and drug response. Because only a limited number of transporter substrates are currently known, the function of a large number of SLC transporters is elusive. Here, we describe the proof-of-concept of a novel strategy to identify SLC transporter substrates exemplarily for the proton-coupled peptide transporter (PEPT) 2 (SLC15A2) and multidrug and toxin extrusion (MATE) 1 transporter (SLC47A1), which are important renal transporters of drug reabsorption and excretion, respectively. By combining metabolomic profiling of mice with genetically-disrupted transporters, in silico ligand screening and in vitro transport studies for experimental validation, we identified nucleobases and nucleoside-derived anticancer and antiviral agents (flucytosine, cytarabine, gemcitabine, capecitabine) as novel drug substrates of the MATE1 transporter. Our data confirms the successful applicability of this new approach for the identification of transporter substrates in general, which may prove particularly relevant in drug research.

Shifting Clade Distribution, Reassortment, and Emergence of New Subtypes of Highly Pathogenic Avian Influenza A(H5) Viruses Collected from Vietnamese Poultry from 2012 to 2015.

Whole-genome sequences of representative highly pathogenic avian influenza A(H5) viruses from Vietnam were generated, comprising samples from poultry outbreaks and active market surveillance collected from January 2012 to August 2015. Six hemagglutinin gene clades were characterized. Clade 1.1.2 was predominant in southern Mekong provinces throughout 2012 and 2013 but gradually disappeared and was not detected after April 2014. Clade 2.3.2.1c viruses spread rapidly during 2012 and were detected in the south and center of the country. A number of clade 1.1.2 and 2.3.2.1c interclade reassortant viruses were detected with different combinations of internal genes derived from 2.3.2.1a and 2.3.2.1b viruses, indicating extensive cocirculation. Although reassortment generated genetic diversity at the genotype level, there was relatively little genetic drift within the individual gene segments, suggesting genetic stasis over recent years. Antigenically, clade 1.1.2, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c viruses remained related to earlier viruses and WHO-recommended prepandemic vaccine strains representing these clades. Clade 7.2 viruses, although detected in only low numbers, were the exception, as indicated by introduction of a genetically and antigenically diverse strain in 2013. Clade 2.3.4.4 viruses (H5N1 and H5N6) were likely introduced in April 2014 and appeared to gain dominance across northern and central regions. Antigenic analyses of clade 2.3.4.4 viruses compared to existing clade 2.3.4 candidate vaccine viruses (CVV) indicated the need for an updated vaccine virus. A/Sichuan/26221/2014 (H5N6) virus was developed, and ferret antisera generated against this virus were demonstrated to inhibit some but not all clade 2.3.4.4 viruses, suggesting consideration of alternative clade 2.3.4.4 CVVs.IMPORTANCE Highly pathogenic avian influenza (HPAI) A(H5) viruses have circulated continuously in Vietnam since 2003, resulting in hundreds of poultry outbreaks and sporadic human infections. Despite a significant reduction in the number of human infections in recent years, poultry outbreaks continue to occur and the virus continues to diversify. Vaccination of poultry has been used as a means to control the spread and impact of the virus, but due to the diversity and changing distribution of antigenically distinct viruses, the utility of vaccines in the face of mismatched circulating strains remains questionable. This study assessed the putative amino acid changes in viruses leading to antigenic variability, underscoring the complexity of vaccine selection for both veterinary and public health purposes. Given the overlapping geographic distributions of multiple, antigenically distinct clades of HPAI A(H5) viruses in Vietnam, the vaccine efficacy of bivalent poultry vaccine formulations should be tested in the future.

Investigation of endogenous compounds for assessing the drug interactions in the urinary excretion involving multidrug and toxin extrusion proteins.

