[Veno-venous extracorporeal membrane oxygenation for capillary leak syndrome during induction chemotherapy in acute myeloid leukemia].

A 44-year-old woman was diagnosed with acute myeloid leukemia (RUNX1::RUNX1T1 translocation) and received induction chemotherapy with idarubicin hydrochloride and cytosine arabinoside. The pneumonia that had been present since admission worsened, and a drug-induced skin rash appeared. On day 17, she presented with respiratory failure and shock, complicated by hemoconcentration and hypoalbuminemia. This was considered capillary leak syndrome due to pneumonia and drug allergy, so she was started on pulse steroid therapy and IVIG, and was intubated on the same day. On day 18, venovenous-extracorporeal membrane oxygenation (VV-ECMO) was started due to worsening blood gas parameters despite ventilatory management. Bronchoalveolar lavage fluid was serous, and both blood and sputum cultures yielded negative. The patient was weaned from VV-ECMO on day 26 as the pneumonia improved with recovery of hematopoiesis. She was disoriented, and a CT scan on day 28 revealed cerebral hemorrhage. Her strength recovered with rehabilitation. After induction chemotherapy, RUNX1::RUNX1T1 mRNA was not detected in bone marrow. The patient received consolidation chemotherapy, and has maintained complete remission. Severe respiratory failure during induction chemotherapy for acute leukemia can be fatal, but VV-ECMO may be lifesaving.

Mitotic Spindle Positioning (MISP) Facilitates Colorectal Cancer Progression by Forming a Complex with Opa Interacting Protein 5 (OIP5) and Activating the JAK2-STAT3 Signaling Pathway.

Patients with inflammatory bowel disease (IBD) who experience long-term chronic inflammation of the colon are at an increased risk of developing colorectal cancer (CRC). Mitotic spindle positioning (MISP), an actin-binding protein, plays a role in mitosis and spindle positioning. MISP is found on the apical membrane of the intestinal mucosa and helps stabilize and elongate microvilli, offering protection against colitis. This study explored the role of MISP in colorectal tumorigenesis using a database, human CRC cells, and a mouse model for colitis-induced colorectal tumors triggered by azoxymethane (AOM)/dextran sodium sulfate (DSS) treatment. We found that MISP was highly expressed in colon cancer patient tissues and that reduced MISP expression inhibited cell proliferation. Notably, MISP-deficient mice showed reduced colon tumor formation in the AOM/DSS-induced colitis model. Furthermore, MISP was found to form a complex with Opa interacting protein 5 (OIP5) in the cytoplasm, influencing the expression of OIP5 in a unidirectional manner. We also observed that MISP increased the levels of phosphorylated STAT3 in the JAK2-STAT3 signaling pathway, which is linked to tumorigenesis. These findings indicate that MISP could be a risk factor for CRC, and targeting MISP might provide insights into the mechanisms of colitis-induced colorectal tumorigenesis.

IGLL5 controlled by super-enhancer affects cell survival and MYC expression in mature B-cell lymphoma.

IGLL5 is shown to be located near super-enhancer (SE) in B-cell tumors, and this gene is frequently mutated and a target of translocation in B-cell tumors. These results suggest roles of the IGLL5 in tumorigenesis; however, its functional properties have been unclear. We found that two mature B-cell lymphoma cell lines expressed IGLL5 mRNA with Cλ1 segment. JQ1 treatment resulted in down-expression of IGLL5, indicating that IGLL5 is controlled by SE. IGLL5 knockdown induced cell death with down-expression of MYC. Our results suggested that IGLL5 might have a role in survival of mature B-cell tumors and involvement in MYC expression. (100 words).

The neutrophil-osteogenic cell axis promotes bone destruction in periodontitis.

