Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity.

Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyn's kinase activity. Conversely, Lyn did not inhibit NF-κB signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn(-/-) dendritic cells and the development of SLE-like symptoms in Lyn(-/-) mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.

Multivalent mannose-conjugated siRNA causes robust gene silencing in pancreatic macrophages in vivo.

Nucleic acid therapeutics have been utilized for gene regulation, and their recent advancement has led to approval of novel drugs for liver-related disorders. However, systemic extrahepatic delivery remains challenging. Here, we report newly designed mannose-conjugated oligonucleotides for delivering oligonucleotides to macrophages by leveraging the mannose receptor, C-type 1 (MRC1, CD206), which is abundantly expressed in macrophages. We investigated the relationship between cellular uptake and multivalency (mono to tetra) of mannose ligands or linker length and selected a trivalent-mannose ligand. Trivalent-mannose (Man3)-conjugated siRNA induced concentration-dependent gene silencing in both human CD206-overexpressing cells and human macrophages in vitro. After subcutaneous injection into mice, we observed a high distribution of Man3-conjugated oligonucleotides in the liver and pancreata as well as cellular uptake into Kupffer cells and pancreatic macrophages. A single subcutaneous injection of Man3-conjugated siRNA (10 mg/kg) targeting ß2-microglobulin (B2M) silenced B2m mRNA expression by ∼50% and decreased its protein levels in mouse pancreatic macrophages compared to those in saline-treated mice. Of note, multiple subcutaneous injections decreased B2m gene expression and B2M protein levels by ∼80% and ∼85%, respectively. These results show that mannose-conjugation with oligonucleotides is expected to help deliver oligonucleotides to macrophages and regulate gene expression in vivo, particularly in the pancreas.

Lipid nanoparticle-targeted mRNA formulation as a treatment for ornithine-transcarbamylase deficiency model mice.

Ornithine transcarbamylase (OTC) plays a significant role in the urea cycle, a metabolic pathway functioning in the liver to detoxify ammonia. OTC deficiency (OTCD) is the most prevalent urea cycle disorder. Here, we show that intravenously delivered human OTC (hOTC) mRNA by lipid nanoparticles (LNP) was an effective treatment for OTCD by restoring the urea cycle. We observed a homotrimer conformation of hOTC proteins produced by the mRNA-LNP in cells by cryo-electron microscopy. The immunohistochemistry revealed the mitochondria localization of produced hOTC proteins in hepatocytes in mice. In livers of mice intravenously injected with hOTC-mRNA/LNP at 1.0 mg/kg, the delivered hOTC mRNA levels steeply decreased with a half-life (t1/2) of 7.1 h, whereas the produced hOTC protein levels retained for 5 days and then declined with a t1/2 of 2.2 days. In OTCD model mice (high-protein diet-fed Otcspf-ash hemizygous males), a single dose of hOTC-mRNA/LNP at 3.0 mg/kg ameliorated hyperammonemia and weight loss with prolonged survival rate (22 days) compared with that of untreated mice (11 days). Weekly repeated doses at 0.3 and 1.0 mg/kg were well tolerated in wild-type mice and showed a dose-dependent amelioration of survival rate in OTCD mice, thus, showing the therapeutic potential of LNP-formulated hOTC mRNA for OTCD.

Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p.

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.

E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesis.

Continued research toward the development of new antifungals that act via inhibition of glycosylphosphatidylinositol (GPI) biosynthesis led to the design of E1210. In this study, we assessed the selectivity of the inhibitory activity of E1210 against Candida albicans GWT1 (Orf19.6884) protein, Aspergillus fumigatus GWT1 (AFUA_1G14870) protein, and human PIG-W protein, which can catalyze the inositol acylation of GPI early in the GPI biosynthesis pathway, and then we assessed the effects of E1210 on key C. albicans virulence factors. E1210 inhibited the inositol acylation activity of C. albicans Gwt1p and A. fumigatus Gwt1p with 50% inhibitory concentrations (IC(50)s) of 0.3 to 0.6 µM but had no inhibitory activity against human Pig-Wp even at concentrations as high as 100 µM. To confirm the inhibition of fungal GPI biosynthesis, expression of ALS1 protein, a GPI-anchored protein, on the surfaces of C. albicans cells treated with E1210 was studied and shown to be significantly lower than that on untreated cells. However, the ALS1 protein levels in the crude extract and the RHO1 protein levels on the cell surface were found to be almost the same. Furthermore, E1210 inhibited germ tube formation, adherence to polystyrene surfaces, and biofilm formation of C. albicans at concentrations above its MIC. These results suggested that E1210 selectively inhibited inositol acylation of fungus-specific GPI which would be catalyzed by Gwt1p, leading to the inhibition of GPI-anchored protein maturation, and also that E1210 suppressed the expression of some important virulence factors of C. albicans, through its GPI biosynthesis inhibition.

