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Overexpression of EPHA2 has been observed in multiple cancers and reported to be associated with poor prognosis. Here, we produced an afucosylated humanized anti-EPHA2 monoclonal antibody (mAb), DS-8895a for cancer treatment. The antibody recognizes the extracellular juxtamembrane region of EPHA2 and therefore can bind to both full-length and truncated forms of EPHA2, which are anchored to cell membranes and recently reported to be produced by post-translational cleavage in tumors. DS-8895a exhibited markedly increased antibody dependent cellular cytotoxicity (ADCC) in vitro and also inhibited tumor growth in EPHA2-positive human breast cancer MDA-MB-231 and human gastric cancer SNU-16 xenograft mouse models. Moreover, DS-8895a in combination with cisplatin (CDDP) showed better efficacy than each of the monotherapies did in the human gastric cancer model. These results suggest that a novel antibody, DS-8895a has therapeutic potential against EPHA2-expressing tumors.

Mas-related G-protein coupled receptor member D (MRGPRD) is a G protein-coupled receptor (GPCR) which belongs to the Mas-related GPCRs expressed in the dorsal root ganglia (DRG). In this study, we investigated two novel ligands in addition to beta-alanine: (1) beta-aminoisobutyric acid, a physiologically active substance, with which possible relation to tumors has been seen together with beta-alanine; (2) diethylstilbestrol, a synthetic estrogen hormone. In addition to the novel ligands, we found that transfection of MRGPRD leads fibroblast cells to form spheroids, which would be related to oncogenicity. To understand the MRGPRD novel character, oncogenicity, a large chemical library was screened in order to obtain MRGPRD antagonists to utilize in exploring the character. The antagonist in turn inhibited the spheroid proliferation that is dependent on MRGPRD signaling as well as MRGPRD signals activated by beta-alanine. The antagonist, a small-molecule compound we found in this study, is a potential anticancer agent.

Purpose: An anti-HER2 antibody–drug conjugate with a novel topoisomerase I inhibitor, DS-8201a, was generated as a new antitumor drug candidate, and its preclinical pharmacologic profile was assessed. Experimental Design: In vitro and in vivo pharmacologic activities of DS-8201a were evaluated and compared with T-DM1 in several HER2-positive cell lines and patient-derived xenograft (PDX) models. The mechanism of action for the efficacy was also evaluated. Pharmacokinetics in cynomolgus monkeys and the safety profiles in rats and cynomolgus monkeys were assessed. Results: DS-8201a exhibited a HER2 expression-dependent cell growth–inhibitory activity and induced tumor regression with a single dosing at more than 1 mg/kg in a HER2-positive gastric cancer NCI-N87 model. Binding activity to HER2 and ADCC activity of DS-8201a were comparable with unconjugated anti-HER2 antibody. DS-8201a also showed an inhibitory activity to Akt phosphorylation. DS-8201a induced phosphorylation of Chk1 and Histone H2A.X, the markers of DNA damage. Pharmacokinetics and safety profiles of DS-8201a were favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys, supporting DS-8201a as being well tolerated in humans. DS-8201a was effective in a T-DM1–insensitive PDX model with high HER2 expression. DS-8201a, but not T-DM1, demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. Conclusions: DS-8201a exhibited a potent antitumor activity in a broad selection of HER2-positive models and favorable pharmacokinetics and safety profiles. The results demonstrate that DS-8201a will be a valuable therapy with a great potential to respond to T-DM1–insensitive HER2-positive cancers and low HER2–expressing cancers. Clin Cancer Res; 22(20); 5097–108. ©2016 AACR.

e24206Background: DS-1062a is a trophoblast cell surface protein 2 (TROP2)-targeting antibody-drug conjugate (ADC) comprised of a humanized anti-TROP2 monoclonal antibody, enzymatically cleavable p...