The immune-stromal cell interactions play a key role in health and diseases. In periodontitis, the most prevalent infectious disease in humans, immune cells accumulate in the oral mucosa and promote bone destruction by inducing receptor activator of nuclear factor-κB ligand (RANKL) expression in osteogenic cells such as osteoblasts and periodontal ligament cells. However, the detailed mechanism underlying immune-bone cell interactions in periodontitis is not fully understood. Here, we performed single-cell RNA-sequencing analysis on mouse periodontal lesions and showed that neutrophil-osteogenic cell crosstalk is involved in periodontitis-induced bone loss. The periodontal lesions displayed marked infiltration of neutrophils, and in silico analyses suggested that the neutrophils interacted with osteogenic cells through cytokine production. Among the cytokines expressed in the periodontal neutrophils, oncostatin M (OSM) potently induced RANKL expression in the primary osteoblasts, and deletion of the OSM receptor in osteogenic cells significantly ameliorated periodontitis-induced bone loss. Epigenomic data analyses identified the OSM-regulated RANKL enhancer region in osteogenic cells, and mice lacking this enhancer showed decreased periodontal bone loss while maintaining physiological bone metabolism. These findings shed light on the role of neutrophils in bone regulation during bacterial infection, highlighting the novel mechanism underlying osteoimmune crosstalk.

Comparison of the outcomes of patients with primary central nervous system lymphoma between the neurosurgery and hematology/oncology departments based on the relative dose intensity of methotrexate.

Primary central nervous system lymphoma (PCNSL) is an extranodal type of lymphoma, which is treated with methotrexate (MTX)-based induction therapy. Although PCNSL is a hematological malignancy, patients with PCNSL may be treated at neurosurgery or hematology/oncology departments; however, the outcomes of PCNSL treatment have not been compared between these two departments. The present study compared the outcomes of 26 patients with newly diagnosed PCNSL that were treated at the Department of Neurological Surgery or Department of Hematology/Oncology (Wakayama Medical University Hospital, Wakayama, Japan) between January 2011 and December 2021. The relative dose intensity (RDI) and relative treatment intensity of MTX were assessed as indicators of the intensity of chemotherapy. The median RDI of MTX was 67 and 93% in the neurosurgery and hematology/oncology groups, respectively (P<0.001). The proportion of patients that achieved a complete response after high-dose MTX-based therapy was significantly higher in the hematology/oncology group than in the neurosurgery group (P=0.038). The estimated 2-year overall survival was 72 and 100% in the neurosurgery and hematology/oncology groups, respectively (P=0.046). As with the difference in the outcomes observed between pediatrics and hematology departments for adolescents with acute lymphoblastic leukemia, the outcomes of patients with PCNSL may differ between neurosurgery and hematology/oncology departments.

Susceptibility to adriamycin-induced hepatotoxicity in mice depends on PRKDC polymorphism.

Adriamycin (ADR) is an effective chemotherapy drug for various cancers but has serious side effects. ADR-induced liver damage is a common problem during therapy, but the underlying mechanism remains to be fully understood. In contrast, ADR-induced glomerular damage is well studied in rodents, and sensitivity to ADR-induced nephropathy is because of the R2140C polymorphism of Prkdc gene. To investigate whether strain differences or sensitivity to ADR-induced liver damage are related to Prkdc polymorphism, this study compared the sensitivity to ADR-induced liver damage among C57BL/6J (B6J), B6-PrkdcR2140C, and BALB/c mice. Although B6J exhibits resistance to ADR-induced liver injury, BALB/c and B6-PrkdcR2140C are more susceptible to liver injury, which is exacerbated by the presence of R2140C mutation in PRKDC.

Mesenchymal loss of p53 alters stem cell capacity and models human soft tissue sarcoma traits.

