Inhibition Mechanism of SARS-CoV-2 Infection by a Cholesterol Derivative, Nat-20(S)-yne.

The coronavirus disease 2019 (COVID-19) is caused by the etiological agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19, with the recurrent epidemics of new variants of SARS-CoV-2, remains a global public health problem, and new antivirals are still required. Some cholesterol derivatives, such as 25-hydroxycholesterol, are known to have antiviral activity against a wide range of enveloped and non-enveloped viruses, including SARS-CoV-2. At the entry step of SARS-CoV-2 infection, the viral envelope fuses with the host membrane dependent of viral spike (S) glycoproteins. From the screening of cholesterol derivatives, we found a new compound 26,27-dinorcholest-5-en-24-yne-3ß,20-diol (Nat-20(S)-yne) that inhibited the SARS-CoV-2 S protein-dependent membrane fusion in a syncytium formation assay. Nat-20(S)-yne exhibited the inhibitory activities of SARS-CoV-2 pseudovirus entry and intact SARS-CoV-2 infection in a dose-dependent manner. Among the variants of SARS-CoV-2, inhibition of infection by Nat-20(S)-yne was stronger in delta and Wuhan strains, which predominantly invade into cells via fusion at the plasma membrane, than in omicron strains. The interaction between receptor-binding domain of S proteins and host receptor ACE2 was not affected by Nat-20(S)-yne. Unlike 25-hydroxycholesterol, which regulates various steps of cholesterol metabolism, Nat-20(S)-yne inhibited only de novo cholesterol biosynthesis. As a result, plasma membrane cholesterol content was substantially decreased in Nat-20(S)-yne-treated cells, leading to inhibition of SARS-CoV-2 infection. Nat-20(S)-yne having a new mechanism of action may be a potential therapeutic candidate for COVID-19.

Kaposi's sarcoma-associated herpesvirus replication and transcription activator protein activates CD274/PD-L1 gene promoter.

Kaposi's sarcoma-associated herpesvirus (KSHV) is responsible for the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. The expression of immunosuppressive genes, such as IL-10 and CD274/PD-L1 is observed during KSHV-associated pathogenesis, and the modulation of the host immune system by KSHV contributes to establishing viral persistence in the host. Understanding the mechanism that allows the virus to evade host cell immunity would be helpful in order to develop therapeutic strategies for KSHV malignancy. In this study, we show that KSHV replication and transcriptional activator (K-RTA), an essential activator of the viral lytic cycle, transactivates the CD274/PD-L1 gene promoter. Mechanistically, we demonstrate that the binding of K-RTA to the cellular specificity protein 1 (SP1) is critical for K-RTA-mediated CD274/PD-L1 promoter activation. These findings suggest that K-RTA cooperates with intracellular SP1 to activate the expression of CD274/PD-L1, which helps the virus regulate immune checkpoints to escape and survive.

The combination of levodopa with levodopa-metabolizing enzyme inhibitors prevents severe fever with thrombocytopenia syndrome virus infection in vitro more effectively than single levodopa.

Severe fever with thrombocytopenia syndrome is a hemorrhagic fever caused by a tick-borne infection. The causative agent, Dabie bandavirus, is also called the severe fever with thrombocytopenia syndrome virus (SFTSV). Ogawa et al. (2022) reported that levodopa, an antiparkinsonian drug with an o-dihydroxybenzene backbone, which is important for anti-SFTSV activity, inhibited SFTSV infection. Levodopa is metabolized by dopa decarboxylase (DDC) and catechol-O-methyltransferase (COMT) in vivo. We evaluated the anti-SFTSV efficacy of two DDC inhibitors, benserazide hydrochloride and carbidopa, and two COMT inhibitors, entacapone and nitecapone, which also have an o-dihydroxybenzene backbone. Only DDC inhibitors inhibited SFTSV infection with pretreatment of the virus (half-maximal inhibitory concentration [IC50]: 9.0-23.6 µM), whereas all the drugs inhibited SFTSV infection when infected cells were treated (IC50: 21.3-94.2 µM). Levodopa combined with carbidopa and/or entacapone inhibited SFTSV infection in both conditions: pretreatment of the virus (IC50: 2.9-5.8 µM) and treatment of infected cells (IC50: 10.7-15.4 µM). The IC50 of levodopa in the above-mentioned study for pretreatment of the virus and treatment of infected cells were 4.5 and 21.4 µM, respectively. This suggests that a synergistic effect was observed, especially for treatment of infected cells, although the effect is unclear for pretreatment of the virus. This study demonstrates the anti-SFTSV efficacy of levodopa-metabolizing enzyme inhibitors in vitro. These drugs may increase the time for which the levodopa concentration is maintained in vivo. The combination of levodopa and levodopa-metabolizing enzyme inhibitors might be a candidate for drug repurposing.