Background HER3 (human epidermal growth factor receptor 3) is a member of HER family, and overexpressed in breast cancer, NSCLC, melanoma, gastric cancer and pancreatic cancer patients` tissues. U3-1402a is an antibody-drug conjugate (ADC) comprised of a fully human anti-HER3 monoclonal immunoglobulin G1 (IgG1) antibody (U3-1287) covalently conjugated via a cleavable peptide linker to exatecan derivative (DXd). The DXd is released after internalization of U3-1402a and leads to apoptosis of the target tumor cells by the inhibition of topoisomerase I. This ADC achieves a high drug-to-antibody-ratio (DAR 7 to 8) with homogeneous conjugation with the topoisomerase I inhibitor. The aim of this study was to preclinically evaluate the efficacy of U3-1402a in breast cancer models. Materials and methods In order to evaluate the pharmacological potential of U3-1402a, in vitro and in vivo studies were performed. In vitro growth inhibition assay evaluated the sensitivity of U3-1402a in HER3-positive human breast cancer cell line (HCC1569) and HER3-negative human cervical carcinoma cell line (C33A). Cells were treated with U3-1402a or MAAA-1181 (payload of U3-1402a) depending on its concentration (U3-1402a: 0.153 to 10 000 ng/mL, MAAA-1181: 2.44 to 160,000 pg/mL). In vivo growth inhibition study evaluated the dose-dependent sensitivity of U3-1402a in HER3-positive breast cancer xenograft model, MDA-MB-453. In addition, several xenograft models with different HER3 expression were tested with its sensitivity to U3-1402a. These models were HCC1569 (human breast cancer cell line, HER3 IHC score 3+), MDA-MB-453 (human breast cancer cell line, HER3 IHC score 2+), NIBIO-G016 (human gastric cancer patient-derived xenograft, HER3 IHC score 1+) and MDA-MB-231 (human breast cancer cell line, HER3 IHC score 0). R esults In vitro study, U3-1402a exhibited anti-tumor killing activity in HER3-positive human breast cancer cell line, HCC1569. C-33A human cervical carcinoma cell line was not sensitive to U3-1402a even MAAA-1181 itself exhibited anti-tumor killing activity to this cell line. In vivo study, U3-1402a showed dose-dependent anti-tumor killing activity in a HER3-positive breast cancer MDA-MB-453 xenograft model. Finally, in vivo tumor regression was only observed in HER3 2+ and 3+ models. Conclusions U3-1402a preclinically exhibited its efficacy in breast cancer model in vitro and in vivo. In vivo efficacy was correlated with HER3 expression. These studies suggest that U3-1402a, a novel HER3-targeting ADC, would be efficacious in a broader patient population with HER3 expression like breast cancer, melanoma, NSCLC, gastric cancer and pancreatic cancer. Citation Format: Suguru Ueno, Kenji Hirotani, Reimar Abraham, Sabine Blum, Birgit Frankenberger, Mauricio Redondo-Muller, Johannes Bange, Yusuke Ogitani, Akiko Zembutsu, Koji Morita, Takashi Nakada, Shuji Majima, Yuki Abe, Toshinori Agatsuma. U3-1402a, a novel HER3-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2017-3092

1031 Background: DS-8201a, a HER2-targeting antibody–drug conjugate (ADC), with a topoisomerase I inhibitor, exatecan drivative (DX-8951 derivative, DXd) has been shown to have antitumor effects in preclinical xenograft models and clinical trials, but the involvement of the immune system in the antitumor efficacy of DS-8201a has not been elucidated yet. Methods: The antitumor efficacy of DS-8201a individually and in combination with an anti-PD-1 antibody was determined in a syngeneic mouse model with human HER2-expressing CT26.WT (CT26.WT-hHER2) cells. Mice whose tumors had been cured by DS-8201a treatment were rechallenged with CT26.WT-hHER2 cells; their splenocytes were co-cultured with CT26.WT-hHER2 or CT26.WT-mock cells, and IFN-g secretion by these cells was determined. To investigate effects of DXd and DS-8201a on dendritic cells (DCs), the expression of DC markers on bone marrow derived DCs (BMDCs) and intratumoral DCs was analyzed by flow cytometry. Furthermore, MHC class I and PD-L1 expression on tumor cells was analyzed. Results: At a weekly dosage of 10 mg/kg, DS-8201a showed significant antitumor effects in the mouse model. Mice whose tumors had been cured by DS-8201a treatment rejected the rechallenge with CT26.WT-hHER2 cells, and splenocytes from these mice were activated by both CT26.WT-hHER2 and CT26.WT-mock cells. In the mouse model, DS-8201a treatment raised a population of intratumoral DCs (CD45+CD11c+MHC class II+) and increased DC expression of CD86, a DC activation marker; DXd also up-regulated CD86 expression on BMDCs in vitro. Furthermore, DS-8201a up-regulated PD-L1 and MHC class I expression on tumor cells. Notably, antitumor effects of the combination of DS-8201a with an anti-PD-1 antibody were better than those of monotherapy. Conclusions: DS-8201a elicits immune responses via mechanisms other than cytotoxic effects on tumor cells. This finding suggests additional benefits of combining DS-8201a with an immune checkpoint inhibitor (ICI). The combination of DS-8201a and an anti-PD-1 antibody was effective in tumor suppression, indicating that DS-8201a may be successfully combined with an ICI in human clinical applications.

