Functional evaluation of rare OASL variants by analysis of SLE patient-derived iPSCs.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by genetic heterogeneity and an interferon (IFN) signature. The overall landscapes of the heritability of SLE remains unclear. OBJECTIVES: To identify and elucidate the biological functions of rare variants underlying SLE, we conducted analyses of patient-derived induced pluripotent stem cells (iPSCs) in combination with genetic analysis. METHODS: Two familial SLE patient- and two healthy donor (HD)-derived iPSCs were established. Type 1 IFN-secreting dendritic cells (DCs) were differentiated from iPSCs. Genetic analyses of SLE-iPSCs, and 117 SLE patients and 107 HDs in the ImmuNexUT database were performed independently. Genome editing of the variants on iPSCs was performed with the CRISPR/Cas9 system. RESULTS: Type 1 IFN secretion was significantly increased in DCs differentiated from SLE-iPSCs compared to HD-iPSCs. Genetic analyses revealed a rare variant in the 2'-5'-Oligoadenylate Synthetase Like (OASL) shared between SLE-iPSCs and another independent SLE patient, and significant accumulation of OASL variants among SLE patients (HD 0.93%, SLE 6.84%, OR 8.387) in the database. Genome editing of mutated OASL 202Q to wild-type 202 R or wild-type OASL 202 R to mutated 202Q resulted in reduced or enhanced Type 1 IFN secretion of DCs. Three other OASL variants (R60W, T261S and A447V) accumulated in SLE patients had also capacities to enhance Type 1 IFN secretion in response to dsRNA. CONCLUSIONS: We established a patient-derived iPSC-based strategy to investigate the linkage of genotype and phenotype in autoimmune diseases. Detailed case-based investigations using patient-derived iPSCs provide information to unveil the heritability of the pathogenesis of autoimmune diseases.

Virtual Screening and Quantum Chemistry Analysis for SARS-CoV-2 RNA-Dependent RNA Polymerase Using the ChEMBL Database: Reproduction of the Remdesivir-RTP and Favipiravir-RTP Binding Modes Obtained from Cryo-EM Experiments with High Binding Affinity.

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the pathogenic cause of coronavirus disease 2019 (COVID-19). The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is a potential target for the treatment of COVID-19. An RdRp complex:dsRNA structure suitable for docking simulations was prepared using a cryo-electron microscopy (cryo-EM) structure (PDB ID: 7AAP; resolution, 2.60 Å) that was reported recently. Structural refinement was performed using energy calculations. Structure-based virtual screening was performed using the ChEMBL database. Through 1,838,257 screenings, 249 drugs (37 approved, 93 clinical, and 119 preclinical drugs) were predicted to exhibit a high binding affinity for the RdRp complex:dsRNA. Nine nucleoside triphosphate analogs with anti-viral activity were included among these hit drugs, and among them, remdesivir-ribonucleoside triphosphate and favipiravir-ribonucleoside triphosphate adopted a similar docking mode as that observed in the cryo-EM structure. Additional docking simulations for the predicted compounds with high binding affinity for the RdRp complex:dsRNA suggested that 184 bioactive compounds could be anti-SARS-CoV-2 drug candidates. The hit bioactive compounds mainly consisted of a typical noncovalent major groove binder for dsRNA. Three-layer ONIOM (MP2/6-31G:AM1:AMBER) geometry optimization calculations and frequency analyses (MP2/6-31G:AMBER) were performed to estimate the binding free energy of a representative bioactive compound obtained from the docking simulation, and the fragment molecular orbital calculation at the MP2/6-31G level of theory was subsequently performed for analyzing the detailed interactions. The procedure used in this study represents a possible strategy for discovering anti-SARS-CoV-2 drugs from drug libraries that could significantly shorten the clinical development period for drug repositioning.

Anti-Chikungunya Virus Monoclonal Antibody That Inhibits Viral Fusion and Release.

Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a ß-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-µm filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.

Menaquinone-4 Accelerates Calcification of Human Aortic Valve Interstitial Cells in High-Phosphate Medium through PXR.

Recently, we confirmed that in human aortic valve interstitial cells (HAVICs) isolated from patients with aortic valve stenosis (AVS), calcification is induced in high inorganic phosphate (high-Pi) medium by warfarin (WFN). Because WFN is known as a vitamin K antagonist, reducing the formation of blood clots by vitamin K cycle, we hypothesized that vitamin K regulates WFN-induced HAVIC calcification. Here, we sought to determine whether WFN-induced HAVIC calcification in high-Pi medium is inhibited by menaquinone-4 (MK-4), the most common form of vitamin K2 in animals. HAVICs obtained from patients with AVS were cultured in α-modified Eagle's medium containing 10% FBS, and when the cells reached 80%-90% confluency, they were further cultured in the presence or absence of MK-4 and WFN for 7 days in high-Pi medium (3.2 mM Pi). Intriguingly, in high-Pi medium, MK-4 dose-dependently accelerated WFN-induced HAVIC calcification and also accelerated the calcification when used alone (at 10 nM). Furthermore, MK-4 enhanced alkaline phosphatase (ALP) activity in HAVICs, and 7 days of MK-4 treatment markedly upregulated the gene expression of the calcification marker bone morphogenetic protein 2 (BMP2). Notably, MK-4-induced calcification was potently suppressed by two pregnane X receptor (PXR) inhibitors, ketoconazole and coumestrol; conversely, PXR activity was weakly increased, but in a statistically significant and dose-dependent manner, by MK-4. Lastly, in physiologic-Pi medium, MK-4 increased BMP2 gene expression and accelerated excess BMP2 (30 ng/ml)-induced HAVIC calcification. These results suggest that MK-4, namely vitamin K2, accelerates calcification of HAVICs from patients with AVS like WFN via PXR-BMP2-ALP pathway. SIGNIFICANCE STATEMENT: For aortic valve stenosis (AVS) induced by irreversible valve calcification, the most effective treatment is surgical aortic or transcatheter aortic valve replacement, but ∼20% of patients are deemed unsuitable because of its invasiveness. For effective drug treatment strategies for AVS, the mechanisms underlying aortic valve calcification must be elucidated. Here, we show that menaquinone-4 accelerates warfarin-induced calcification of AVS-patient human aortic valve interstitial cells in high inorganic phosphate medium; this effect is mediated by pregnane X receptor-bone morphogenetic protein 2-alkaline phosphatase signaling, which could be targeted for novel drug development.