4-Oxooctahydroquinoline-1(2H)-carboxamides as hepatitis B virus (HBV) capsid core protein assembly modulators.

Hepatitis B virus (HBV) core protein, the building block of the HBV capsid, plays multiple roles in viral replication, and is an attractive target for development of antiviral agents with a new mechanism of action. In addition to the heteroaryldihydropyrimidines (HAPs), sulfamoylbenzamides (SBAs), dibenzothiazepine derivatives (DBTs), and sulfamoylpyrrolamides (SPAs) that inhibit HBV replication by modulation of viral capsid assembly and are currently under clinical trials for the treatment of chronic hepatitis B (CHB), other chemical structures with activity to modulate HBV capsid assembly have also been explored. Here we describe our continued optimization of a benzamide originating from our high throughput screening.

Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis.

The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic "HAP" pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically "normal" capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis.

Mechanisms Governing Metabolic Heterogeneity in Breast Cancer and Other Tumors.

Reprogramming of metabolic priorities promotes tumor progression. Our understanding of the Warburg effect, based on studies of cultured cancer cells, has evolved to a more complex understanding of tumor metabolism within an ecosystem that provides and catabolizes diverse nutrients provided by the local tumor microenvironment. Recent studies have illustrated that heterogeneous metabolic changes occur at the level of tumor type, tumor subtype, within the tumor itself, and within the tumor microenvironment.

Prospects for the Global Elimination of Hepatitis B.

Chronic hepatitis B virus (HBV) infection is the leading cause of liver cirrhosis and hepatocellular carcinoma, estimated to be globally responsible for ∼800,000 deaths annually. Although effective vaccines are available to prevent new HBV infection, treatment of existing chronic hepatitis B (CHB) is limited, as the current standard-of-care antiviral drugs can only suppress viral replication without achieving cure. In 2016, the World Health Organization called for the elimination of viral hepatitis as a global public health threat by 2030.

Hepatoselective Dihydroquinolizinone Bis-acids for HBsAg mRNA Degradation.

Chronic hepatitis B (CHB) is characterized by high levels of hepatitis B virus (HBV) surface antigen (HBsAg) in blood circulation. A major goal of CHB interventions is reducing or eliminating this antigenemia; however, there are currently no approved methods that can do this. A novel family of compounds with a dihydroquinolizinone (DHQ) scaffold has been shown to reduce circulating levels of HBsAg in animals, representing a first for a small molecule.

Restoration of a functional antiviral immune response to chronic HBV infection by reducing viral antigen load: if not sufficient, is it necessary?

The prolonged viral antigen stimulation is the driving force for the development of immune tolerance to chronic hepatitis B virus (HBV) infection. The sustained reduction of viral proteins may allow for the recovery and efficient activation of HBV-specific T and B cells by immune-stimulating agents, checkpoint blockades and/or therapeutic vaccinations. Recently, several therapeutic approaches have been shown to significantly reduce intrahepatic viral proteins and/or circulating HBV surface antigen (HBsAg) with variable impacts on the host antiviral immune responses in animal models or human clinical trials.

Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis.

Genome-wide association studies (GWAS) for kidney function identified hundreds of risk regions; however, the causal variants, target genes, cell types, and disease mechanisms remain poorly understood. Here, we performed transcriptome-wide association studies (TWAS), summary Mendelian randomization, and MetaXcan to identify genes whose expression mediates the genotype effect on the phenotype. Our analyses identified Dachshund homolog 1 (DACH1), a cell-fate determination factor.

Identification of hepatitis B virus core protein residues critical for capsid assembly, pgRNA encapsidation and resistance to capsid assembly modulators.

Assembly of hepatitis B virus (HBV) capsids is driven by the hydrophobic interaction of core protein (Cp) at dimer-dimer interface. Binding of core protein allosteric modulators (CpAMs) to a hydrophobic "HAP" pocket formed between the inter-dimer interface strengths the dimer-dimer interaction and misdirects the assembly of Cp dimers into non-capsid Cp polymers or morphologically normal capsids devoid of viral pregenomic (pg) RNA and DNA polymerase. In this study, we performed a systematic mutagenesis analysis to identify Cp amino acid residues at Cp dimer-dimer interface that are critical for capsid assembly, pgRNA encapsidation and resistance to CpAMs.