A series of hexadeoxyribonucleotides (6-mers), d(TGGGAG), substituted with a variety of aromatic groups at the 5'-end were synthesized and tested for anti-human immunodeficiency virus type 1 (HIV-1) activity. While unmodified d(TGGGAG) (31) had no anti-HIV-1 activity, compound 23 with a 3,4-di(benzyloxy)benzyl (DBB) group at the 5'-end potently inhibited the HIV-1IIIB-induced cytopathicity of MT-4 cells in vitro (IC50 = 0.37 microM) without cytotoxicity up to 40 microM. A thermal denaturation study on the 5'-end-substituted 6-mers by means of the circular dichroism (CD) spectra demonstrated that the aromatic substituent attached at the 5'-end of the 6-mer strongly enhanced the formation of a parallel helical structure consisting of four strands (quadruplex). On the contrary, compound 36, in which one of the guanosines of 23 was replaced by a thymidine, did not form a quadruplex, thus exhibiting no anti-HIV-1 activity. Moreover, both compound 15, with a tert-butyldiphenylsilyl group solely at its 3'-end, and compound 21, with a relatively small substituent, a benzyl group, at the 5'-end, formed quadruplexes but had no anti-HIV-1 activity. These findings led us to the conclusion that both the quadruplex structure and the aromatic substituent with adequate size at the 5'-end are crucial for the interaction of the 5'-end-substituted 6-mers with the V3 loop as well as the CD4 binding site on viral gp120, resulting in anti-HIV-1 activity.

We have determined that hexadeoxyribonucleotides (5′TGGGAG3′), with modified aromatic groups such as a trityl group at the 5′-end, have anti-HIV-1 activity in vitro. The 6-mer bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5′-end had the most potent activity and the least cytotoxicity. When the 3′-end of the 5′-(3,4-DBB)-modified 6-mer was substituted with a 2-hydroxyethylphosphate, a 2-hydroxyethylthiophosphate, or a methylphosphate group at the 3′-end, anti-HIV-1 activity increased. Moreover, among various 3′- and 5′-end-modified 6-mers that were tested, the 6-mer (R-95288) bearing a 3,4-DBB group at the 5′-end and a 2-hydroxyethylphosphate group at the 3′-end was the most stable, when incubated with mouse, rat, or human plasma. Therefore, R-95288 was chosen as the best candidate for possible use in therapy on the basis of its anti-HIV-1 activity.

The phosphodiester hexadeoxyribonucleotide R-95288 is a potent anti-human immunodeficiency virus type 1 (HIV-1) agent in vitro which consists or a TGGGAG nucleoside sequence with dibenzyloxybenzyl and hydroxyethyl substituents at the 5′- and 3′-ends, respectively. In this study, the antiviral activity of R-95288 against various strains of HIV-1 in vitro was assessed and its mechanism of action was analysed. R-95288 inhibited replication of all strains of HIV-1 used including laboratory strains with the syncytium-inducing (SI) phenotype and clinical isolates with both SI and non-SI (NSI) phenotypes. The 50% inhibitory concentrations (IC50s) were 0.62–18 μg mL−1 (0.21–6.2 μM). R-95288 inhibited the binding and fusion of HIV-1-infected T cells with CD4+ cells. In addition, R-95288 specifically blocked the binding of monoclonal antibodies, recognizing the anti-V3 loop or the CD4-binding site of the virus envelope glycoprotein gp120. Furthermore, the target site of R-95288 within the V3 loop was found in the putative heparin-binding region by binding inhibition assays using various anti-V3 loop antibodies. These results suggest that R-95288 can inhibit various strains of HIV-1, possibly by specific interaction with gp120.

