A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.

A toolkit for a modern gynecologic oncology tissue bank.

OBJECTIVES: Tissue banking procedures have evolved to keep pace with precision medicine, technology, emerging understanding of racial disparities, and regulatory requirements. However, there is little published guidance regarding strategies to create and maintain a successful biorepository. Our objective is to describe the infrastructure and protocols used by our Gynecologic Oncology Tissue Bank. METHODS: Our Tissue Bank was founded in 1992. In August 2022, internal funding was used to modernize the Tissue Bank. We hired three full-time employees, implemented universal screening of patients treated by gynecologic oncology faculty, updated consenting protocols, and standardized communication with providers. Tumor tissue, blood derivatives, ascites, and pleural fluid were collected from eligible, consenting patients and processed. Patient-derived cell lines and organoids were generated. For quality control purposes, one formalin-fixed, paraffin-embedded (FFPE) sample per tissue site was analyzed by a board-certified pathologist. All samples were labeled and tracked in an OpenSpecimen collection protocol and clinically annotated in a secure database. RESULTS: From August 2022 to October 2023, 227 patients (83% white, 15% Black, 1% Asian) were enrolled and 4249 specimens were collected. Adherent cell lines were generated from 15 patients with ovarian cancer and cell suspensions for organoid generation were collected from 46 patients with ovarian cancer. A recharge center was established to self-sustain the Tissue Bank. Samples have been shared with academic and commercial collaborators. CONCLUSIONS: Our Tissue Bank has enrolled a large number of diverse patients, collected numerous specimen types, and collaborated widely. The procedures described here provide guidance for other institutions establishing similar resources.

SQ1274, a novel microtubule inhibitor, inhibits ovarian and uterine cancer cell growth.

OBJECTIVE: Paclitaxel, a microtubule inhibitor, is subject to tumor resistance while treating high-grade serous ovarian and uterine cancer. This study aims to directly compare the effects of SQ1274, a novel microtubule inhibitor that binds to the colchicine-binding site on tubulin, and paclitaxel in high-grade serous ovarian and uterine cancer cell lines both in vitro and in vivo. METHODS: We assessed the sensitivity of ovarian (OVCAR8) and uterine (ARK1) cancer cell lines to SQ1274 and paclitaxel using XTT assays. We used western blot and quantitative real-time PCR to analyze changes in AXL RNA and protein expression by SQ1274 and paclitaxel. Differences in cell-cycle arrest and apoptosis were investigated using flow cytometry. Finally, we treated ovarian and uterine xenograft models with vehicle, paclitaxel, or SQ1274. RESULTS: First, we demonstrate that SQ1274 has a much lower IC50 than paclitaxel in both ARK1 (1.26 nM vs. 15.34 nM, respectively) and OVCAR8 (1.34 nM vs. 10.29 nM, respectively) cancer cell lines. Second, we show SQ1274 decreases both RNA and protein expression of AXL. Third, we show that SQ1274 causes increased cell-cycle arrest and apoptosis compared to paclitaxel. Finally, we report that SQ1274 more effectively inhibits tumor growth in vivo compared to paclitaxel. CONCLUSIONS: SQ1274 presents as a viable alternative to paclitaxel for treating ovarian and uterine cancer. This study supports the development of SQ1274 as a chemotherapeutic to treat ovarian and uterine cancer.

Synergistic Interactions between Hepatitis B Virus RNase H Antagonists and Other Inhibitors.

Combination therapies are standard for management of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections; however, no such therapies are established for human hepatitis B virus (HBV). Recently, we identified several promising inhibitors of HBV RNase H (here simply RNase H) activity that have significant activity against viral replication in vitro Here, we investigated the in vitro antiviral efficacy of combinations of two RNase H inhibitors with the current anti-HBV drug nucleoside analog lamivudine, with HAP12, an experimental core protein allosteric modulator, and with each other. Anti-HBV activities of the compounds were tested in a HepG2-derived cell line by monitoring intracellular core particle DNA levels, and cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The antiviral efficiencies of the drug combinations were evaluated using the median-effect equation derived from the mass-action law principle and combination index theorem of Chou and Talalay. We found that combinations of two RNase H inhibitors from different chemical classes were synergistic with lamivudine against HBV DNA synthesis. Significant synergism was also observed for the combination of the two RNase H inhibitors. Combinations of RNase H inhibitors with HAP12 had additive antiviral effects. Enhanced cytotoxicity was not observed in the combination experiments. Because of these synergistic and additive effects, the antiviral activity of combinations of RNase H inhibitors with drugs that act by two different mechanisms and with each other can be achieved by administering the compounds in combination at doses below the respective single drug doses.