PURPOSE: Multidrug and toxin extrusion proteins (MATEs) are multispecific organic cation transporters mediating the efflux of various cationic drugs into the urine. The present study aimed at identifying endogenous compounds in human plasma and urine specimens as biomarkers to evaluate drug interactions involving MATEs in the kidney without administration of their exogenous probe drugs. METHODS: An untargeted metabolomic analysis was performed using urine and plasma samples from healthy volunteers and mice treated with or without the potent MATE inhibitor, pyrimethamine. Plasma and urinary concentrations of candidate markers were measured using liquid chromatography-mass spectrometry. Transport activities were determined in MATE- or OCT2-expressing HEK293 cells. The deuterium-labeled compounds of candidates were administered to mice for pharmacokinetics study. RESULTS: Urinary excretion of eleven compounds including thiamine and carnitine was significantly lower in the pyrimethamine-treatment group in humans and mice, whereas no endogenous compound was noticeably accumulated in the plasma. The renal clearance of thiamine and carnitine was decreased by 70%-84% and 90%-94% (p < 0.05), respectively, in human. The specific uptake of thiamine was observed in MATE1-, MATE2-K- or OCT2-expressing HEK293 cells with Km of 3.5 ± 1.0, 3.9 ± 0.8 and 59.9 ± 6.7 µM, respectively. The renal clearance of carnitine-d 3 was decreased by 62% in mice treated with pyrimethamine. CONCLUSIONS: Our findings indicate that MATEs account for the efflux of thiamine and perhaps carnitine as well as drugs into the urine. The urinary excretion of thiamine is useful to detect drug interaction involving MATEs in the kidney.

The effect of ABCG2 genotype on the population pharmacokinetics of sunitinib in patients with renal cell carcinoma.

BACKGROUND: Sunitinib, a multitargeted tyrosine kinase inhibitor, offers favorable therapeutic outcomes to patients with advanced renal cell carcinoma. However, to maximize the clinical benefits, an effective therapeutic management strategy with dose optimization is essential. The objectives of this analysis were to describe the pharmacokinetics (PK) of sunitinib by a population PK approach and to quantitatively evaluate the effect of potential predictive factors including ABCG2 genotype on the PK of sunitinib. METHODS: Plasma concentration-time profiles at 3 consecutive days including a total of 245 sunitinib plasma concentrations were available from 19 Japanese patients with renal cell carcinoma. Blood samples were collected on days 2, 8, and 15 after the start of the therapy. Population PK analysis was performed using NONMEM 7.2. Body weight, gender, and genotype of ABCG2 421C>A were evaluated as potential covariates. Interoccasion variability (IOV) among the 3 sampling days was also assessed as a random effect parameter. RESULTS: The sunitinib PK profiles were best described by a 1-compartment model with first-order absorption. The ABCG2 421C>A genotype was identified as a significant covariate for the prediction of oral clearance (CL/F). No significant improvement in model fit was observed by including body weight and/or gender. A systematic difference in estimated population CL/F was observed between days 2 and 8, which was quantified as approximately 30% decrease over time. This difference was described as a covariate for CL/F in the model. IOV included as a random effect parameter significantly improved the model fit. CONCLUSIONS: This analysis provides a population PK model of sunitinib with the ABCG2 421C>A genotype as a predictive covariate for CL/F. It also suggests that IOV and change of CL/F over time need to be considered to predict the sunitinib PK more accurately. These findings will be implemented to optimize the pharmacotherapy of sunitinib.

Prevalence and distribution of avian influenza a(H5N1) virus clade variants in live bird markets of Vietnam, 2011-2013.

Active surveillance for avian influenza (Al) viruses in poultry sold at live bird markets (LBMs) was conducted in 44 of 63 provinces throughout Vietnam over two periods from September 2011 to February 2012 and October 2012 to June 2013. The study objectives were to assess the prevalence of avian influenza type A, H5, and H5N1 subtype viruses and characterize the geographical and temporal distribution of H5N1 virus genetic variants across the country. Monthly sampling was conducted in 394 LBMs located in 372 communes. A total of 9790 oropharyngeal swabs from poultry were screened for influenza A virus by real-time reverse-transcriptase PCR Virus isolation was attempted on all positive samples in embryonated chicken eggs, and the HA1 region of each H5 virus isolate was sequenced. Market prevalence of H5 subtype virus was 32.2% (127/394) over the cumulative 15 mo of surveillance. Phylogenetic analyses indicated that clade 1.1 viruses persisted in the south, whereas three genetically distinct subgroups of dade 2.3.2.1 were found simultaneously in northern, central, and southern Vietnam. Clade 2.3.2.1c viruses first appeared in July 2012 and spread rapidly to the center and south of Vietnam in late 2012, where they were predominant among clade 2.3.2.1 viruses and were detected in both active LBM surveillance and poultry outbreaks. Given the overlapping geographic distribution of dade variants and the antigenic divergence previously described for these dades, current AI poultry vaccines used in Vietnam may require bivalent formulations containing representatives of both dade 1.1 and dade 2.3.2.1 viruses.