It has been shown in previous reports that a guanine-rich phosphodiester oligonucleotide bearing a dimethoxytrityl (DmTr) residue on its 5'-terminal. DmTr-TGGGAGGTGGGTCTG (SA-1042), is an inhibitor of HIV-1 infection in vitro. SA-1042 interfered with the attachment of gp120 to the CD4 receptor and the subsequent entry stage of viral infection. We investigated the structure-activity relationship of the DmTr-conjugated oligomer by using 15-mer oligonucleotides with various nucleotide sequences. Results show that location of guanine nucleosides at the 5'-terminal and modification of the 5'-terminal with DmTr are essential for anti-HIV-1 activity. First, substitution of the guanine nucleoside close to the 5'-terminal of SA-1042 with another nucleotide prevented antiviral activity. Second, the existence of at least three consecutive guanine nucleosides adjacent to the 5'-terminal was required for the activity. Finally, modification of the 5'-terminal was essential for the activity. Based on these findings, the hexanucleotide, DmTr-TGGGAG, was identified as a potent inhibitor of HIV-1 infection. The hexamer was found to be capable of inhibiting the binding of gp120 to its receptor CD4 molecule, and it was also capable of inhibiting accessibility of anti-V3 monoclonal antibody to its ligand V3 peptide.

We analyzed the anti-HIV-1 activity of an oligonucleotide derivative, R-95288, in severe combined immunodeficient (SCID/beige) mice transplanted with normal human peripheral blood leukocytes (PBLs), designated hu-PBL-SCID/beige mice. The human chimeric mice were inoculated with HIV-1(CC1) 3 weeks after the transplantation and sacrificed 2 weeks later. Virus infection was determined by coculture of splenocytes with fresh human PBLs and also by detection of HIV- specific DNA sequences using the polymerase chain reaction. No evidence of infection was observed in mice treated with R-95288 (100 mg/kg/day) using intraperitoneal delivery by osmotic minipumps starting 1 day before virus challenge. In contrast, virus infection was observed in over 80% of the saline-treated control mice. In addition, partial inhibition of HIV-1 infection was obtained in mice treated subcutaneously with R-95288 (100 mg/kg/day). Toxicity towards the engrafted human cells was not observed by flow cytometric analysis. Moreover, R-95288 failed to inhibit lymphocyte proliferation (CC50 > 400 microg/ml), while 90% inhibition of HIV-1 replication was achieved at 3.1 microg/ml in vitro. These results suggest the ability of R-95288 to protect the human chimeric mice against HIV-1 infection.

For the construction of new combinatorial libraries, a lead compound was created by replacing the core structure of a hit compound discovered by screening for cytotoxic agents against a tumorigenic cell line. The newly designed compound maintained biological activity and allowed alternative library construction for antitumor drugs.

Fibroblast Growth Factor Receptor 4 (FGFR4) is the fourth member of the Fibroblast Growth Factor Receptor (FGFR) family of receptor tyrosine kinases. All of the FGFR's have been implicated in cancer development due to increased activation of their enzymatic activity either by gene mutation, over-expression or inadvertent ligand-mediated stimulation. One important alteration that may lead to FGFR4 activation in cancer is the overexpression of its ligand FGF19 in 20-40% of primary liver cancer. Here, we report the development of U3-1784, a phage display-derived fully human antibody that specifically binds to FGFR4 but not to isoforms of FGFR1-3. The antibody binds to an epitope in the putative ligand binding domain of the receptor and consequently inhibits ligand binding and downstream signaling. In a panel of 10 tumor models derived from hepatocellular carcinoma, U3-1784 significantly inhibits the growth of FGF19-expressing models up to 90% whereas models without FGF19 expression are insensitive. These results strongly suggest that the FGFR4/FGF19 axis is an oncogenic driver in hepatocellular carcinoma. U3-1784 is currently in phase I clinical trials. Citation Format: Rene Bartz, Keisuke Fukuchi, Tanja Lange, Katrin Gruner, Toshiaki Ohtsuka, Ichiro Watanabe, Shinko Hayashi, Mauricio Redondo-Müller, Mizuki Takahashi, Toshinori Agatsuma, Johannes Bange, Reimar Abraham. U3-1784, a human anti-FGFR4 antibody for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3852.