Hydroxylated tropolones inhibit hepatitis B virus replication by blocking viral ribonuclease H activity.

Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 µM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 µM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50]=25 to 79 µM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.

Stromal DDR2 Promotes Ovarian Cancer Metastasis through Regulation of Metabolism and Secretion of Extracellular Matrix Proteins.

Ovarian cancer is the leading cause of gynecologic cancer-related deaths. The propensity for metastasis within the peritoneal cavity is a driving factor for the poor outcomes associated with this disease, but there is currently no effective therapy targeting metastasis. In this study, we investigate the contribution of stromal cells to ovarian cancer metastasis and identify normal stromal cell expression of the collagen receptor, discoidin domain receptor 2 (DDR2), that acts to facilitate ovarian cancer metastasis. In vivo, global genetic inactivation of Ddr2 impairs the ability of Ddr2-expressing syngeneic ovarian cancer cells to spread throughout the peritoneal cavity. Specifically, DDR2 expression in mesothelial cells lining the peritoneal cavity facilitates tumor cell attachment and clearance. Subsequently, omentum fibroblast expression of DDR2 promotes tumor cell invasion. Mechanistically, we find DDR2-expressing fibroblasts are more energetically active, such that DDR2 regulates glycolysis through AKT/SNAI1 leading to suppressed fructose-1,6-bisphosphatase and increased hexokinase activity, a key glycolytic enzyme. Upon inhibition of DDR2, we find decreased protein synthesis and secretion. Consequently, when DDR2 is inhibited, there is reduction in secreted extracellular matrix proteins important for metastasis. Specifically, we find that fibroblast DDR2 inhibition leads to decreased secretion of the collagen crosslinker, LOXL2. Adding back LOXL2 to DDR2 deficient fibroblasts rescues the ability of tumor cells to invade. Overall, our results suggest that stromal cell expression of DDR2 is an important mediator of ovarian cancer metastasis. IMPLICATIONS: DDR2 is highly expressed by stromal cells in ovarian cancer that can mediate metastasis and is a potential therapeutic target in ovarian cancer.

RAD51 Foci as a Biomarker Predictive of Platinum Chemotherapy Response in Ovarian Cancer.

PURPOSE: To determine the ability of RAD51 foci to predict platinum chemotherapy response in high-grade serous ovarian cancer (HGSOC) patient-derived samples. EXPERIMENTAL DESIGN: RAD51 and γH2AX nuclear foci were evaluated by immunofluorescence in HGSOC patient-derived cell lines (n = 5), organoids (n = 11), and formalin-fixed, paraffin-embedded tumor samples (discovery n = 31, validation n = 148). Samples were defined as RAD51-High if >10% of geminin-positive cells had ≥5 RAD51 foci. Associations between RAD51 scores, platinum chemotherapy response, and survival were evaluated. RESULTS: RAD51 scores correlated with in vitro response to platinum chemotherapy in established and primary ovarian cancer cell lines (Pearson r = 0.96, P = 0.01). Organoids from platinum-nonresponsive tumors had significantly higher RAD51 scores than those from platinum-responsive tumors (P < 0.001). In a discovery cohort, RAD51-Low tumors were more likely to have a pathologic complete response (RR, 5.28; P < 0.001) and to be platinum-sensitive (RR, ∞; P = 0.05). The RAD51 score was predictive of chemotherapy response score [AUC, 0.90; 95% confidence interval (CI), 0.78-1.0; P < 0.001). A novel automatic quantification system accurately reflected the manual assay (92%). In a validation cohort, RAD51-Low tumors were more likely to be platinum-sensitive (RR, ∞; P < 0.001) than RAD51-High tumors. Moreover, RAD51-Low status predicted platinum sensitivity with 100% positive predictive value and was associated with better progression-free (HR, 0.53; 95% CI, 0.33-0.85; P < 0.001) and overall survival (HR, 0.43; 95% CI, 0.25-0.75; P = 0.003) than RAD51-High status. CONCLUSIONS: RAD51 foci are a robust marker of platinum chemotherapy response and survival in ovarian cancer. The utility of RAD51 foci as a predictive biomarker for HGSOC should be tested in clinical trials.