The function of SARS-CoV-2 spike protein is impaired by disulfide-bond disruption with mutation at cysteine-488 and by thiol-reactive N-acetyl-cysteine and glutathione.

Viral spike proteins play important roles in the viral entry process, facilitating attachment to cellular receptors and fusion of the viral envelope with the cell membrane. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor angiotensin converting enzyme-2 (ACE2) via its receptor-binding domain (RBD). The cysteine residue at position 488, consisting of a disulfide bridge with cysteine 480 is located in an important structural loop at ACE2-binding surface of RBD, and is highly conserved among SARS-related coronaviruses. We showed that the substitution of Cys-488 with alanine impaired pseudotyped SARS-CoV-2 infection, syncytium formation, and cell-cell fusion triggered by SARS-CoV-2 spike expression. Consistently, in vitro binding of RBD and ACE2, spike-mediated cell-cell fusion, and pseudotyped viral infection of VeroE6/TMPRSS2 cells were inhibited by the thiol-reactive compounds N-acetylcysteine (NAC) and a reduced form of glutathione (GSH). Furthermore, we demonstrated that the activity of variant spikes from the SARS-CoV-2 alpha and delta strains were also suppressed by NAC and GSH. Taken together, these data indicate that Cys-488 in spike RBD is required for SARS-CoV-2 spike functions and infectivity, and could be a target of anti-SARS-CoV-2 therapeutics.

L-DOPA, a treatment for Parkinson's disease, and its enantiomer D-DOPA inhibit severe fever with thrombocytopenia syndrome virus infection in vitro.

Severe fever with thrombocytopenia syndrome (SFTS) is a hemorrhagic fever. Patients mainly develop fever, thrombocytopenia, and leukopenia. A high case fatality rate of 16.2-47% has been reported. Vaccines and antivirals that are effective against SFTS virus (SFTSV) are not yet available in clinical practice. We previously showed that o-dihydroxybenzene is the important chemical core structure for anti-SFTSV activity. In this study, we evaluated the anti-SFTSV efficacy of 3-Hydroxy-L-tyrosine (L-DOPA), a treatment for Parkinson's disease and its enantiomer, 3-hydroxy-D-tyrosine (D-DOPA), both of which have an o-dihydroxybenzene backbone. SFTSV was preincubated with L- or D-DOPA and then inhibition of viral infection as well as viral attachment to host cells were evaluated by viral quantification. Both L- and D-DOPA inhibited SFTSV infection in a dose-dependent manner, mainly by blocking viral attachment to host cells. The half-maximal inhibitory concentration (IC50) of L-DOPA was 4.46-5.09 µM. IC50 of D-DOPA was 4.23-6.72 µM. IC50 of L-DOPA is very close to its maximum blood concentration after oral administration as a therapy for Parkinson's disease. D-DOPA, which IC50 was almost the same as that of L-DOPA, might not cause side effect. Thus, our present study demonstrated that L- and D-DOPA are potentially useful candidates for anti-SFTSV drugs.

SARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization and Intracellular S1/S2 Processing.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor-angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry. SARS-CoV-2 spike protein expression in the ACE2-expressing cell surface induces cell-cell membrane fusion, thus forming syncytia. To exert its fusogenic activity, the spike protein is typically processed at a specific site (the S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the spike-ACE2 interacting surface, is critical for the fusogenic and infectious roles of the SARS-CoV-2 spike protein. We have demonstrated that the C488 residue of the spike protein is involved in subcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed by anti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover, brefeldin-A-mediated endoplasmic-reticulum-to-Golgi traffic suppression also suppressed spike protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing and syncytia formation, the C488 residue of spike protein is required for functional spike protein processing.

Direct Inhibition of SARS-CoV-2 Spike Protein by Peracetic Acid.

Peracetic acid (PAA) disinfectants are effective against a wide range of pathogenic microorganisms, including bacteria, fungi, and viruses. Several studies have shown the efficacy of PAA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, its efficacy in SARS-CoV-2 variants and the molecular mechanism of action of PAA against SARS-CoV-2 have not been investigated. SARS-CoV-2 infection depends on the recognition and binding of the cell receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the spike protein. Here, we demonstrated that PAA effectively suppressed pseudotyped virus infection in the Wuhan type and variants, including Delta and Omicron. Similarly, PAA reduced the authentic viral load of SARS-CoV-2. Computational analysis suggested that the hydroxyl radicals produced by PAA cleave the disulfide bridges in the RBD. Additionally, the PAA treatment decreased the abundance of the Wuhan- and variant-type spike proteins. Enzyme-linked immunosorbent assay showed direct inhibition of RBD-ACE2 interactions by PAA. In conclusion, the PAA treatment suppressed SARS-CoV-2 infection, which was dependent on the inhibition of the interaction between the spike RBD and ACE2 by inducing spike protein destabilization. Our findings provide evidence of a potent disinfection strategy against SARS-CoV-2.

Effect of TNIK upregulation on JQ1-resistant human colorectal cancer HCT116 cells.

JQ1 disrupts the binding of bromodomain and extra-terminal (BET) family of proteins to acetylated histones, modulates the expression of various genes, and inhibits the proliferation of cancer cells. We established two JQ1-resistant sublines from human colorectal cancer HCT116 cells. These resistant cells showed an 8- to 9-fold higher resistance to JQ1, and a 2- to 4-fold higher resistance to various anti-cancer agents, such as doxorubicin, etoposide, mitoxantrone, SN-38, cisplatin, and methotrexate than the parental HCT116 cells. The JQ1-resistant cells expressed higher levels of TRAF2 and NCK-interacting protein kinase (TNIK), cyclin D1 (CCND1), cyclin E1 (CCNE1), and their corresponding mRNAs than the parental cells. TNIK is a regulator of Wnt/ß-catenin signaling and is known to transactivate CCND1. Transient transfection of HCT116 cells with a TNIK expression plasmid resulted in the upregulation of cyclin D1, cyclin E1, and their corresponding mRNAs, as well as an increase in CCNE1 promoter activity. Furthermore, luciferase assay revealed that the JQ1-resistant cells showed high CCNE1 promoter activity. These results suggest that TNIK also transactivates CCNE1. Three stable TNIK transfectant clones of HEK293 cells expressed 1.5- to 2-fold higher levels of TNIK, cyclin D1, and cyclin E1 than the parental cells. The 293/TNIK-6 cells, which expressed the highest level of TNIK among the transfectants, showed a 2.3-fold higher resistance to JQ1 than the parental cells. These results suggest the possible involvement of TNIK in cellular resistance to JQ1.

IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) causes a persistent infection, presenting latent and lytic replication phases during its life cycle. KSHV-related diseases are associated with deregulated expression of inflammatory cytokines, including IL-6 and IL-10, but the mechanisms underlying this dysregulation are unclear. Herein, we report a molecular mechanism for KSHV-induced IL-10 gene expression. KSHV replication and transcription activator (K-RTA) is a molecular switch for the initiation of expression of viral lytic genes, and we describe, for the first time, that K-RTA significantly activates the promoter of the human IL-10 gene. Of note, mutations involving a basic region of K-RTA reduced the association of K-RTA with the IL-10 promoter. Moreover, the host-cell transcription factors, specificity proteins (SP) 1 and 3, play a pivotal cooperative role in K-RTA-mediated transactivation of the IL-10 promoter. K-RTA can interact with SP1 and SP3 directly in vitro, and electrophoresis mobility shift assays (EMSAs) revealed co-operative interaction involving K-RTA, SP1, and SP3 in binding to the IL-10 promoter. As DNase I footprinting assays indicated that K-RTA did not affect SP3 binding to the IL-10 promoter, SP3 can function to recruit K-RTA to the IL-10 promoter. These findings indicate that K-RTA can directly contribute to IL-10 up-regulation via a functional interplay with the cellular transcription factors SP1 and SP3.

Novel pharmacological effects of poly (ADP-ribose) polymerase inhibitor rucaparib on the lactate dehydrogenase pathway.

Poly (ADP-ribose) polymerases (PARPs) are involved in various cellular events, including DNA repair. PARP inhibitors including olaparib and rucaparib, have been specially developed against breast and ovarian cancers deficient in DNA repair systems. In this study, we found that PARP1-defective olaparib-resistant A2780 cells (ola-R cells) cells were still sensitive to two PARP inhibitors, rucaparib and veliparib. Metabolomic analysis revealed that rucaparib suppressed the lactate dehydrogenase (LDH)-mediated conversion of pyruvic acid to lactic acid in A2780 cells, although olaparib did not. The inhibition of LDH by siRNA-mediated knockdown or by LDH inhibitors suppressed the growth of ovarian cancer cells. Our results suggested that the suppression of the LDH-associated pathway contributed to the pharmacological effects of rucaparib.

Functional Effects of AKT3 on Aurora Kinase Inhibitor-induced Aneuploidy.

The suppression of mitotic Aurora kinases (AURKs) by AURK inhibitors frequently causes cytokinetic failure, leading to polyploidy or aneuploidy, indicating the critical role of AURK-mediated phosphorylation during cytokinesis. We demonstrate the deregulated expression of AKT3 in Aurora kinase inhibitor (AURKi)-resistant cells, which we established from human colorectal cancer HCT 116 cells. The AKT family, which includes AKT1, -2, and -3, plays multiple roles in antiapoptotic functions and drug resistance and is involved in cell growth and survival pathways. We found that an AKT inhibitor, AZD5363, showed synergistic effect with an AURKi, VX-680, on two AKT3-expressing AURKi-resistant cell lines, and AKT3 knockdown sensitized cells to VX-680. Consistent with these activities, AKT3 expression suppressed AURKi-induced apoptosis and conferred resistance to AURKi. Thus, AKT3 expression affects cell sensitivity to AURKi. Moreover, we found that AKT3 expression suppressed AURKi-induced aneuploidy, and inversely AKT3 knockdown enhanced it. In addition, partial co-localization of AKT3 with AURKB was observed during anaphase. Overall, this study suggests that AKT3 could repress the antiproliferative effects of AURKi, with a novel activity particularly suppressing the aneuploidy induction.

Protein kinase C alpha-mediated phosphorylation of PIM-1L promotes the survival and proliferation of acute myeloid leukemia cells.

FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) is a constitutively active mutant of FLT3 and causes 20%-30% of acute myeloid leukemia (AML) cases. FLT3-ITD upregulates the proviral integration site for Moloney murine leukemia virus 1 (PIM-1) expression and promotes the proliferation of AML cells. In this study, we investigated the role of protein kinase C (PKC)-mediated phosphorylation on the expression and function of PIM-1L. Drug screening in leukemia cell lines revealed that sotrastaurin (a PKC inhibitor) suppressed the proliferation of the FLT3-ITD-positive AML cell line MV4-11 but not of K562, HL60, or KG-1a cells, similar to SGI-1776 (a PIM-1/FLT3 inhibitor) and quizartinib (an FLT3 inhibitor). Sotrastaurin decreased the expression of pro-survival protein myeloid cell leukemia (MCL-1) and the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), both of which are downstream effectors of PIM-1. PKCα directly phosphorylated Ser65 of PIM-1L, which is a long isoform of PIM-1. The PKCα-mediated phosphorylation stabilized PIM-1L. The phosphorylation-mimicked mutant, PIM-1L-S65D, was more stable and showed higher kinase activity than PIM-1L-S65A. Expression of PIM-1L-wildtype or -S65D reduced sotrastaurin-mediated apoptosis and growth inhibition in MV4-11 transfectants. These results suggest that PKCα directly upregulates PIM-1L, resulting in promotion of the survival and proliferation of AML cells.

Effect of AKT3 expression on MYC- and caspase-8-dependent apoptosis caused by polo-like kinase inhibitors in HCT 116 cells.

Polo-like kinase (PLK) is a cell-cycle regulator that is overexpressed in several cancer cell types. Polo-like kinase is considered a novel target for cancer therapies, and several PLK inhibitors (PLKis), including BI 2536, BI 6727, and GSK461364, have been developed. In this study, we established five BI 2536-resistant cell lines from human colorectal cancer HCT 116 cells, to explore the resistance mechanism and identify predictable biomarkers of PLKis. We showed that PLKi-induced caspase-8 activation was attenuated in the BI 2536-resistant cell lines. We also showed that the expression of P-glycoprotein (P-GP) and AKT3 was upregulated, whereas that of MYC was downregulated in some BI 2536-resistant cell lines. Expression of P-GP conferred resistance to PLKis, and PLKi-induced apoptosis was dependent on MYC and caspase-8 in HCT 116 cells. We also showed for the first time that AKT3 suppressed BI 6727-induced caspase-8 activation and conferred resistance to PLKis. Collectively, these results indicate that MYC, caspase-8, P-GP, and AKT3 play critical roles in PLKi-induced apoptosis. Therefore, they are candidate biomarkers of the pharmacological efficacy of PLKis.

Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies.

A current challenge is the emergence of SARS-CoV-2 variants, such as BQ.1.1 and XBB.1.5, that can evade immune defenses, thereby limiting antibody drug effectiveness. Emergency-use antibody drugs, including the widely effective bebtelovimab, are losing their benefits. One potential approach to address this issue are bispecific antibodies which combine the targeting abilities of two antibodies with distinct epitopes. We engineered neutralizing bispecific antibodies in the IgG-scFv format from two initially non-neutralizing antibodies, CvMab-6 (which binds to the receptor-binding domain [RBD]) and CvMab-62 (targeting a spike protein S2 subunit epitope adjacent to the known anti-S2 antibody epitope). Furthermore, we created a bispecific antibody by incorporating the scFv of bebtelovimab with our anti-S2 antibody, demonstrating significant restoration of effectiveness against bebtelovimab-resistant BQ.1.1 variants. This study highlights the potential of neutralizing bispecific antibodies, which combine existing less effective anti-RBD antibodies with anti-S2 antibodies, to revive the effectiveness of antibody therapeutics compromised by immune-evading variants.

FBXO15 regulates P-glycoprotein/ABCB1 expression through the ubiquitin--proteasome pathway in cancer cells.