Soft tissue sarcomas (STSs) are a heterogeneous group of tumors that originate from mesenchymal cells. p53 is frequently mutated in human STS. In this study, we found that the loss of p53 in mesenchymal stem cells (MSCs) mainly causes adult undifferentiated soft tissue sarcoma (USTS). MSCs lacking p53 show changes in stem cell properties, including differentiation, cell cycle progression, and metabolism. The transcriptomic changes and genetic mutations in murine p53-deficient USTS mimic those seen in human STS. Furthermore, single-cell RNA sequencing revealed that MSCs undergo transcriptomic alterations with aging-a risk factor for certain types of USTS-and that p53 signaling decreases simultaneously. Moreover, we found that human STS can be transcriptomically classified into six clusters with different prognoses, different from the current histopathological classification. This study paves the way for understanding MSC-mediated tumorigenesis and provides an efficient mouse model for sarcoma studies.

Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis.

SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3-/-mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3-/-embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3-/-mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3-/-mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3-/-mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3-/- mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases.

Molecular and Pathological Analyses of IARS1-Deficient Mice: An IARS Disorder Model.

Most mitochondrial diseases are hereditary and highly heterogeneous. Cattle born with the V79L mutation in the isoleucyl-tRNA synthetase 1 (IARS1) protein exhibit weak calf syndrome. Recent human genomic studies about pediatric mitochondrial diseases also identified mutations in the IARS1 gene. Although severe prenatal-onset growth retardation and infantile hepatopathy have been reported in such patients, the relationship between IARS mutations and the symptoms is unknown. In this study, we generated hypomorphic IARS1V79L mutant mice to develop an animal model of IARS mutation-related disorders. We found that compared to wild-type mice, IARSV79L mutant mice showed a significant increase in hepatic triglyceride and serum ornithine carbamoyltransferase levels, indicating that IARS1V79L mice suffer from mitochondrial hepatopathy. In addition, siRNA knockdown of the IARS1 gene decreased mitochondrial membrane potential and increased reactive oxygen species in the hepatocarcinoma-derived cell line HepG2. Furthermore, proteomic analysis revealed decreased levels of the mitochondrial function-associated protein NME4 (mitochondrial nucleoside diphosphate kinase). Concisely, our mutant mice model can be used to study IARS mutation-related disorders.

Robust protein-based engineering of hepatocyte-like cells from human mesenchymal stem cells.

BACKGROUND: Cells of interest can be prepared from somatic cells by forced expression of lineage-specific transcription factors, but it is required to establish a vector-free system for their clinical use. Here, we report a protein-based artificial transcription system for engineering hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells (MSCs). METHODS: MSCs were treated for 5 days with 4 artificial transcription factors (4F), which targeted hepatocyte nuclear factor (HNF)1α, HNF3γ, HNF4α, and GATA-binding protein 4 (GATA4). Then, engineered MSCs (4F-Heps) were subjected to epigenetic analysis, biochemical analysis and flow cytometry analysis with antibodies to marker proteins of mature hepatocytes and hepatic progenitors such as delta-like homolog 1 (DLK1) and trophoblast cell surface antigen 2 (TROP2). Functional properties of the cells were also examined by injecting them to mice with lethal hepatic failure. RESULTS: Epigenetic analysis revealed that a 5-day treatment of 4F upregulated the expression of genes involved in hepatic differentiation, and repressed genes related to pluripotency of MSCs. Flow cytometry analysis detected that 4F-Heps were composed of small numbers of mature hepatocytes (at most 1%), bile duct cells (~19%) and hepatic progenitors (~50%). Interestingly, ~20% of 4F-Heps were positive for cytochrome P450 3A4, 80% of which were DLK1-positive. Injection of 4F-Heps significantly increased survival of mice with lethal hepatic failure, and transplanted 4F-Heps expanded to more than 50-fold of human albumin-positive cells in the mouse livers, well consistent with the observation that 4F-Heps contained DLK1-positive and/or TROP2-positive cells. CONCLUSION: Taken together with observations that 4F-Heps were not tumorigenic in immunocompromised mice for at least 2 years, we propose that this artificial transcription system is a versatile tool for cell therapy for hepatic failures.