Oligodeoxyribonucleotides (ODN) linked at their 5'-end with dimethoxytrityl (DmTr) residue were examined for antiviral activities against human immunodeficiency virus type 1 (HIV-1). We found that guanine-rich oligonucleotides exhibit anti-HIV activity upon 5'-end modification with DmTr. One oligonucleotide, DmTr-TGGGAGGTGGGTCTG (SA-1042), showed potent anti-HIV activity in vitro. A greater than 95% reduction of infectivity was observed if the cells were treated with 10 micrograms/ml of SA-1042 at the time of viral infection, PCR analysis confirmed that there was a significant reduction of provirus in the cells exposed to virus in the presence of SA-1042. By contrast, no inhibition was observed if the cells were treated with the oligomer 1 h after virus adsorption. SA-1042 prevented syncytium formation between chronically infected cells and CD4 positive uninfected cells. Furthermore, the oligomer interfered the interaction of purified gp120 to the CD4 receptor. By contrast, SA-1042 had no inhibitory effect on chronically HIV-infected cells. These results strongly suggest that the DMTr-ODNs with appropriate base sequences antagonize HIV-1 infection during the stage of virus-cell interaction.

To elucidate the function of MAS-related GPCR, member D (MRGD) in cancers, we investigated the in vitro and in vivo oncogenic function of MRGD using murine fibroblast cell line NIH3T3 in which MRGD is stably expressed. The expression pattern of MRGD in clinical samples was also analyzed. We found that overexpression of MRGD in NIH3T3 induced focus formation and multi-cellular spheroid formation, and promoted tumors in nude mice. In other words, overexpression of MRGD in NIH3T3 induced the loss of contact inhibition, anchorage-independent growth and in vivo tumorigenesis. Furthermore, it was found that the ligand of MRGD, beta-alanine, enhanced spheroid formation in MRGD-expressing NIH3T3 cells. From investigation of clinical cancer tissues, we found high expression of MRGD in several lung cancers by immunohistochemistry as well as real time PCR. Based on these results, MRGD could be involved in tumorigenesis and could also be a novel anticancer drug target.

Lymphokine-activated killer (LAK) cells were induced from C57BL/6 mouse spleen cells and the effects of culture time on the expression of cell surface phenotypes and cytotoxic activity of LAK cells were determined. The expression of CD2 remarkably decreased after culture of LAK cells for 30 days, while LFA-1, a principal adhesion molecule in LAK cells, and CD3 were not changed by the culture. LAK cells cultured for 90 days completely lost CD2. In accordance with the decrease of CD2, the cytotoxic activity of LAK cells declined but a certain leven was retained even after the complete loss of CD2. The established LAK cell clones were also strongly positive for the expression of LFA-1 but negative for CD2. When the LAK cell clones were transfected with the CD2 cDNA, they started to express CD2 on their cell surface and to show greater binding ability and stronger cytotoxicity to target tumor cells. These results indicated that CD2 plays a role as an adhesion molecule responsible for target cell killing in murine LAK cells.