Ago-allosteric modulators of human glucagon-like peptide 2 receptor.

Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP-2R), a class-B G protein-coupled receptor (GPCR). Few synthetic agonists have been reported so far for class-B GPCRs. Here, we report the first scaffold compounds of ago-allosteric modulators for human GLP-2R, derived from methyl 2-{[(2Z)-2-(2,5-dichlorothiophen-3-yl)-2-(hydroxyimino)ethyl]sulfanyl}benzoate (compound 1).

[Contribution to development of remedies for COVID-19: focusing on Eritoran].

Eritoran (E5564) is Eisai's in-house discovered and developed investigational Toll-Like Receptor 4 (TLR4) antagonist created with natural product organic synthesis technology. It is a structural analogue of Lipid A, which is an activator of endotoxins of bacteria. It has been previously observed to be safe in 14 clinical studies including a large Phase 3 randomized trial in severe sepsis. In order to evaluate therapeutic efficacy by eritoran, we are participating in the international network REMAP-CAP-COVID (Randomized, Embedded, Multi-factorial, Adaptive Platform-Community Acquired Pneumonia COVID) which aims for novel coronavirus medicine development through drug repurposing, and began an international collaborative clinical trial in October 2020 which is designated for confirmed novel coronavirus patients who are hospitalized and are in a progressing disease state. It is hoped that through suppressing the most upstream TLR4 activity which controls production of multiple cytokines by eritoran, the cytokine storm in patients can be suppressed and pneumonia can thus be prevented from becoming severe. On the other hand, E6011 is the only humanized anti-fractalkine (FKN) monoclonal antibody in the world created by KAN Research Institute. E6011 inhibits the tight binding of CD16-positive monocytes (a cell population that highly expresses the FKN receptor CX3CR1) to vascular endothelial cells, which are important for the local inflammatory response. This is expected to suppress the formation and exacerbation of vasculopathy in COVID-19.

Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates.

mRNA and lipid nanoparticles have emerged as powerful systems for the preparation of vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The emergence of novel variants or the necessity of cold chain logistics for approved mRNA vaccines undermines the investigation of next-generation systems that could preserve both potency and stability. However, the correlation between lipid nanoparticle composition and activity is not fully explored. Here, we screened a panel of ionizable lipids in vivo and identified lead lipid nanoparticles with a branched-tail lipid structure. Buffer optimization allowed the determination of lyophilization conditions, where lipid nanoparticle-encapsulated mRNA encoding SARS-CoV-2 spike protein could induce robust immunogenicity in mice after 1 month of storage at 5°C and 25°C. Intramuscularly injected lipid nanoparticles distributed in conventional dendritic cells in mouse lymph nodes induced balanced T helper (Th) 1/Th2 responses against SARS-CoV-2 spike protein. In nonhuman primates, two doses of 10 or 100 µg of mRNA induced higher spike-specific binding geometric mean titers than those from a panel of SARS-CoV-2-convalescent human sera. Immunized sera broadly inhibited the viral entry receptor angiotensin-converting enzyme 2 (ACE2) from binding to the spike protein in all six strains tested, including variants of concern. These results could provide useful information for designing next-generation mRNA vaccines.

Irf5 siRNA-loaded biodegradable lipid nanoparticles ameliorate concanavalin A-induced liver injury.

RNA interference-based gene silencing drugs are attracting attention for treating various diseases. Lipid nanoparticles (LNPs) are carriers that efficiently deliver small interfering RNA (siRNA) to the cytoplasm of target cells. Recently, we developed potent and well-tolerated biodegradable LNPs with asymmetric ionizable lipids. Here, we evaluated the effect of LNPs on immune cells in mice. After intravenous administration, LNPs were efficiently incorporated into several tissue-resident macrophages, including liver macrophages, through an apolipoprotein E (ApoE)-independent mechanism. Administration of LNP-encapsulated siRNA against Irf5, encoding the transcription factor critical for inflammatory responses, sharply reduced its expression in macrophages in vivo, and persisted for as long as 7 days. The therapeutic potential of Irf5 siRNA-loaded LNPs in inflammatory diseases was tested in a concanavalin A (Con A)-induced hepatitis model, whose pathogenic mechanisms are dependent on cytokine secretion from macrophages. We found that Con A-induced liver injury was significantly attenuated after LNP injection. Serum aspartate transaminase, alanine aminotransferase, and inflammatory cytokine levels were significantly reduced in mice injected with Irf5 siRNA-loaded LNPs compared to those injected with control siRNA-loaded LNPs. Our results suggest that administering biodegradable LNPs to deliver siRNA is a promising strategy for treating inflammatory disorders.

Genetic and chemical inhibition of IRF5 suppresses pre-existing mouse lupus-like disease.