Pattern of chromosomal aberrations persisting over 30 years in a Chernobyl Nuclear Power Plant accident survivor: study using mFISH.

The long-term in vivo cytogenetic effects of high-dose radiation exposure can be traced in accidentally irradiated persons, and particularly useful for developing strategies of monitoring and therapy of such patients, as well as for elucidating the fundamental aspects of hematopoiesis and radiobiology. Using 24-color fluorescent in situ hybridization (mFISH), we analysed the frequency and the spectrum of chromosomal aberrations (CA) in peripheral blood lymphocytes of the Chernobyl Nuclear Power Plant (NPP) accident victim 30, 31, 32 and 33 years after acute accidental exposure to high-dose gamma radiation of the whole body. Totally, 993 metaphase cells were analyzed (or 219, 272, 258, 244 cells each year), of which 297 were aberrant. Our study demonstrated a constant aberrant cell frequency at 28% in 2016-2018 years, while in 2019, a significant increase up to 35% occurred due to contribution of significantly elevated frequency of simple aberrations in the absence of evident recent genotoxic factors. Four clonal aberrations were detected, three of which persisted for more than one year at a frequency up to 2.5% of analyzed cells. The distribution of 731 breakpoints per individual chromosomes was nearly proportional to their physical length, excepting Chromosomes 13 and 20, which were significantly breakpoint-deficient compared to the genome median rate. Monitoring of the long-term effects on chromosomal instability caused by radiation exposure is important for understanding and predicting the long-term effects of ionizing radiation.

GAS6-AXL Inhibition by AVB-500 Overcomes Resistance to Paclitaxel in Endometrial Cancer by Decreasing Tumor Cell Glycolysis.

Chemotherapy is often ineffective in advanced-stage and aggressive histologic subtypes of endometrial cancer. Overexpression of the receptor tyrosine kinase AXL has been found to be associated with therapeutic resistance, metastasis, and poor prognosis. However, the mechanism of how inhibition of AXL improves response to chemotherapy is still largely unknown. Thus, we aimed to determine whether treatment with AVB-500, a selective inhibitor of GAS6-AXL, improves endometrial cancer cell sensitivity to chemotherapy particularly through metabolic changes. We found that both GAS6 and AXL expression were higher by immunohistochemistry in patient tumors with a poor response to chemotherapy compared with tumors with a good response to chemotherapy. We showed that chemotherapy-resistant endometrial cancer cells (ARK1, uterine serous carcinoma and PUC198, grade 3 endometrioid adenocarcinoma) had improved sensitivity and synergy with paclitaxel and carboplatin when treated in combination with AVB-500. We also found that in vivo intraperitoneal models with ARK1 and PUC198 cells had decreased tumor burden when treated with AVB-500 + paclitaxel compared with paclitaxel alone. Treatment with AVB-500 + paclitaxel decreased AKT signaling, which resulted in a decrease in basal glycolysis. Finally, multiple glycolytic metabolites were lower in the tumors treated with AVB-500 + paclitaxel than in tumors treated with paclitaxel alone. Our study provides strong preclinical rationale for combining AVB-500 with paclitaxel in aggressive endometrial cancer models.

GAS6/AXL Inhibition Enhances Ovarian Cancer Sensitivity to Chemotherapy and PARP Inhibition through Increased DNA Damage and Enhanced Replication Stress.