Expression of P-glycoprotein (P-gp)/ABCB1 on cancer cell surfaces is a critical determinant of anticancer drug resistance. Regulators of P-gp expression and function are key molecules controlling drug resistance. Here we report the mechanism underlying the ubiquitin-proteasome pathway-mediated degradation of P-gp. The proteasome inhibitor MG132 increased the P-gp level, enhanced its ubiquitination, and delayed the disappearance of the ubiquitinated P-gp. To search for regulators of P-gp ubiquitination, MALDI-time of flight mass spectrometry analyses were carried out, and 22 candidates were identified as P-gp binding partners. Among them, FBXO15/Fbx15 is known as an F-box protein in the ubiquitin E3 ligase complex, Skp1-Cullin1-FBXO15 (SCF(Fbx15) ); therefore, we further studied the involvement of FBXO15 on P-gp degradation. Coprecipitation assays revealed that FBXO15 bound to P-gp. We screened ubiquitin-conjugating enzyme E2s that bind to FBXO15 and P-gp; Ube2r1/Cdc34/Ubc3 was found to be a binding partner. Exogenous FBXO15 expression enhanced P-gp ubiquitination, but FBXO15 knockdown suppressed it. FBXO15 knockdown increased P-gp expression without affecting its mRNA level. Ube2r1 knockdown decreased P-gp ubiquitination, and simultaneous knockdown of Ube2r1 with FBXO15 further suppressed the ubiquitination. Ube2r1 knockdown increased P-gp expression, suggesting that Ube2r1 is a partner of FBXO15 in P-gp ubiquitination. FBXO15 knockdown enhanced vincristine resistance and lowered intracellular levels of rhodamine 123. These data suggest that FBXO15 and Ube2r1 regulate P-gp expression through the ubiquitin-proteasome pathway.

p38MAPK and Rho-dependent kinase are involved in anoikis induced by anicequol or 25-hydroxycholesterol in DLD-1 colon cancer cells.

Anchorage-independent growth is evidence of the malignant transformation of cells. We previously reported the characterization of anicequol, a novel inhibitor of the anchorage-independent growth of tumor cells, and here we show that the effects of 25-hydroxycholesterol (25-HC) on colon cancer cells were very similar to those of anicequol. By analyzing the effects of inhibitors and performing RNA interference experiments, we found that p38 mitogen-activated protein kinase (p38MAPK) was involved in anicequol- and 25-HC-induced anoikis in DLD-1 cells. In addition, Rho-associated, coiled-coil containing protein kinase (ROCK) was also associated with anoikis induced by anicequol or 25-HC. Taken together, our findings suggest that activation of the p38MAPK and ROCK pathways might provide a new therapeutic strategy against cancer, and raise the possibility that tumor metastasis is influenced by 25-HC under physiological conditions.

Regulating the licensing of DNA replication origins in metazoa.

Eukaryotic DNA replication is a highly conserved process; the proteins and sequence of events that replicate animal genomes are remarkably similar to those that replicate yeast genomes. Moreover, the assembly of prereplication complexes at DNA replication origins ('DNA licensing') is regulated in all eukaryotes so that no origin fires more than once in a single cell cycle. And yet there are significant differences between species both in the selection of replication origins and in the way in which these origins are licensed to operate. Moreover, these differences impart advantages to multicellular animals and plants that facilitate their development, such as better control over endoreduplication, flexibility in origin selection, and discrimination between quiescent and proliferative states.

Expression of human ABCB5 confers resistance to taxanes and anthracyclines.

Human ABCB5, an ATP-binding cassette (ABC) transporter gene, has two major mRNA species. One transcript encodes an 812 amino acid polypeptide, ABCB5ß, with a transmembrane domain and a nucleotide-binding domain. We isolated the cDNA of another ABCB5 mRNA that encodes a 1257 amino acid polypeptide. The translated ABCB5 protein is a full-sized ABC transporter that has an internally duplicated structure with two transmembrane domains and two nucleotide-binding domains. The 5' and 3' parts of the ABCB5 mRNA were expressed in the prostate and testis. HEK293 cells transfected with the ABCB5 cDNA expressed a 150-160kDa protein. The ABCB5 transfectants showed approximately 1.5-fold higher resistance to doxorubicin, and 2- to 3-fold higher resistance to paclitaxel and docetaxel. Cellular uptake of radiolabeled paclitaxel and docetaxel in the transfectants was lower than that in the parental HEK293 cells. Treatment of the transfectants with ABCB5-targeted siRNA lowered their resistance to docetaxel. Revertant cells that express a reduced amount of ABCB5 also showed a lowered level of docetaxel resistance. These results indicated that the expression of ABCB5 conferred resistance to taxanes and anthracyclines. Membrane vesicles prepared from ABCB5 baculovirus-infected Sf21 cells showed higher vanadate-sensitive ATPase activity than the Sf21 control vesicles. The k(m) and V(max) values of ATPase activity in the ABCB5 vesicles were 1.8mM and 65nmol/min/mg protein, respectively. ABCB5 ATPase activity was 1.25-fold higher in the presence of 100µM docetaxel than it was in the absence of docetaxel. These results indicates that the full-length ABCB5 protein has ATPase activity that is sensitive to docetaxel.