Peripheral-dominant liver fibrosis and tumor distribution in a mouse model of congestive hepatopathy.

AIM: Congestive hepatopathy often leads to liver fibrosis and hepatocellular carcinoma. Imaging modalities provided clinical evidence that elevation of liver stiffness and tumor occurrence are mainly induced in the periphery of the liver in patients with congestive hepatopathy. However, clinical relevance of liver stiffness and liver fibrosis is unclear because liver congestion itself increases liver stiffness in congestive hepatopathy. It also unclear which factors configure such regional disparity of tumor development in patients with congestive hepatopathy. To answer these questions, we evaluated the macroscopic spatial distribution of liver fibrosis and tumors in the murine model of congestive hepatopathy. METHODS: Chronic liver congestion was induced by partial ligation of the suprahepatic inferior vena cava. Distribution of liver congestion, fibrosis, and tumors in partial ligation of the suprahepatic inferior vena cava mice were assessed by histological findings, laser microdissection (LMD)-based qPCR and enhanced computed tomography. LMD-based RNA-sequencing was performed to identify causal factors that promote tumor development in congestive hepatopathy. RESULTS: Liver fibrosis was mainly induced in the periphery of the liver and co-localized with distribution of liver congestion. Liver tumors were also induced in the periphery of the liver where liver congestion and fibrosis occurred. LMD-based RNA-sequencing revealed the upregulation of extracellular matrix/collagen fibril-, wound healing-, angiogenesis-, morphogenesis-, and cell motility-related signaling pathways in periphery of liver compared with liver center. CONCLUSIONS: Our findings showed the experimental relevance of liver congestion, fibrosis, and tumor development in congestive hepatopathy, and may provide important locational information. Macroscopic regional disparity observed in this murine model should be considered to manage patients with congestive hepatopathy.

ETS1 governs pathological tissue-remodeling programs in disease-associated fibroblasts.

Fibroblasts, the most abundant structural cells, exert homeostatic functions but also drive disease pathogenesis. Single-cell technologies have illuminated the shared characteristics of pathogenic fibroblasts in multiple diseases including autoimmune arthritis, cancer and inflammatory colitis. However, the molecular mechanisms underlying the disease-associated fibroblast phenotypes remain largely unclear. Here, we identify ETS1 as the key transcription factor governing the pathological tissue-remodeling programs in fibroblasts. In arthritis, ETS1 drives polarization toward tissue-destructive fibroblasts by orchestrating hitherto undescribed regulatory elements of the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL) as well as matrix metalloproteinases. Fibroblast-specific ETS1 deletion resulted in ameliorated bone and cartilage damage under arthritic conditions without affecting the inflammation level. Cross-tissue fibroblast single-cell data analyses and genetic loss-of-function experiments lent support to the notion that ETS1 defines the perturbation-specific fibroblasts shared among various disease settings. These findings provide a mechanistic basis for pathogenic fibroblast polarization and have important therapeutic implications.

A protein kinase D inhibitor suppresses AKT on T cells and antagonizes cancer immunotherapy by anti-PD-1.

Antibodies that block the interaction between PD-1 and PD-1 ligands (anti-PD-1) are in clinical use for the treatment of cancer, yet their efficacy is limited. Pre-approved therapies that enhance the effect of anti-PD-1 in combination are beneficial. Small-molecule inhibitors that attenuate T cell receptor signaling are reported to prevent T cell exhaustion and induce memory T cells with stem cell potential, resulting in a durable effector T cell response in combination with anti-PD-1. In search of such targets, we focused on protein kinase D (PKD), which is suggested to be suppressive in both tumor growth and TCR signaling. We report that CRT0066101, a PKD inhibitor (PKDi), suppressed the growth of mouse tumors at a sub-micromolar concentration in vitro. Despite its inhibitory effects on tumors, a single treatment of tumor-bearing mice with PKDi did not inhibit, but rather accelerated tumor growth, and reversed the therapeutic effect of anti-PD-1. Mice treated with PKDi showed reduced T cell infiltration and defects in the generation of effector T cells, compared to those treated with anti-PD-1, suggesting that PKDi inhibited ongoing antitumor responses. Mechanistically, PKDi inhibited phosphorylation of AKT, a primary checkpoint that is reactivated by anti-PD-1. In conclusion, PKD is fundamentally required for T cell reactivation by anti-PD-1; therefore, inhibition of PKD is not appropriate for combination therapy with anti-PD-1. On the other hand, a single dose of PKDi was shown to strongly suppress experimental autoimmunity in mice, indicating that PKDi could be useful for the treatment of immune-related adverse events that are frequently reported in anti-PD-1 therapy.