The therapeutic effect of a combined modality of lymphokine-activated killer (LAK) cells and tumor necrosis factor alpha (TNF alpha) on MBL-2 tumor in C57BL/6 mice was studied. Murine LAK cells induced from splenocytes by interleukin 2 (IL2) could lyse MBL-2 target cells in vitro. but no enhancement of the LAK activity was found by the treatment of LAK cells with TNF alpha in vitro. However, the treatment of MBL-2 with TNF alpha enhanced the sensitivity to LAK cells. Moreover, administration of TNF alpha to mice bearing solid MBL-2 tumor led to increased tumor vascular permeability within 1 h, and resulted in the enhanced accumulation of systemically transferred LAK cells in tumor tissue. Based on these results, we treated MBL-2-bearing mice with TNF alpha and then with LAK cells 1 h later. No therapeutic effect was observed when tumor-bearing mice were treated with TNF alpha alone or LAK cells plus IL2. However, adoptive immunotherapy using LAK cells and TNF alpha had therapeutic effects, i.e., growth inhibition of tumor nodules and prolongation of survival. These results indicated that appropriately timed pretreatment of tumor-bearing mice with TNF alpha augmented the anti-tumor efficacy of LAK cells.

To assess the properties of lymphokine-activated killer (LAK) cells, we established mouse LAK cell clones from LAK cell lines induced from C57BL/6 mouse spleen cells. Although these clones expressed similar phenotypes to the parent LAK cells, Lyt-2 was expressed in a restricted portion of the clones. All clones were found to express T3 CD2 and T cell receptor (TcR) alpha beta on their cell surface. Rearrangement patterns of TcR beta were the same among the clones derived from the same parent cell line but differed in those from different cell lines as determined by using C beta 1 and J beta 2 probes. The molecules responsible for LAK-target cell binding were examined by using a monoclonal antibody (mAb) against lymphocyte function associated antigen 1 (LFA-1). This mAb (termed KBA) showed inhibitory effects on both LAK-target cell binding and cytolytic activity of LAK cell clones, indicating a principal role of LFA-1 in LAK cell clones. The magnitude of perforin mRNA expression in LAK cell clones was unrelated to their cytolytic activities.

Release of granule enzyme(s) (BLT esterase) in the antibody dependent lymphokine-activated killer (LAK) cell-mediated cytotoxic reaction (LAK ADCC) was studied using LAK cells induced from murine splenocytes and thymocytes, various human tumor cells and relevant monoclonal antibodies (mAbs) to the tumor cells. BLT esterase was not significantly released from LAK cells in direct LAK cell-mediated cytotoxic reactions (LAK CMC). However, cultures of LAK cells and IgG-coated target tumor cells resulted in release of the enzyme concomitantly with target cell lysis, although esterase release proceeded faster than target cell lysis. Anti-LFA-1 mAb showed an inhibitory effect on LAK CMC but not on either LAK ADCC or BLT esterase release in the ADCC. These results indicate that exocytosis of granule enzyme from LAK cells is triggered by stimulation of Fc receptor on LAK cells and that LAK CMC and LAK ADCC differ in their lytic mechanism in terms of the release of BLT esterase.

The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9H-pyrido[2,3-b]indole acetate (MeA alpha C) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeA alpha C were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.

Based on 2-methyl-4-nitro-2H-pyrazole-3-carboxylic acid[2-(cyclohexanecarbonylamino)benzothiazol-6-yl]amide (1), which shows selective cytotoxicity against tumorigenic cell lines, 2,6-dichloro-N-[2-(cyclopropanecarbonylamino)benzothiazol-6-yl]benzamide (13b) was designed and synthesized as a biologically stable derivative containing no nitro group. The highly potent derivative 13b exhibited excellent in vivo inhibitory effect on tumor growth.

As a result of a hit-to-lead program using a technique of solution-phase parallel synthesis, a highly potent (2,4-dimethoxyphenyl)-[6-(3-fluorophenyl)-4-hydroxy-3-methylbenzofuran-2-yl]methanone (15b) was synthesized as an optimized derivative of 4-hydroxy-3-methyl-6-phenylbenzofuran-2-carboxylic acid ethyl ester (1), which was discovered as a screening hit from small-molecule libraries and exhibited selective cytotoxicity against a tumorigenic cell line.