Development and application of a vaccination planning tool for avian influenza.

The vaccination planning tool for avian influenza supports evidence-based planning and preparedness for vaccinating poultry at national and regional levels. This study describes the development, testing, and application of a vaccination planning tool for H5N1 highly pathogenic avian influenza (HPAI) used in two South Asian countries. The tool consists of eight planning clusters, 37 planning elements, and 303 referenced planning criteria. Both countries attained a score of 52% among planning clusters as a measure of preparedness. The highest and lowest planning cluster scores included vaccination strategies and financial readiness, respectively. The comprehensive vaccination program was identified as the most-useful planning cluster for assessing preparedness, and 86% of participants indicated that the objectives of the planning tool were achieved. Based on these results, the planning tool provides a structured approach for decision makers to develop their national vaccination program for HPAI as part of an overall strategy for the progressive reduction and control of endemic influenza viruses in poultry.

Sugar-induced conformational change found in the HA-33/HA-17 trimer of the botulinum toxin complex.

Large-sized botulinum toxin complex (L-TC) is formed by conjugation of neurotoxin, nontoxic nonhemagglutinin and hemagglutinin (HA) complex. The HA complex is formed by association of three HA-70 molecules and three HA-33/HA-17 trimers, comprised of a single HA-17 and two HA-33 proteins. The HA-33/HA-17 trimer isolated from serotype D L-TC has the ability to bind to and penetrate through the intestinal epithelial cell monolayer in a sialic acid-dependent manner, and thus it plays an important role in toxin delivery through the intestinal cell wall. In this study, we determined the solution structure of the HA-33/HA-17 trimer by using small-angle X-ray scattering (SAXS). The SAXS image of HA-33/HA-17 exhibited broadly similar appearance to the crystal image of the complex. On the other hand, in the presence of N-acetylneuraminic acid, glucose and galactose, the solution structure of the HA-33/HA-17 trimer was drastically altered compared to the structure in the absence of the sugars. Sugar-induced structural change of the HA-33/HA-17 trimer may contribute to cell binding and subsequent transport across the intestinal cell layer.

Random phage display-based screening of peptides that bind to botulinum neurotoxin binding protein, nontoxic nonhemagglutinin.

Botulinum neurotoxin (BoNT) binds to nontoxic nonhemagglutinin (NTNHA) protein in a pH-dependent manner, and yields the protease-resistant BoNT/NTNHA complex. Here, we screened short peptides that bind to the serotype D NTNHA (NTNHA-D) using random phage display technique. NTNHA was fixed onto electrode of quartz crystal microbalance (QCM) apparatus, and then the phages displaying random heptapeptides were exposed to the NTNHA-D under the acidic condition. After rinsing with acidic buffer, the released phages under the alkaline condition were collected. The binding and release of the phage were monitored by the frequency shift on the QCM. As a result of the screening, 16 were selected as peptides that bind to NTNHA-D. The selected peptides do not share any conserved sequence, but tend to be rich in basic and/or hydrophobic amino acid. This would explain the binding manner of the BoNT to the NTNHA protein.

Lipid Composition Analysis and Characterization of Acyl Sterol Glycosides in Adzuki and Soybean Cultivars by Non-Targeted LC-MS.

Beans, a globally significant economic and nutritional food crop, are rich in polyphenolic chemicals with potential health advantages, providing high protein, fiber, minerals, and vitamins. However, studies on the global profiling of lipids in beans are limited. We applied a non-targeted lipidomic approach based on high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (HPLC/LTQ-Orbitrap-MS) to comprehensively profile and compare the lipids in six distinct bean cultivars, namely, adzuki red beans-adzuki cultivar (ARB-AC), adzuki red beans-Benidainagon cultivar (ARB-BC), adzuki red beans-Erimoshouzu cultivar (ARB-EC), soybean-Fukuyutaka cultivar 2021 (SB-FC21), soybean-Fukuyutaka cultivar 2022 (SB-FC22), and soybean-Oosuzu cultivar (SB-OC). MS/MS analysis defined 144 molecular species from four main lipid groups. Multivariate principal component analysis indicated unique lipid compositions in the cultivars except for ARB-BC and ARB-EC. Evaluation of the concentrations of polyunsaturated fatty acid to saturated fatty acid ratio among all the cultivars showed that SB-FC21 and SB-FC22 had the highest value, suggesting they are the most beneficial for health. Furthermore, lipids such as acyl sterol glycosides were detected and characterized for the first time in these bean cultivars. Hierarchical cluster correlations revealed the predominance of ceramides in ARB-EC, lysophospholipids in SB-FC21, and glycerophospholipids in SB-OC. This study comprehensively investigated lipids and their compositions in beans, indicating their potential utility in the nutritional evaluation of beans as functional foods.