Isolation of human monoclonal antibodies with neutralizing activity to a broad spectrum of SARS-CoV-2 viruses including the Omicron variants.

Monoclonal antibody therapy is a promising option for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and a cocktail of antibodies (REGN-COV) has been administered to infected patients with a favorable outcome. However, it is necessary to continue generating novel sets of monoclonal antibodies with neutralizing activity because viral variants can emerge that show resistance to the currently utilized antibodies. Here, we isolated a new cocktail of antibodies, EV053273 and EV053286, from peripheral blood mononuclear cells derived from convalescent patients infected with wild-type SARS-CoV-2. EV053273 exerted potent antiviral activity against the Wuhan wild-type virus as well as the Alpha and Delta variants in vitro, whereas the antiviral activity of EV053286 was moderate, but it had a wide-range of suppressive activity on the wild-type virus as well as the Alpha, Beta, Delta, Kappa, Omicron BA.1, and BA.2 variants. With the combined use of EV053273 and EV053286, we observed similar inhibitory effects on viral replication as with REGN-COV in vitro. We further assessed their activity in vivo by using a mouse model infected with a recently established viral strain with adopted infectious activity in mice. Independent experiments revealed that the combined use of EV053273 and EV053286 or the single use of each monoclonal antibody efficiently blocked infection in vivo. Together with data showing that these two monoclonal antibodies could neutralize REGN-COV escape variants and the Omicron variant, our findings suggest that the EV053273 and EV053286 monoclonal antibody cocktail is a novel clinically applicable therapeutic candidate for SARS-CoV-2 infection.

SLC15A4 mediates M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress.

The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage's respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells' ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.

Stepwise cell fate decision pathways during osteoclastogenesis at single-cell resolution.

Osteoclasts are the exclusive bone-resorbing cells, playing a central role in bone metabolism, as well as the bone damage that occurs under pathological conditions1,2. In postnatal life, haematopoietic stem-cell-derived precursors give rise to osteoclasts in response to stimulation with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, both of which are produced by osteoclastogenesis-supporting cells such as osteoblasts and osteocytes1-3. However, the precise mechanisms underlying cell fate specification during osteoclast differentiation remain unclear. Here, we report the transcriptional profiling of 7,228 murine cells undergoing in vitro osteoclastogenesis, describing the stepwise events that take place during the osteoclast fate decision process. Based on our single-cell transcriptomic dataset, we find that osteoclast precursor cells transiently express CD11c, and deletion of receptor activator of nuclear factor-κB specifically in CD11c-expressing cells inhibited osteoclast formation in vivo and in vitro. Furthermore, we identify Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) as the molecular switch triggering terminal differentiation of osteoclasts, and deletion of Cited2 in osteoclast precursors in vivo resulted in a failure to commit to osteoclast fate. Together, the results of this study provide a detailed molecular road map of the osteoclast differentiation process, refining and expanding our understanding of the molecular mechanisms underlying osteoclastogenesis.

OPG Production Matters Where It Happened.