Over 80% of women with high-grade serous ovarian cancer (HGSOC) develop tumor resistance to chemotherapy and die of their disease. There are currently no FDA-approved agents to improve sensitivity to first-line platinum- and taxane-based chemotherapy or to PARP inhibitors. Here, we tested the hypothesis that expression of growth arrest-specific 6 (GAS6), the ligand of receptor tyrosine kinase AXL, is associated with chemotherapy response and that sequestration of GAS6 with AVB-S6-500 (AVB-500) could improve tumor response to chemotherapy and PARP inhibitors. We found that GAS6 levels in patient tumor and serum samples collected before chemotherapy correlated with ovarian cancer chemoresponse and patient survival. Compared with chemotherapy alone, AVB-500 plus carboplatin and/or paclitaxel led to decreased ovarian cancer-cell survival in vitro and tumor burden in vivo. Cells treated with AVB-500 plus carboplatin had more DNA damage, slower DNA replication fork progression, and fewer RAD51 foci than cells treated with carboplatin alone, indicating AVB-500 impaired homologous recombination (HR). Finally, treatment with the PARP inhibitor olaparib plus AVB-500 led to decreased ovarian cancer-cell survival in vitro and less tumor burden in vivo. Importantly, this effect was seen in HR-proficient and HR-deficient ovarian cancer cells. Collectively, our findings suggest that GAS6 levels could be used to predict response to carboplatin and AVB-500 could be used to treat platinum-resistant, HR-proficient HGSOC. IMPLICATIONS: GAS6/AXL is a novel target to sensitize ovarian cancers to carboplatin and olaparib. Additionally, GAS6 levels can be associated with response to carboplatin treatment.

Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

Inhibition of AXL and VEGF-A Has Improved Therapeutic Efficacy in Uterine Serous Cancer.

Endometrial cancer remains the most prevalent gynecologic cancer with continued rising incidence. A less common form of this cancer is uterine serous cancer, which represents 10% of endometrial cancer cases. However, this is the most aggressive cancer. The objective was to assess whether inhibiting the receptor tyrosine kinase AXL with AVB-500 in combination with bevacizumab would improve response in uterine serous cancer. To prove this, we conducted multiple angiogenesis assays including tube formation assays and angiogenesis invasion assays. In addition, we utilized mouse models with multiple cells lines and subsequently analyzed harvested tissue through immunohistochemistry CD31 staining to assess microvessel density. The combination treatment arms demonstrated decreased angiogenic potential in each assay. In addition, intraperitoneal mouse models demonstrated a significant decrease in tumor burden in two cell lines. The combination of AVB-500 and bevacizumab reduced tumor burden in vivo and reduced morphogenesis and migration in vitro which are vital to the process of angiogenesis.

Amide-containing α-hydroxytropolones as inhibitors of hepatitis B virus replication.

The Hepatitis B Virus (HBV) ribonuclease H (RNaseH) is a promising but unexploited drug target. Here, we synthesized and analyzed a library of 57 amide-containing α-hydroxytropolones (αHTs) as potential leads for HBV drug development. Fifty percent effective concentrations ranged from 0.31 to 54 µM, with selectivity indexes in cell culture of up to 80. Activity against the HBV RNaseH was confirmed in semi-quantitative enzymatic assays with recombinant HBV RNaseH. The compounds were overall poorly active against human ribonuclease H1, with 50% inhibitory concentrations of 5.1 to >1,000 µM. The αHTs had modest activity against growth of the fungal pathogen Cryptococcus neoformans, but had very limited activity against growth of the Gram - bacterium Escherichia coli and the Gram + bacterium Staphylococcus aureus, indicating substantial selectivity for HBV. A molecular model of the HBV RNaseH templated against the Ty3 RNaseH was generated. Docking the compounds to the RNaseH revealed the anticipated binding pose with the divalent cation coordinating motif on the compounds chelating the two Mn++ ions modeled into the active site. These studies reveal that that amide αHTs can be strong, specific HBV inhibitors that merit further assessment toward becoming anti-HBV drugs.

Efficacy of hepatitis B virus ribonuclease H inhibitors, a new class of replication antagonists, in FRG human liver chimeric mice.