One Health Surveillance Highlights Circulation of Viruses with Zoonotic Potential in Bats, Pigs, and Humans in Viet Nam.

A One Health cross-sectoral surveillance approach was implemented to screen biological samples from bats, pigs, and humans at high-risk interfaces for zoonotic viral spillover for five viral families with zoonotic potential in Viet Nam. Over 1600 animal and human samples from bat guano harvesting sites, natural bat roosts, and pig farming operations were tested for coronaviruses (CoVs), paramyxoviruses, influenza viruses, filoviruses and flaviviruses using consensus PCR assays. Human samples were also tested using immunoassays to detect antibodies against eight virus groups. Significant viral diversity, including CoVs closely related to ancestors of pig pathogens, was detected in bats roosting at the human-animal interfaces, illustrating the high risk for CoV spillover from bats to pigs in Viet Nam, where pig density is very high. Season and reproductive period were significantly associated with the detection of bat CoVs, with site-specific effects. Phylogeographic analysis indicated localized viral transmission among pig farms. Our limited human sampling did not detect any known zoonotic bat viruses in human communities living close to the bat cave and harvesting bat guano, but our serological assays showed possible previous exposure to Marburg virus-like (Filoviridae), Crimean-Congo hemorrhagic fever virus-like (Bunyaviridae) viruses and flaviviruses. Targeted and coordinated One Health surveillance helped uncover this viral pathogen emergence hotspot.

The Concise Guide to PHARMACOLOGY 2023/24: Transporters.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16182. Transporters are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Developmental expression of renal organic anion transporters in rat kidney and its effect on renal secretion of phenolsulfonphthalein.

Organic anion transporters (OAT1 and OAT3) and multidrug resistance-associated proteins (MRP2 and MRP4) play important roles in anionic drug secretion in renal proximal tubules. Changes in the expression of such transporters are considered to affect the tubular secretion of anionic drugs. The purpose of this study was to elucidate the developmental changes in the expression of OAT1, OAT3, MRP2, and MRP4 and their effects on the tubular secretion of drugs. The mRNA level of each transporter was measured by real-time PCR, and the protein expression was evaluated by Western blotting and immunohistochemical analysis. In addition, the tubular secretion of phenolsulfonphthalein (PSP) in infant (postnatal day 14) and adult rats was estimated based on in vivo clearance study. The protein expression of organic anion transporters were very low at postnatal day 0 and gradually increased with age. In postnatal day 14 rats, the expression of OAT1 and OAT3 seemed to be at almost mature levels, while MRP2 and MRP4 seemed to be at immature levels. Immunohistochemical analysis in the kidney of postnatal day 0 rats revealed OATs on the basolateral membrane and MRPs on the brush-border membrane. At postnatal day 0, the distribution of these transporters was restricted to the inner cortical region, while after postnatal day 14, it was identical to that in adult kidney. An in vivo clearance study revealed that the tubular secretion of PSP was significantly lower in postnatal day 14 rats than adult rats. These results indicate that age-dependent changes in organic anion transporter expression affect the tubular secretion of anionic drugs in pediatric patients.

Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein.

Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X(35)-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.

Small-angle X-ray scattering reveals structural dynamics of the botulinum neurotoxin associating protein, nontoxic nonhemagglutinin.