Osteoprotegerin (OPG) is a circulating decoy receptor for RANKL, a multifunctional cytokine essential for the differentiation of tissue-specific cells in bone and immune systems such as osteoclasts, medullary thymic epithelial cells (mTECs), and intestinal microfold cells (M cells). However, it is unknown whether OPG functions only at the production site or circulates to other tissues acting in an endocrine fashion. Here we explore the cellular source of OPG by generating OPG-floxed mice and show that locally produced OPG, rather than circulating OPG, is crucial for bone and immune homeostasis. Deletion of OPG in osteoblastic cells leads to severe osteopenia without affecting serum OPG. Deletion of locally produced OPG increases mTEC and M cell numbers while retaining the normal serum OPG level. This study shows that OPG limits its functions within the tissue where it was produced, illuminating the importance of local regulation of the RANKL system.

Fibroblasts as a source of self-antigens for central immune tolerance.

Fibroblasts are one of the most common but also neglected types of stromal cells, the heterogeneity of which underlies the specific function of tissue microenvironments in development and regeneration. In the thymus, autoreactive T cells are thought to be negatively selected by reference to the self-antigens expressed in medullary epithelial cells, but the contribution of other stromal cells to tolerance induction has been poorly examined. In the present study, we report a PDGFR+ gp38+ DPP4- thymic fibroblast subset that is required for T cell tolerance induction. The deletion of the lymphotoxin ß-receptor in thymic fibroblasts caused an autoimmune phenotype with decreased expression of tissue-restricted and fibroblast-specific antigens, offering insight into the long-sought target of lymphotoxin signaling in the context of the regulation of autoimmunity. Thus, thymic medullary fibroblasts play an essential role in the establishment of central tolerance by producing a diverse array of self-antigens.

GRL-0920, an Indole Chloropyridinyl Ester, Completely Blocks SARS-CoV-2 Infection.

We assessed various newly generated compounds that target the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and various previously known compounds reportedly active against SARS-CoV-2, employing RNA quantitative PCR (RNA-qPCR), cytopathicity assays, and immunocytochemistry. Here, we show that two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, exerted potent activity against SARS-CoV-2 in cell-based assays performed using VeroE6 cells and TMPRSS2-overexpressing VeroE6 cells. While GRL-0820 and the nucleotide analog remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred. No significant anti-SARS-CoV-2 activity was found for several compounds reportedly active against SARS-CoV-2 such as lopinavir, nelfinavir, nitazoxanide, favipiravir, and hydroxychroloquine. In contrast, GRL-0920 exerted potent activity against SARS-CoV-2 (50% effective concentration [EC50] = 2.8 µM) and dramatically reduced the infectivity, replication, and cytopathic effect of SARS-CoV-2 without significant toxicity as examined with immunocytochemistry. Structural modeling shows that indole and chloropyridinyl of the derivatives interact with two catalytic dyad residues of Mpro, Cys145 and His41, resulting in covalent bonding, which was verified using high-performance liquid chromatography-mass spectrometry (HPLC/MS), suggesting that the indole moiety is critical for the anti-SARS-CoV-2 activity of the derivatives. GRL-0920 might serve as a potential therapeutic for coronavirus disease 2019 (COVID-19) and might be optimized to generate more-potent anti-SARS-CoV-2 compounds.IMPORTANCE Targeting the main protease (Mpro) of SARS-CoV-2, we identified two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, active against SARS-CoV-2, employing RNA-qPCR and immunocytochemistry and show that the two compounds exerted potent activity against SARS-CoV-2. While GRL-0820 and remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred as examined with immunocytochemistry. In contrast, GRL-0920 completely blocked the infectivity and cytopathic effect of SARS-CoV-2 without significant toxicity. Structural modeling showed that indole and chloropyridinyl of the derivatives interacted with two catalytic dyad residues of Mpro, Cys145 and His41, resulting in covalent bonding, which was verified using HPLC/MS. The present data should shed light on the development of therapeutics for COVID-19, and optimization of GRL-0920 based on the present data is essential to develop more-potent anti-SARS-CoV-2 compounds for treating COVID-19.