The transcription factor IRF5 has been implicated as a therapeutic target for the autoimmune disease systemic lupus erythematosus (SLE). However, IRF5 activation status during the disease course and the effects of IRF5 inhibition after disease onset are unclear. Here, we show that SLE patients in both the active and remission phase have aberrant activation of IRF5 and interferon-stimulated genes. Partial inhibition of IRF5 is superior to full inhibition of type I interferon signaling in suppressing disease in a mouse model of SLE, possibly due to the function of IRF5 in oxidative phosphorylation. We further demonstrate that inhibition of IRF5 via conditional Irf5 deletion and a newly developed small-molecule inhibitor of IRF5 after disease onset suppresses disease progression and is effective for maintenance of remission in mice. These results suggest that IRF5 inhibition might overcome the limitations of current SLE therapies, thus promoting drug discovery research on IRF5 inhibitors.

MPR1 as a novel selection marker in Saccharomyces cerevisiae.

L-Azetidine-2-carboxylic acid (AZC) is a toxic four-membered ring analogue of L-proline that is transported into cells by proline transporters. AZC and L-proline in the cells are competitively incorporated into nascent proteins. When AZC is present in a minimum medium, misfolded proteins are synthesized in the cells, thereby inhibiting cell growth. The MPR1 gene has been isolated from the budding yeast Saccharomyces cerevisiae Sigma1278b as a multicopy suppressor of AZC-induced growth inhibition. MPR1 encodes a novel acetyltransferase that detoxifies AZC via N-acetylation. Since MPR1 is absent in the laboratory strain of S. cerevisiae S288C, it could be a positive selection marker that confers AZC resistance in the S288C background strains. To examine the usefulness of MPR1, we constructed some plasmid vectors that harboured MPR1 under the control of various promoters and introduced them into the S288C-derived strains. The expression of MPR1 conferred AZC resistance that was largely dependent on the expression level of MPR1. In an additional experiment, the galactose-inducible MPR1 and ppr1(+), the fission yeast Schizosaccharomyces pombe homologue of MPR1, were used for gene disruption by homologous recombination, and here AZC-resistant colonies were also successfully selected. We concluded that our MPR1-AZC system provides a powerful tool for yeast transformation.

T-type Calcium Channels Determine the Vulnerability of Dopaminergic Neurons to Mitochondrial Stress in Familial Parkinson Disease.

Parkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients.

Status of KRAS in iPSCs Impacts upon Self-Renewal and Differentiation Propensity.

Oncogenic KRAS mutations in hematopoietic stem cells cause RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). KRAS plays essential roles in stemness maintenance in some types of stem cells. However, its roles in pluripotent stem cells (PSCs) are poorly understood. Here, we investigated the roles of KRAS on stemness in the context of induced PSCs (iPSCs). We used KRAS mutant (G13C/WT) and wild-type isogenic (WT/WT) iPSCs from the same RALD patients, as well as wild-type (WTed/WT) and heterozygous knockout (Δed/WT) iPSCs, both obtained by genome editing from the same G13C/WT clone. Compared with WT iPSCs, G13C/WT iPSCs displayed enforced retention of self-renewal and suppressed capacity for neuronal differentiation, while Δed/WT iPSCs showed normalized cellular characteristics similar to those of isogenic WTed/WT cells. The KRAS-ERK pathway, but not the KRAS-PI3K pathway, was shown to govern these G13C/WT-specific phenotypes, indicating the strong impact of the KRAS-ERK signaling upon self-renewal and differentiation propensity in human iPSCs.

Functional Comparison of Neuronal Cells Differentiated from Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Different Oxygen and Medium Conditions.

Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2, resulting in high responsiveness of neural cells to glutamate, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ( S)-3,5-dihydroxyphenylglycine (an agonist for mGluR1/5), as revealed by calcium imaging assays. NMDA receptors, AMPA receptors, mGluR1, and mGluR5 were functionally validated by using the specific antagonists MK-801, NBQX, JNJ16259685, and 2-methyl-6-(phenylethynyl)-pyridine, respectively. Multielectrode array analysis showed that spontaneous firing occurred earlier in cells cultured in 2% O2 than in 20% O2. Optimization of O2 tension and culture medium for neural differentiation of hiPSCs can efficiently generate physiologically relevant cells for screening systems.

A novel truncated glucagon-like peptide 2 (GLP-2) as a tool for analyzing GLP-2 receptor agonists.

Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP-2R), a class-B G protein-coupled receptor. The GLP-2R antagonist GLP-2(3-33) has relatively high partial agonistic activity, and there are as yet no ideal known potent GLP-2R antagonists. We therefore prepared several truncated forms of human GLP-2 and characterized them by binding and reporter assays to find antagonists more potent than GLP-2(3-33). We found that GLP-2(11-33) was the most potent orthosteric GLP-2R antagonist, with binding activity almost equal to those of GLP-2 and GLP-2(3-33) and weaker intrinsic agonistic activity than GLP-2(3-33). GLP-2(11-33) retained weak agonistic activity toward human, cynomolgus monkey, dog, and Syrian hamster GLP-2Rs. However, it had no agonistic activity toward rat GLP-2R. GLP-2(11-33) potentiated the agonistic activity of an ago-allosteric modulator of GLP-2R, compound 1 (N-[1-(2,5-dichlorothiophen-3-yl)-2-(phenylsulfanyl)ethylidene]hydroxylamine), synergistically toward human GLP-2R. In the case of rat GLP-2R, GLP-2(11-33) decreased the agonistic activity of compound 1, although GLP-2 and GLP-2(3-33) increased this activity additively. These findings suggest that the binding sites of the ago-allosteric modulator and GLP-2 overlap, at least in rat GLP-2R. GLP-2(11-33) is a novel, useful tool for analyzing the mode of action of agonists and ago-allosteric modulators of GLP-2R.

Species-specific differences in agonistic activity of ago-allosteric modulators toward glucagon-like peptide 2 receptor.

Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP- 2R), a class-B G protein-coupled receptor (GPCR) coupled with Gα(s). Few small-molecule agonists had been reported for class-B GPCRs, but we recently reported the first scaffold compounds of ago-allosteric modulators for human GLP-2R. Methyl 2-{[(2Z)-2-(2,5-dichlorothiophen- 3-yl)-2-(hydroxyimino)ethyl]sulfanyl}benzoate (compound 1) and its de-esterified derivative (compound 2) induced placental alkaline phosphatase (PLAP) activity in HEK293 cells overexpressing human GLP-2R and PLAP driven by cAMP response element. In this study, we observed that rat, Syrian hamster, and dog GLP-2Rs also responded to compounds 1 and 2 in the same reporter system. However, no agonistic activity of the compounds toward mouse GLP-2R was detected. Mutagenesis studies showed that mutant human GLP-2Rs with Pro392Leu substitution of mouse GLP-2R for human GLP-2R amino acid residues nullified the PLAP activity of compound 2, although these mutant receptors responded to GLP-2. This finding suggests that the Pro392 residue of human GLP-2R is essential for the agonistic activity of compound 2.

GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast.

Glycosylphosphatidylinositol (GPI) is a conserved post-translational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to beta1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029-1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and Deltagwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas Deltagwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe, and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25-32), is due to the properties of Gwt1p itself and not to other membrane components.

Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly.

Glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins are required for the adhesion of pathogenic fungi, such as Candida albicans, to human epithelium. Small molecular inhibitors of the cell surface presentation of GPI-anchored mannoproteins would be promising candidate drugs to block the establishment of fungal infections. Here, we describe a medicinal genetics approach to identifying the gene encoding a novel target protein that is required for the localization of GPI-anchored cell wall mannoproteins. By means of a yeast cell-based screening procedure, we discovered a compound, 1-[4-butylbenzyl]isoquinoline (BIQ), that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae. Treatment of C. albicans cells with this compound resulted in reduced adherence to a rat intestine epithelial cell monolayer. A previously uncharacterized gene YJL091c, named GWT1, was cloned as a dosage-dependent suppressor of the BIQ-induced phenotypes. GWT1 knock-out cells showed similar phenotypes to BIQ-treated wild-type cells in terms of cell wall structure and transcriptional profiles. Two different mutants resistant to BIQ each contained a single missense mutation in the coding region of the GWT1 gene. These results all suggest that the GWT1 gene product is the primary target of the compound.

Synthesis and evaluation of novel pyrimido-acridone, -phenoxadine, and -carbazole as topoisomerase II inhibitors.

As part of a series of studies to discover new topoisomerase II inhibitors, novel pyrimidoacridones, pyrimidophenoxadines, and pyrimidocarbazoles were synthesized, and in vitro and in vivo antitumor activities and DNA-protein and/or DNA-topoisomerase II cross-linking activity as an indicator of topoisomerase II-DNA cleavable complex formation were evaluated. The pyrimidocarbazoles possessed high in vitro and in vivo potencies. Compound 26 (ER-37326), 8-acetyl-2-[2-(dimethylamino)ethyl]-1H-pyrimido[5,6,1-jk]carbazole-1,3(2H)-dione, showed in vitro growth inhibitory activity with respective IC(50) values of 0.049 microM and 0.35 microM against mouse leukemia P388 and human oral cancer KB. In vivo, this compound inhibited the tumor growth of mouse sarcoma M5076 implanted into mice with T/C values of 42% and 13% at 3.13 and 6.25 mg/kg/d respectively without significantly affecting the body weight. In addition, compound 26 (ER-37326) increased the formation of DNA-topoisomerase II cross-linking in P388 cells.