Inhibitors of Human ABCG2: From Technical Background to Recent Updates With Clinical Implications

The ATP-binding cassette transporter G2 (ABCG2; also known as breast cancer resistance protein, BCRP) has been suggested to be involved in clinical multidrug resistance (MDR) in cancer like other ABC transporters such as ABCB1 (P-glycoprotein). As an efflux pump exhibiting a broad substrate specificity localized on cellular plasma membrane, ABCG2 excretes a variety of endogenous and exogenous substrates including chemotherapeutic agents, such as mitoxantrone and several tyrosine kinase inhibitors. Moreover, in the normal tissues, ABCG2 is expressed on the apical membranes and plays a pivotal role in tissue protection against various xenobiotics. For this reason, ABCG2 is recognized to be an important determinant of the pharmacokinetic characteristics of its substrate drugs. Although the clinical relevance of reversing the ABCG2-mediated MDR has been inconclusive, an appropriate modulation of ABCG2 function during chemotherapy should logically enhance the efficacy of anti-cancer agents by overcoming the MDR phenotype and/or improving their pharmacokinetics. To confirm this possibility, considerable efforts have been devoted to developing ABCG2 inhibitors, although there is no clinically available substance for this purpose. As a clue for addressing this issue, this mini-review provides integrated information covering the technical backgrounds necessary to evaluate the ABCG2 inhibitory effects on the target compounds and a current update on the ABCG2 inhibitors. This essentially includes our recent findings, as we serendipitously identified febuxostat, a well-used agent for hyperuricemia as a strong ABCG2 inhibitor, that possesses some promising potentials. We hope that an overview described here will add value to further studies involving in the multidrug transporters

Bafilomycin A1-sensitive pathway is required for the maturation of cystic fibrosis transmembrane conductance regulator

AbstractCystic fibrosis (CF) is the most common lethal genetic disease in Caucasians caused by the trafficking defects of CF transmembrane conductance regulator (CFTR), which is a cAMP-dependent Cl− channel at the plasma membrane. The trafficking pathway of CFTR is thought to be non-conventional because CFTR maturation is inhibited by the dysfunction of syntaxin 13, which is involved in protein recycling via endosomal pathway. In this study, to clarify whether the endosomal trafficking is required for CFTR maturation, we utilized a specific vacuolar H+-ATPase inhibitor, bafilomycin A1 (BafA1), which inhibits the protein trafficking from early endosome. Our data showed that low concentration of BafA1 (50nM) decreased the expression of mature CFTR but induced the accumulation of immature CFTR in the juxta-nuclear region containing an early endosome marker. Pulse-chase analysis showed that BafA1 inhibited the maturation of CFTR, but it slightly stabilized immature CFTR. These results indicate that BafA1-sensitive pathway is required for CFTR maturation and emphasize that endosomal trafficking pathway might be involved in the maturation of CFTR

Interploidy gene flow involving the sexual-asexual cycle facilitates the diversification of gynogenetic triploid Carassius fish

クローン繁殖フナは稀に有性生殖をしながら繁栄 --遺伝的に多様なクローンフナが存在する謎を解明--. 京都大学プレスリリース. 2021-11-19.Asexual vertebrates are rare and at risk of extinction due to their restricted adaptability through the loss of genetic recombination. We explore the mechanisms behind the generation and maintenance of genetic diversity in triploid asexual (gynogenetic) Carassius auratus fish, which is widespread in East Asian fresh waters and exhibits one of the most extensive distribution among asexual vertebrates despite its dependence on host sperm. Our analyses of genetic composition using dozens of genetic markers and genome-wide transcriptome sequencing uncover admixed genetic composition of Japanese asexual triploid Carassius consisting of both the diverged Japanese and Eurasian alleles, suggesting the involvement of Eurasian lineages in its origin. However, coexisting sexual diploid relatives and asexual triploids in Japan show regional genetic similarity in both mitochondrial and nuclear markers. These results are attributed to a unique unidirectional gene flow from diploids to sympatric triploids, with the involvement of occasional sexual reproduction. Additionally, the asexual triploid shows a weaker population structure than the sexual diploid, and multiple triploid lineages coexist in most Japanese rivers. The generated diversity via repeated interploidy gene flow as well as an increased establishment of immigrants is assumed to offset the cost of asexual reproduction and might contribute to the successful broad distribution of this asexual vertebrate

Inhibition of post-translational N-glycosylation by HRD1 that controls the fate of ABCG5/8 transporter