(3-Amino-6-thiophen-2-yl-thieno[2,3-b]pyridin-2-yl)phenylmethanone (3) was discovered as a new type of cytotoxic agent selective against a tumorigenic cell line. The molecular structure of a previously reported compound, (4-hydroxy-3-methyl-6-phenylbenzofuran-2-yl)phenylmethanone (2), had remarkably similar bioisosteric substructures to that of compound 3. Although the relationship between the molecular structure and biological activity of each derivative synthesized from these two hit compounds (2 and 3) were studied, unexpectedly no correlation was observed. However, after further synthetic study from 3, one of the most potent derivative (10k) having a different SAR profile from 2, was discovered.

Based on the structure of 4-hydroxy-3-methyl-6-phenylbenzofuran-2-carboxylic acid ethyl ester (1), which exhibits selective cytotoxicity against a tumorigenic cell line, (2,4-dimethoxyphenyl)-(4-hydroxy-3-methyl-6-phenylbenzofuran-2-yl)-methanone (18m) was designed and synthesized as a biologically stable derivative containing no ester group. Although the potency of 18m was almost the same as our initial hit compound 1, 18m is expected to last longer in the human body as an anticancer agent.

We have found that a hexadeoxyribonucleotide (5′TGGGAG3′, R-95288), Koizumi, M. et al. Bioorganic & Medicinal Chemistry , 1997 , 5, 2235, bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5′-end and a 2-hydroxyethylphosphate at the 3′-end, has high anti-HIV-1 activity and the least cytotoxicity in vitro and in vivo. In order to synthesize more potent hexadeoxyribonucleotides, we substituted phosphodiester (PO) bonds in the 6-mer with the least phosphorothioate (PS), phosphoramidate (PN), or methylphosphonate (PMe) bonds. When more than two PN or PMe bonds were introduced into a 6-mer, the phosphate-modified 6-mers had weak or no anti-HIV-1 activity, in spite of quadruplex structure formation. However, when PS bonds were substituted for PO bonds, anti-HIV-1 activity of their 6-mers did not dramatically decrease, compared with compounds substituted with PN or PMe bonds. The results suggest that the formation of a quadruplex structure is not always sufficient for anti-HIV-1 activity of the 6-mer, and that net negative charges derived from PO or PS bonds in the quadruplex are important for anti-HIV-1 activity. Moreover, among various phosphate-modified ODNs, we found that the anti-HIV-1 activity of ODN PS7 with only one PS bond was the same as that of R-95288, both having a high stability in human plasma.

Previous studies have shown that a guanine-rich oligonucleotide SA-1042, DmTr-TGGGAGGTGGGTCTG, neutralizes HIV-1 infectivity, blocks syncytium formation and inhibits the binding of recombinant gp120 to immobilized soluble CD4 in vitro (Furukawa et al., 1994). We have now investigated the precise mode of action of SA-1042. We show here that SA-1042 specifically antagonizes the binding of anti-V3 loop antibodies or anti-CD4 binding-site antibodies to recombinant gp120, and also blocks the binding of an anti-V3 loop antibody to the V3 peptide (gp120IIIB: aa302-324). In contrast, SA-1042 does not inhibit gp120 binding of monoclonal antibodies directed to other regions of gp120, such as the conserved N-terminal regions (gp120IIIB: aa35-108 or gp120IIIB: aa72-130) or the C-terminal region (gp120IIIB: aa481-496). Furthermore, SA-1042 does not interfere with the binding of monoclonal antibodies directed to other molecules, gp41, CD4, CD11a, CD18, CD26, CD44 or CD54. These data suggest that SA-1042 exerts its antiviral effects by targeting the V3 loop as well as the CD4 binding site on gp120.

Male F344 rats were treated with hepatocarcinogenic heterocyclic aromatic amines such as amino acid- and protein-pyrolysate components (Trp P-1, Trp P-2, Glu P-1, Glu P-2, A alpha C, MeA alpha C, IQ and MeIQx) and changes in microsomal cytochrome P-450 isozymes in the livers were examined by means of immuno-Western blotting using anti-rat cytochrome P-450 monoclonal antibodies. The results suggested that all chemicals tested induce cytochrome P-448 isozymes, particularly cytochrome P-448H (P-450IA2), which efficiently mediate mutagenic activation of the carcinogens. This was substantiated by the enzymatic analyses with the substrates showing different characters to rat cytochrome P-450 isozyme-mediated mutagenesis.