An Update on Glioblastoma Biology, Genetics, and Current Therapies: Novel Inhibitors of the G Protein-Coupled Receptor CCR5.

The mechanisms governing therapeutic resistance of the most aggressive and lethal primary brain tumor in adults, glioblastoma, have increasingly focused on tumor stem cells. These cells, protected by the periarteriolar hypoxic GSC niche, contribute to the poor efficacy of standard of care treatment of glioblastoma. Integrated proteogenomic and metabolomic analyses of glioblastoma tissues and single cells have revealed insights into the complex heterogeneity of glioblastoma and stromal cells, comprising its tumor microenvironment (TME).

A New Method for Improving Extraction Efficiency and Purity of Urine and Plasma Cell-Free DNA.

This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance.

Hepatitis B virus nucleocapsid uncoating: biological consequences and regulation by cellular nucleases.

Upon infection of hepatocyte, Hepatitis B virus (HBV) genomic DNA in nucleocapsid is transported into the nucleus and converted into a covalently closed circular (ccc) DNA to serve as the template for transcription of viral RNAs. Viral DNA in the cytoplasmic progeny nucleocapsid is another resource to fuel cccDNA amplification. Apparently, nucleocapsid disassembly, or viral genomic DNA uncoating, is an essential step for cccDNA synthesis from both infection and intracellular amplification pathways, and has a potential to activate DNA sensors and induce an innate immune response in infected hepatocytes.

piRNA-823 Is Involved in Cancer Stem Cell Regulation Through Altering DNA Methylation in Association With Luminal Breast Cancer.

Cancer stem cells (CSCs) are believed to be the main source of cancer relapse and metastasis. PIWI-interacting small non-coding RNAs (piRNAs) have been recently recognized to be relevant to cancer biology. Whether and how piRNAs regulate human CSCs remain unknown.

Highly multiplexed 2-dimensional imaging mass cytometry analysis of HBV-infected liver.

Studies of human hepatitis B virus (HBV) immune pathogenesis are hampered by limited access to liver tissues and technologies for detailed analyses. Here, utilizing imaging mass cytometry (IMC) to simultaneously detect 30 immune, viral, and structural markers in liver biopsies from patients with hepatitis B e antigen+ (HBeAg+) chronic hepatitis B, we provide potentially novel comprehensive visualization, quantitation, and phenotypic characterizations of hepatic adaptive and innate immune subsets that correlated with hepatocellular injury, histological fibrosis, and age. We further show marked correlations between adaptive and innate immune cell frequencies and phenotype, highlighting complex immune interactions within the hepatic microenvironment with relevance to HBV pathogenesis.

A putative amphipathic alpha helix in hepatitis B virus small envelope protein plays a critical role in the morphogenesis of subviral particles.

Hepatitis B virus (HBV) small (S) envelope protein has the intrinsic ability to direct the formation of small spherical subviral particles (SVPs) in eukaryotic cells. However, the molecular mechanism underlying the morphogenesis of SVPs from the monomeric S protein initially synthesized at the endoplasmic reticulum (ER) membrane remains largely elusive. Structure prediction and extensive mutagenesis analysis suggested that the amino acid residues spanning W156 to R169 of S protein form an amphipathic alpha helix and play essential roles in SVP production and S protein metabolic stability.

Leronlimab, a humanized monoclonal antibody to CCR5, blocks breast cancer cellular metastasis and enhances cell death induced by DNA damaging chemotherapy.

Background: Triple-negative breast cancer (BCa) (TNBC) is a deadly form of human BCa with limited treatment options and poor prognosis. In our prior analysis of over 2200 breast cancer samples, the G protein-coupled receptor CCR5 was expressed in > 95% of TNBC samples. A humanized monoclonal antibody to CCR5 (leronlimab), used in the treatment of HIV-infected patients, has shown minimal side effects in large patient populations.

Synthesis of 4-oxotetrahydropyrimidine-1(2H)-carboxamides derivatives as capsid assembly modulators of hepatitis B virus.