N-glycosylation of proteins in endoplasmic reticulum is critical for protein quality control. We showed here a post-translational N-glycosylation affected by the HRD1 E3 ubiquitin ligase. Both WT- and E3-defective C329S-HRD1 decreased the level of high mannose form of ABCG8, a protein that heterodimerizes with ABCG5 to control sterol balance. Meanwhile, HRD1 increased the non-glycosylated ABCG8 regardless of its E3 activity, thereby suppressing full maturation of ABCG5/8 transporter. Pulse chase and mutational analysis indicated that HRD1 inhibits STT3B-dependent post-translational N-glycosylation of ABCG8. Whereas, HRD1 had only slight effect on the N-glycosylation status of ABCG5; rather it accelerated ABCG5 degradation in an E3 activity-dependent manner. Finally, RMA1, another E3 ubiquitin ligase, accelerated the degradation of both ABCG5 and ABCG8 via E3 activity-dependent manner. HRD1 and RMA1 may therefore be negative regulators of disease-associated transporter ABCG5/ABCG8. The findings also highlight the unexpected E3 activity-independent role of HRD1 in the regulation of N-glycosylation

A Proposal for Practical Diagnosis of Renal Hypouricemia : Evidenced from Genetic Studies of Nonfunctional Variants of URAT1/SLC22A12 among 30,685 Japanese Individuals

Background: Renal hypouricemia (RHUC) is characterized by a low serum uric acid (SUA) level and high fractional excretion of uric acid (FEUA). Further studies on FEUA in hypouricemic individuals are needed for a more accurate diagnosis of RHUC. Methods: In 30,685 Japanese health-examination participants, we genotyped the two most common nonfunctional variants of URAT1 (NFV-URAT1), W258X (rs121907892) and R90H (rs121907896), in 1040 hypouricemic individuals (SUA ≤ 3.0 mg/dL) and 2240 individuals with FEUA data. The effects of NFV-URAT1 on FEUA and SUA were also investigated using linear and multiple regression analyses. Results: Frequency of hypouricemic individuals (SUA ≤ 3.0 mg/dL) was 0.97% (male) and 6.94% (female) among 30,685 participants. High frequencies of those having at least one allele of NFV-URAT1 were observed in 1040 hypouricemic individuals. Furthermore, NFV-URAT1 significantly increased FEUA and decreased SUA, enabling FEUA and SUA levels to be estimated. Conversely, FEUA and SUA data of hypouricemic individuals are revealed to be useful to predict the number of NFV-URAT1. Conclusions: Our findings reveal that specific patterns of FEUA and SUA data assist with predicting the number of nonfunctional variants of causative genes for RHUC, and can also be useful for practical diagnosis of RHUC even before genetic tests

Identification of a dysfunctional exon-skipping splice variant in GLUT9/SLC2A9 causal for renal hypouricemia type 2

Renal hypouricemia (RHUC) is a pathological condition characterized by extremely low serum urate and overexcretion of urate in the kidney; this inheritable disorder is classified into type 1 and type 2 based on causative genes encoding physiologically-important urate transporters, URAT1 and GLUT9, respectively; however, research on RHUC type 2 is still behind type 1. We herein describe a typical familial case of RHUC type 2 found in a Slovak family with severe hypouricemia and hyperuricosuria. Via clinico-genetic analyses including whole exome sequencing and in vitro functional assays, we identified an intronic GLUT9 variant, c.1419+1G>A, as the causal mutation that could lead the expression of p.Gly431GlufsTer28, a functionally-null variant resulting from exon 11 skipping. The causal relationship was also confirmed in another unrelated Macedonian family with mild hypouricemia. Accordingly, non-coding regions should be also kept in mind during genetic diagnosis for hypouricemia. Our findings provide a better pathogenic understanding of RHUC and pathophysiological importance of GLUT9

Subtype-specific gout susceptibility loci and enrichment of selection pressure on ABCG2 and ALDH2 identified by subtype genome-wide meta-analyses of clinically defined gout patients