DNA ligand designed to antagonize EBNA1 represses Epstein-Barr virus-induced immortalization.

Epstein-Barr virus (EBV) transforms human B lymphocytes into immortalized cells in vitro and is associated with various malignancies in vivo. EBNA1, which is expressed in the majority of EBV-infected cells, recognizes specific DNA sequences at the cis-acting latent origin of plasmid replication (oriP) element of the EBV genome. EBNA1 plays a critical role in the viral episome maintenance and transactivates viral transforming genes in latently infected cells. Therefore, DNA-targeting agents that can disrupt the EBNA1-oriP interaction will offer novel functional inhibitors of EBNA1. Pyrrole-imidazole polyamides, sequence-specific DNA ligands, can be designed to interfere with the binding of various transcriptional factors. Here, we synthesized pyrrole-imidazole polyamides targeting EBNA1-bound DNA sequences and developed an inhibitor for the EBNA1-oriP interaction. A pyrrole-imidazole polyamide, designated as DSE-3, bound adjacent to the EBNA1 recognition sequences located in the dyad symmetry element of oriP, and selectively inhibited EBNA1-oriP binding both in vitro and in vivo. DSE-3 also inhibited the proliferation of established lymphoblastoid cell lines by eradicating EBV episomes from the cells. In addition, DSE-3 repressed the expression of viral transforming genes after infecting human peripheral blood mononuclear cells with EBV and, as a consequence, inhibited EBV-mediated B-cell immortalization. These results suggest that EBNA1 functions will be an attractive pharmacological target for EBV-associated diseases.

Novel regulatory role for Kaposi's sarcoma-associated herpesvirus-encoded vFLIP in chemosensitization to bleomycin.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is associated with malignancy. KSHV-derived vFLIP is structurally related to cellular FLIP and binds to NEMO/IκB kinase (IKKγ) to activate NF-κB signaling. NF-κB activation is postulated to confer chemoresistance to various anticancer drugs. However, here we showed that vFLIP expression uniquely sensitized HEK293 cells to bleomycin and its derivatives. Chemosensitization to bleomycin by vFLIP accompanied accumulation of γ-H2AX and G2/M-arrest of cells, while bleomycin-induced DNA damage checkpoints, such as phosphorylation of Chk2 and foci formation of Rad51, were similarly detected in both parental and vFLIP-expressing cells, suggesting that primary DNA damage was not affected by vFLIP. Paradoxically, while NF-κB activity was little affected by bleomycin treatment, vFLIP-stimulated NF-κB activity was suppressed by it. Additionally, cAMP-response element (CRE)- and p53-dependent transcriptional reporter activity was negatively regulated by vFLIP in the presence of bleomycin. Interestingly, a negative regulatory phosphatase essential for G2 checkpoint recovery and for dephosphorylation of γ-H2AX, Wip1/PPM1D, whose gene promoter is regulated by p53, CRE and NF-κB, was selectively downregulated in vFLIP-expressing cells after bleomycin treatment. These results suggest that vFLIP-mediated transcriptional regulation such as Wip1/PPM1D repression is involved in chemosensitization to bleomycin.