Chronic hepatitis B virus infection cannot be cured by current therapies, so new treatments are urgently needed. We recently identified novel inhibitors of the hepatitis B virus ribonuclease H that suppress viral replication in cell culture. Here, we employed immunodeficient FRG KO mice whose livers had been engrafted with primary human hepatocytes to ask whether ribonuclease H inhibitors can suppress hepatitis B virus replication in vivo. Humanized FRG KO mice infected with hepatitis B virus were treated for two weeks with the ribonuclease H inhibitors #110, an α-hydroxytropolone, and #208, an N-hydroxypyridinedione. Hepatitis B virus viral titers and S and e antigen plasma levels were measured. Treatment with #110 and #208 caused significant reductions in plasma viremia without affecting hepatitis B virus S or e antigen levels, and viral titers rebounded following treatment cessation. This is the expected pattern for inhibitors of viral DNA synthesis. Compound #208 suppressed viral titers of both hepatitis B virus genotype A and C isolates. These data indicate that Hepatitis B virus replication can be suppressed during infection in an animal by inhibiting the viral ribonuclease H, validating the ribonuclease H as a novel target for antiviral drug development.

Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture.

BACKGROUND AIMS: Spontaneous mutagenesis often leads to appearance of genetic changes in cells. Although human multipotent mesenchymal stromal cells (hMSC) are considered as genetically stable, there is a risk of genomic and structural chromosome instability and, therefore, side effects of cell therapy associated with long-term effects. In this study, the karyotype, genetic variability and clone formation analyses have been carried out in the long-term culture MSC from human gingival mucosa. METHODS: The immunophenotype of MSC has been examined using flow cytofluorometry and short tandem repeat (STR) analysis has been carried out for authentication. The karyotype has been examined using GTG staining and mFISH, while the assessment of the aneuploidy 8 frequency has been performed using centromere specific chromosome FISH probes in interphase cells. RESULTS: The immunophenotype and STR loci combination did not change during the process of cultivation. From passage 23 the proliferative activity of cultured MSCs was significantly reduced. From passage 12 of cultivation, clones of cells with stable chromosome aberrations have been identified and the biggest of these (12%) are tetrasomy of chromosome 8. The random genetic and structural chromosomal aberrations and the spontaneous level of chromosomal aberrations in the hMSC long-term cultures were also described. CONCLUSIONS: The spectrum of spontaneous chromosomal aberrations in MSC long-term cultivation has been described. Clonal chromosomal aberrations have been identified. A clone of cells with tetrasomy 8 has been detected in passage 12 and has reached the maximum size by passage 18 before and decreased along with the reduction of proliferative activity of cell line by passage 26. At later passages, the MSC line exhibited a set of cells with structural variants of the karyotype with a preponderance of normal diploid cells. The results of our study strongly suggest a need for rigorous genetic analyses of the clone formation in cultured MSCs before use in medicine.

In Vitro Enzymatic and Cell Culture-Based Assays for Measuring Activity of HBV RNaseH Inhibitors.

HBV is a small, enveloped DNA virus that replicates by reverse transcription via an RNA intermediate. Current anti-HBV treatment regiments that include interferon α and nucleos(t)ide analogs have insufficient efficiency, are of long duration and can be accompanied by systemic side effects. Though HBV RNaseH is essential for viral replication, it is unexploited as a drug target against HBV. RNaseH inhibitors that actively block viral replication would represent an important addition to the potential new drugs for treating HBV infection. Here we describe two methods to measure the activity of RNaseH inhibitors. DNA oligonucleotide-directed RNA cleavage assay allows low-throughput screening of compounds for potential anti-HBV RNaseH activity in vitro. Analysis of preferential inhibition of plus-polarity DNA strand synthesis by HBV RNaseH inhibitors in a cell culture model of HBV replication can be used to validate the efficiency of these compounds to block viral replication.

Efficacy and cytotoxicity in cell culture of novel α-hydroxytropolone inhibitors of hepatitis B virus ribonuclease H.

