The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Development of a Highly Sensitive Cytotoxicity Assay System for CYP3A4-Mediated Metabolic Activation

ABSTRACT: Drug-induced hepatotoxicity, which is a rare but serious adverse reaction to a large number of pharmaceutical drugs, is sometimes associated with reactive metabolites produced by drug-metabolizing enzymes. In the present study, we constructed a cell-based system to evaluate the cytotoxicity of reactive metabolites produced by CYP3A4 using human hepatoma cells infected with an adenovirus vector expressing human CYP3A4 (AdCYP3A4). When seven hepatoma cell lines (HepG2, Hep3B, HLE, HLF, Huh6, Huh7, and Fa2N4 cells) were infected with AdCYP3A4, HepG2 cells showed the highest CYP3A4 protein expression and testosterone 6␤-hydroxylase activity (670 pmol ⅐ min ؊1 ⅐ mg ؊1 ). With the use of AdCYP3A4-infected HepG2 cells, the cytotoxicities of 23 drugs were evaluated by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, and the cell viability when treated with 11 drugs (amiodarone, desipramine, felbamate, isoniazid, labetalol, leflunomide, nefazodone, nitrofurantoin, tacrine, terbinafine, and tolcapone) was significantly decreased. Moreover, the transfection of siRNA for nuclear factor erythroid 2-related factor 2 (Nrf2) to decrease the cellular expression level of Nrf2 exacerbated the cytotoxicity of some drugs (troglitazone, flutamide, acetaminophen, clozapine, terbinafine, and desipramine), suggesting that the genes regulated by Nrf2 are associated with the detoxification of the cytotoxicities mediated by CYP3A4. We constructed a highly sensitive cell-based system to detect the drug-induced cytotoxicity mediated by CYP3A4. This system would be beneficial in preclinical screening in drug development and increase our understanding of the druginduced cytotoxicity associated with CYP3A4

Essential structural factors of annonaceous acetogenins as potent inhibitors of mitochondrial complex I

AbstractThe annonaceous acetogenins are the most potent of the known inhibitors of bovine heart mitochondrial complex I. These inhibitors act, at the terminal electron transfer step of the enzyme, in a similar way to the usual complex I inhibitors, such as piericidin A and rotenone; however, structural similarities are not apparent between the acetogenins and these known complex I inhibitors. A systematic set of isolated natural acetogenins was prepared and examined for their inhibitory actions with bovine heart mitochondrial complex I to identify the essential structural factors of these inhibitors for the exhibition of potent activity. Despite their very potent activity, the structural requirements of the acetogenins are not particularly rigid and remain somewhat ambiguous. The most common structural units, such as adjacent bis-tetrahydrofuran (THF) rings and hydroxyl groups in the 4- and/or 10-positions, were not essential for exhibiting potent activity. The stereochemistry surrounding the THF rings, surprisingly, seemed to be unimportant, which was corroborated by an exhaustive conformational space search analysis, indicating that the model compounds, with different stereochemical arrangements around the THF moieties, were in fairly good superimposition. Proper length and flexibility of the alkyl spacer moiety, which links the THF and the α,β-unsaturated γ-lactone ring moieties, were essential for the potent activity. This probably results from some sort of specific conformation of the spacer moiety which regulates the two ring moieties to locate into an optimal spatial position on the enzyme. It is, therefore, suggested that the structural specificity of the acetogenins, required for optimum inhibition, differs significantly from that of the common complex I inhibitors in which essential structural units are compactly arranged and conveniently defined. The structure–activity profile for complex I inhibition is discussed in comparison with those for other biological activities

Identification of the active metabolite of ticlopidine from rat in vitro metabolites

1. Ticlopidine is a well-known anti-platelet agent, but is not active by itself in vitro. We identified a metabolite with anti-platelet activity, which was generated after incubation of 2-oxo-ticlopidine with phenobarbital-induced rat liver homogenate in vitro. 2. An active moiety (UR-4501) was isolated by high-performance liquid chromatography after large-scale preparation of metabolites. 3. The chemical structure of UR-4501 was determined by a combination of liquid chromatography mass/mass spectrometry (LC/MS/MS) and nuclear magnetic resonance (NMR) analysis. 4. UR-4501 produced a concentration-dependent inhibition (3–100 μM) of ADP (10 μM)-induced human platelet aggregation, whereas 2-oxo-ticlopidine (3–100 μM) did not elicit inhibitory responses. 5. UR-4501 (10–100 μM) strongly inhibited ADP- and collagen-induced aggregation and slightly inhibited thrombin-induced aggregation. 6. The inhibition of rat washed platelet aggregation by UR-4501 (100 μM) persisted, even after the platelets had been washed twice. 7. These results suggest that UR-4501 is the molecule responsible for the in vivo activities of ticlopidine