Objectives Genome-wide meta-analyses of clinically defined gout were performed to identify subtype-specific susceptibility loci. Evaluation using selection pressure analysis with these loci was also conducted to investigate genetic risks characteristic of the Japanese population over the last 2000–3000 years. Methods Two genome-wide association studies (GWASs) of 3053 clinically defined gout cases and 4554 controls from Japanese males were performed using the Japonica Array and Illumina Array platforms. About 7.2 million single-nucleotide polymorphisms were meta-analysed after imputation. Patients were then divided into four clinical subtypes (the renal underexcretion type, renal overload type, combined type and normal type), and meta-analyses were conducted in the same manner. Selection pressure analyses using singleton density score were also performed on each subtype. Results In addition to the eight loci we reported previously, two novel loci, PIBF1 and ACSM2B, were identified at a genome-wide significance level (p<5.0×10–8) from a GWAS meta-analysis of all gout patients, and other two novel intergenic loci, CD2-PTGFRN and SLC28A3-NTRK2, from normal type gout patients. Subtype-dependent patterns of Manhattan plots were observed with subtype GWASs of gout patients, indicating that these subtype-specific loci suggest differences in pathophysiology along patients’ gout subtypes. Selection pressure analysis revealed significant enrichment of selection pressure on ABCG2 in addition to ALDH2 loci for all subtypes except for normal type gout. Conclusions Our findings on subtype GWAS meta-analyses and selection pressure analysis of gout will assist elucidation of the subtype-dependent molecular targets and evolutionary involvement among genotype, phenotype and subtype-specific tailor-made medicine/prevention of gout and hyperuricaemia

Genome-wide association study revealed novel loci which aggravate asymptomatic hyperuricaemia into gout

Objective The first ever genome-wide association study (GWAS) of clinically defined gout cases and asymptomatic hyperuricaemia (AHUA) controls was performed to identify novel gout loci that aggravate AHUA into gout. Methods We carried out a GWAS of 945 clinically defined gout cases and 1003 AHUA controls followed by 2 replication studies. In total, 2860 gout cases and 3149 AHUA controls (all Japanese men) were analysed. We also compared the ORs for each locus in the present GWAS (gout vs AHUA) with those in the previous GWAS (gout vs normouricaemia). Results This new approach enabled us to identify two novel gout loci (rs7927466 of CNTN5 and rs9952962 of MIR302F) and one suggestive locus (rs12980365 of ZNF724) at the genome-wide significance level (p<5.0×10– 8). The present study also identified the loci of ABCG2, ALDH2 and SLC2A9. One of them, rs671 of ALDH2, was identified as a gout locus by GWAS for the first time. Comparing ORs for each locus in the present versus the previous GWAS revealed three ‘gout vs AHUA GWAS’-specific loci (CNTN5, MIR302F and ZNF724) to be clearly associated with mechanisms of gout development which distinctly differ from the known gout risk loci that basically elevate serum uric acid level. Conclusions This meta-analysis is the first to reveal the loci associated with crystal-induced inflammation, the last step in gout development that aggravates AHUA into gout. Our findings should help to elucidate the molecular mechanisms of gout development and assist the prevention of gout attacks in high-risk AHUA individuals

Spontaneous Production of Glutathione-Conjugated Forms of 1,2-Dichloropropane: Comparative Study on Metabolic Activation Processes of Dihaloalkanes Associated with Occupational Cholangiocarcinoma

Recently, epidemiological studies revealed a positive relationship between an outbreak of occupational cholangiocarcinoma and exposure to organic solvents containing 1,2-dichloropropane (1,2-DCP). In 1,2-DCP-administered animal models, we previously found biliary excretion of potentially oncogenic metabolites consisting of glutathione-(GSH-) conjugated forms of 1,2-DCP (GS-DCPs); however, the GS-DCP production pathway remains unknown. To enhance the understanding of 1,2-DCPrelated risks to human health, we examined the reactivity of GSH with 1,2-DCP in vitro and compared it to that with dichloromethane (DCM), the other putative substance responsible for occupational cholangiocarcinoma. Our results showed that 1,2-DCP was spontaneously conjugated with GSH, whereas this spontaneous reaction was hardly detected between DCM and GSH. Further analysis revealed that glutathione S-transferase theta 1 (GSTT1) exhibited less effect on the 1,2-DCP reaction as compared with that observed for DCM. Although GSTT1-mediated bioactivation of dihaloalkanes could be a plausible explanation for the production of reactive metabolites related to carcinogenesis based on previous studies, this catalytic pathway might not mainly contribute to 1,2-DCP-related occupational cholangiocarcinoma. Considering the higher catalytic activity of GSTT1 on DCM as compared with that on 1,2-DCP, our findings suggested differences in the activation processes associated with 1,2-DCP and DCM metabolism