Chronic Hepatitis B virus (HBV) infection is a major worldwide public health problem. Current direct-acting anti-HBV drugs target the HBV DNA polymerase activity, but the equally essential viral ribonuclease H (RNaseH) activity is unexploited as a drug target. Previously, we reported that α-hydroxytropolone compounds can inhibit the HBV RNaseH and block viral replication. Subsequently, we found that our biochemical RNaseH assay underreports efficacy of the α-hydroxytropolones against HBV replication. Therefore, we conducted a structure-activity analysis of 59 troponoids against HBV replication in cell culture. These studies revealed that antiviral efficacy is diminished by larger substitutions on the tropolone ring, identified key components in the substitutions needed for high efficacy, and revealed that cytotoxicity correlates with increased lipophilicity of the α-hydroxytropolones. These data provide key guidance for further optimization of the α-hydroxytropolone scaffold as novel HBV RNaseH inhibitors.

Inhibition of hepatitis B virus replication by N-hydroxyisoquinolinediones and related polyoxygenated heterocycles.

We previously reported low sensitivity of the hepatitis B virus (HBV) ribonuclease H (RNaseH) enzyme to inhibition by N-hydroxyisoquinolinedione (HID) compounds. Subsequently, our biochemical RNaseH assay was found to have a high false negative rate for predicting HBV replication inhibition, leading to underestimation of the number of HIDs that inhibit HBV replication. Here, 39 HID compounds and structurally related polyoxygenated heterocycles (POH), N-hydroxypyridinediones (HPD), and flutimides were screened for inhibition of HBV replication in vitro. Inhibiting the HBV RNaseH preferentially blocks synthesis of the positive-polarity DNA strand and causes accumulation of RNA:DNA heteroduplexes. Eleven HIDs and one HPD preferentially inhibited HBV positive-polarity DNA strand accumulation. EC50s ranged from 0.69 µM to 19 µM with therapeutic indices from 2.4 to 71. Neither the HIDs nor the HPD had an effect on the ability of the polymerase to elongate DNA strands in capsids. HBV RNaseH inhibition by the HIDs was confirmed with an improved RNaseH assay and by detecting accumulation RNA:DNA heteroduplexes in HBV capsids from cells treated with a representative HID. Therefore, the HID scaffold is more promising for anti-HBV drug discovery than we originally reported, and the HPD scaffold may hold potential for antiviral development. The preliminary structure-activity relationship will guide optimization of the HID/HPDs as HBV inhibitors.

Mitochondrial localization of p53 during adenovirus infection and regulation of its activity by E1B-19K.

Recent results have revealed that the p53 tumor suppressor protein possesses a direct transcription-independent apoptotic activity. During apoptosis induced by genotoxic stress, a small fraction of p53 is targeted to mitochondria where it initiates apoptosis by causing mitochondrial dysfunction. In adenovirus-infected cells, the expression of E1A protein enhances the accumulation of p53 during early phases of infection and during late times after infection, it is targeted for degradation by the combined action of E1B-55K and E4-orf6 proteins. The functional significance of E1A-mediated accumulation of p53 during early phases of viral replication is not known. Our studies with isogenic epithelial cell lines that differ only on the status of p53 indicate that Ad infection induces apoptosis by p53-dependent and -independent pathways and both pathways are suppressed by E1B-19K. We show that during early phase of Ad infection, a fraction of p53 is targeted to the mitochondria. In virus infected cells, a large fraction of the viral antiapoptosis protein E1B-19K is also localized in mitochondria during early and late phases of infection. Coimmunoprecipitation analysis has revealed that p53 and E1B-19K form a complex in mitochondria. The interaction of 19K involves two noncontiguous regions located around amino-acid residues 14-15 and 123-124. On p53, the mutations within the DNA-binding domain reduce interaction with E1B-19K. Our studies also suggest that 19K may additionally complex with the multidomain mitochondrial proapoptotic protein BAK, thereby reducing the level of p53 interaction with BAK. We suggest that p53-induced apoptosis may be important for efficient cell lysis and viral spread and that E1B-19K may neutralize the apoptotic activity of p53 at multiple levels.