In cell culture supernatants, the botulinum neurotoxin (BoNT) exists as part of a toxin complex (TC) in which nontoxic nonhemagglutinin (NTNHA) and/or hemagglutinins (HAs) are assembled onto the BoNT. A series of investigations indicated that formation of the TC is vital for delivery of the toxin to nerve cells through the digestive tract. In the assembly process, BoNT binds to NTNHA yielding M-TC, and it then matures into L-TC by further association with the HAs via NTNHA in the M-TC. Here, we report a crystal structure of the NTNHA from Clostridium botulinum serotype D strain 4947. Additionally, we performed small-angle X-ray scattering (SAXS) analysis of the NTNHA and the M-TC to elucidate the solution structure. The crystal structure of D-4947 NTNHA revealed that BoNT and NTNHA share a closely related structure consisting of three domains. The SAXS image indicated that, even though the N-terminal two-thirds of the NTNHA molecule had an apparently similar conformation in both the crystal and solution structures, the C-terminal third of the molecule showed a more extended structure in the SAXS image than that seen in the crystallographic image. The discrepancy between the crystal and solution structures implies a high flexibility of the C-terminal third domain of NTNHA, which is involved in binding to BoNT. Structural dynamics of the NTNHA molecule revealed by SAXS may explain its binding to BoNT to form the BoNT/NTNHA complex.

Impaired riboflavin transport due to missense mutations in SLC52A2 causes Brown-Vialetto-Van Laere syndrome.

Brown-Vialetto-Van Laere syndrome (BVVLS [MIM 211530]) is a rare neurological disorder characterized by infancy onset sensorineural deafness and ponto-bulbar palsy. Mutations in SLC52A3 (formerly C20orf54), coding for riboflavin transporter 2 (hRFT2), have been identified as the molecular genetic correlate in several individuals with BVVLS. Exome sequencing of just one single case revealed that compound heterozygosity for two pathogenic mutations in the SLC52A2 gene coding for riboflavin transporter 3 (hRFT3), another member of the riboflavin transporter family, is also associated with BVVLS. Overexpression studies confirmed that the gene products of both mutant alleles have reduced riboflavin transport activities. While mutations in SLC52A3 cause decreased plasma riboflavin levels, concordant with a role of SLC52A3 in riboflavin uptake from food, the SLC52A2-mutant individual had normal plasma riboflavin concentrations, a finding in line with a postulated function of SLC52A2 in riboflavin uptake from blood into target cells. Our results contribute to the understanding of human riboflavin metabolism and underscore its role in the pathogenesis of BVVLS, thereby providing a rational basis for a high-dose riboflavin treatment.

Crystallization and preliminary X-ray analysis of the Clostridium botulinum type D nontoxic nonhaemagglutinin.

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex assembled with nontoxic nonhaemagglutinin (NTNHA) and/or haemagglutinin components. Complex formation with NTNHA is considered to be critical in eliciting food poisoning because the complex shields the BoNT from the harsh conditions in the digestive tract. In the present study, NTNHA was expressed in Escherichia coli and crystallized. Diffraction data were collected to 3.9 Å resolution. The crystal belonged to the trigonal space group P321 or P3(1)21/P3(2)21, with unit-cell parameters a = b = 147.85, c = 229.74 Å. The structure of NTNHA will provide insight into the assembly mechanism that produces the unique BoNT-NTNHA complex.

Significance of trough monitoring for tacrolimus blood concentration and calcineurin activity in adult patients undergoing primary living-donor liver transplantation.

PURPOSE: Tacrolimus pharmacokinetics and calcineurin activity in peripheral blood mononuclear cells (PBMCs) were investigated in adult patients undergoing primary living-donor liver transplantation (LDLT) in order to clarify the significance of monitoring the tacrolimus blood trough concentration during the early post-transplantation period. METHODS: Fourteen patients were enrolled in this study, and time-course data following the oral administration of a conventional tacrolimus formulation twice daily were obtained at 1 and 3 weeks post-transplantation. The concentration of tacrolimus in whole blood and calcineurin activity in PBMCs were measured. RESULTS: The apparent clearance of tacrolimus significantly increased at 3 weeks versus 1 week post-transplantation, although the trough concentration did not significantly differ at these time points. The concentration at each sampling time, except at 1 h post-dose, correlated well with the area under the concentration-time curve from 0 to 12 h (AUC(0-12)). Neither the concentration at the trough time point nor AUC(0-12) was correlated with the area under the calcineurin activity-time curve from 0 to 12 h; however, calcineurin activity at the trough time point was strongly correlated with the latter (r (2) > 0.92). CONCLUSIONS: Based on these results, trough concentration monitoring can be considered an appropriate procedure for routine tacrolimus dosage adjustment in adult LDLT patients. Monitoring of calcineurin activity at the trough time point was also found to be potentially useful for predicting the immunological status of the patient during the tacrolimus dosing interval.