We report herein the synthesis and evaluation of phenyl ureas derived from 4-oxotetrahydropyrimidine as novel capsid assembly modulators of hepatitis B virus (HBV). Among the derivatives, compound () and several analogs showed an activity of submicromolar EC against HBV and low cytotoxicities (>50 μM). Structure-activity relationship studies revealed a tolerance for an additional group at position 5 of 4-oxotetrahydropyrimidine.

Phosphodiesterase Type-5 Inhibitor Tadalafil Modulates Steroid Hormones Signaling in a Prostate Cancer Cell Line.

Background: The androgen receptor (AR) plays a key role in normal prostate homeostasis and in prostate cancer (PCa) development, while the role of aromatase (Cyp19a1) is still unclear. We evaluated the effects of a treatment with Tadalafil (TAD) on both these proteins.

Methods: Androgen-sensitive human PCa cell line (LnCAP) was incubated with/without TAD (10 M) and bicalutamide (BCT) (10 M) to evaluate a potential modulation on cell proliferation, protein and mRNA expression of Cyp19a, AR and estrogen receptor-β (ERβ), respectively.

Host RNA quality control as a hepatitis B antiviral target.

Inhibition of the host RNA polyadenylating polymerases, PAPD5 and PAPD7 (PAPD5/7), with dihydroquinolizinone, a small orally available, molecule, results in a rapid and selective degradation of hepatitis B virus (HBV) RNA, and hence reduction in the amounts of viral gene products. DHQ, is a first in class investigational agent and could represent an entirely new category of HBV antivirals. PAPD5 and PAPD7 are non-canonical, cell specified, polyadenylating polymerases, also called terminal nucleotidyl transferases 4B and 4A (TENT4B/A), respectively.

Scalable Synthesis, In Vitro cccDNA Reduction, and In Vivo Antihepatitis B Virus Activity of a Phosphonomethoxydeoxythreosyl Adenine Prodrug.

Standard literature procedures for the chemical synthesis of l-threose nucleosides generally employ l-ascorbic acid as starting material. Herein, we have explored two alternative routes that start from either l-arabitol or l-diethyl tartrate, both affording 2--methyl-l-threofuranose as a key building block for nucleobase incorporation. The access to multigram quantities of this glycosyl donor in a reproducible fashion allows for the preparation of 2'-deoxy-α-l-threofuranosyl phosphonate nucleosides on a large scale.

Methods for extracellular vesicle isolation from cancer cells.

Cells are known to release different types of vesicles such as small extracellular vesicles (sEVs) and large extracellular vesicles (LEVs). sEVs and LEVs play important roles in intercellular communication, pre-metastatic niche formation, and disease progression; both can be detected cell culture media and biological fluids. sEVs and LEVs contain a variety of protein and RNA cargo, and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins.

The Dihydroquinolizinone Compound RG7834 Inhibits the Polyadenylase Function of PAPD5 and PAPD7 and Accelerates the Degradation of Matured Hepatitis B Virus Surface Protein mRNA.

Hepatitis B virus (HBV) mRNA metabolism is dependent upon host proteins PAPD5 and PAPD7 (PAPD5/7). PAPD5/7 are cellular, noncanonical, poly(A) polymerases (PAPs) whose main function is to oligoadenylate the 3' end of noncoding RNA (ncRNA) for exosome degradation. HBV seems to exploit these two ncRNA quality-control factors for viral mRNA stabilization, rather than degradation.

Identification of a 3-β-homoalanine conjugate of brusatol with reduced toxicity in mice.

Brusatol, a quassinoid natural product, is effective against multiple diseases including hematologic malignancies, as we reported recently by targeting the PI3Kγ isoform, but toxicity limits its further development. Herein, we report the synthesis of a series of conjugates of brusatol with amino acids and short peptides at its enolic hydroxyl at C-3. A number of conjugates with smaller amino acids and peptides demonstrated activities comparable to brusatol.

The membrane-associated form of cyclin D1 enhances cellular invasion.

The essential G-cyclin, CCND1, is a collaborative nuclear oncogene that is frequently overexpressed in cancer. D-type cyclins bind and activate CDK4 and CDK6 thereby contributing to G-S cell-cycle progression. In addition to the nucleus, herein cyclin D1 was also located in the cytoplasmic membrane.