Recent advances in structural biology of peptide transporters.

Peptide transporters localized at brush-border membranes of intestinal and renal epithelial cells mediate the membrane transport of di- and tripeptides, and play important roles in protein absorption and the conservation of peptide-bound amino nitrogen. Peptide-like drugs that show structural similarities to di- and tripeptides are also recognized by peptide transporters. The energy for transport of small peptides and peptide-like drugs is provided by the proton gradient across the cell membrane. Since the cloning of H(+)/peptide cotransporter (PEPT1, SLC15A1), there have been advances in the molecular biology, biochemistry, biophysics and structural determination of PEPT1. By integrating these advances, much effort has been made to understand the relationship between structure and function. In silico experimental strategies are classified as (1) construction of kinetic models, (2) computer modeling of PEPT1 structure and (3) homology modeling of PEPT1 with crystal structures of bacterial transporters. The hypotheses regarding the structure-function relationship produced by these strategies have been confirmed by in vitro mutagenesis including cysteine-scanning mutagenesis. Recently, the crystal structure of PepT(So), a functionally similar prokaryotic homolog of the mammalian peptide transporters from Shewanella oneidensis, was classified, and the previous hypotheses regarding the structure-function relationship of PEPT1 have been re-evaluated. This review highlights the recent advances in our knowledge of the structural biology of PEPT1.

Maternal riboflavin deficiency, resulting in transient neonatal-onset glutaric aciduria Type 2, is caused by a microdeletion in the riboflavin transporter gene GPR172B.

Riboflavin, or vitamin B2, is a precursor to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) molecules, required in biological oxidation-reduction reactions. We previously reported a case of a newborn female who had clinical and biochemical features of multiple acyl-CoA dehydrogenation deficiency (MADD), which was corrected by riboflavin supplementation. The mother was then found to be persistently riboflavin deficient, suggesting that a possible genetic defect in riboflavin transport in the mother was the cause of the transient MADD seen in the infant. Two recently-identified riboflavin transporters G protein-coupled receptor 172B (GPR172B or RFT1) and riboflavin transporter 2 (C20orf54 or RFT2) were screened for mutations. Two missense sequence variations, c.209A>G [p.Q70R] and c.886G>A [p.V296M] were found in GPR172B. In vitro functional studies of both missense variations showed that riboflavin transport was unaffected by these variations. Quantitative real-time PCR revealed a de novo deletion in GPR172B spanning exons 2 and 3 in one allele from the mother. We postulate that haploinsufficiency of this riboflavin transporter causes mild riboflavin deficiency, and when coupled with nutritional riboflavin deficiency in pregnancy, resulted in the transient riboflavin-responsive disease seen in her newborn infant. This is the first report of a genetic defect in riboflavin transport in humans.

Effects of metabolic acidosis on expression levels of renal drug transporters.

PURPOSE: In the renal proximal tubular cells, various transporters play important roles in the secretion and reabsorption of drugs. When metabolic acidosis is induced, a number of adaptive changes occur in the kidney. The purpose of this study was to clarify the changes of drug transporters under the acidosis and the effects of these changes on urinary drug excretion. METHODS: Wistar/ST rats were given 1.5% NH4Cl in tap water for 48 h to induce the acidosis. Pharmacokinetics of PSP or metformin was evaluated. In addition, expression levels of drug transporters were examined by Western Blotting. RESULTS: The renal clearance of PSP was markedly decreased, whereas the creatinine clearance and renal clearance of metformin were unchanged. Furthermore, Western blots indicated that the protein expression level of organic anion transporter (OAT) 3 was decreased. In contrast to OAT3 levels, OAT1 and organic cation transporter (OCT) 2 levels were unaffected. An immunohistochemical analysis showed that the OAT3 protein in the proximal tubules was localized in the basolateral membrane both of the normal and the acidosis rats. CONCLUSION: The decrease of renal excretion of anionic drugs during metabolic acidosis might be partly due to a reduction in the level of OAT3 protein.