"PROTAC" modified dihydroquinolizinones (DHQs) that cause degradation of PAPD-5 and inhibition of hepatitis A virus and hepatitis B virus, in vitro.

Dihydroquinolizinones (DHQs) that inhibit cellular polyadenylating polymerases 5 and 7 (PAPD5 & 7), such as RG7834, have been shown to inhibit both hepatitis A (HAV) and hepatitis B virus (HBV) in vitro and in vivo. In this report, we describe RG7834-based proteolysis-targeting chimeras (PROTACs), such as compound 12b, (6S)-9-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-21-oxo-3,6,9,12,15,18-hexaoxa-22-azapentacosan-25-yl)oxy)-6-isopropyl-10-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2,1-a]isoquinoline-3-carboxylic acid. The PROTAC DHQs described here inhibited an HAV reporter virus in vitro with an IC50 of 277 nM. Although the PROTAC DHQs were also inhibitory to HBV, their activities were substantially less potent against HBV in vitro, being in the 10 to 20 µM range, based on the reduction of HBsAg and HBV mRNA levels. Importantly, unlike RG7834, the incubation of cells in vitro with PROTAC DHQ 12b resulted in the degradation of PAPD5, as expected for a PROTAC compound, but curiously not PAPD7. PAPD5 polypeptide degradation was prevented when a proteasome inhibitor, epoxomicin, was used, indicating that proteasome mediated proteolysis was associated with the observed activities of 12b. Taken together, these data show that 12b is the first example of a PROTAC that suppresses both HAV and HBV that is based on a small molecule warhead. The possibility that it has mechanisms that differ from its parent compound, RG7834, and has clinical value, is discussed.

Understanding the role of Cu+/Cu0 sites at Cu2O based catalysts in ethanol production from CO2 electroreduction -A DFT study.

Cu2O based electrocatalysts generally exhibit better selectivity for C2 products (ethylene or ethanol) in electrochemical carbon dioxide reduction. The surface characteristic of the mixed Cu+ and Cu0 chemical state is believed to play an essential role that is still unclear. In the present study, density functional theory (DFT) calculations have been performed to understand the role of copper chemical states in selective ethanol formation using a partially reduced Cu2O surface model consisting of adjacent Cu+/Cu0 sites. We mapped out the free energy diagram of the reaction pathway from CO intermediate to ethanol and discussed the relation between the formation of critical reduction intermediates and the configuration of Cu+/Cu0 sites. The results showed that Cu+ sites facilitate the adsorption and stabilization of *CO, as well as its further hydrogenation to *CHO. More importantly, as compared to the high reaction energy (1.23 eV) of the dimerization of two *CO on Cu+/Cu0 sites, the preferable formation of *CHO on the Cu+ site makes the C-C coupling reaction with *CO on the Cu0 site happen under a relatively lower energy barrier of 0.58 eV. Furthermore, the post C-C coupling steps leading to the formation of the key intermediate *OCHCH2 to C2 compound are all thermodynamically favoured. Noteworthily, it is found that *OCHCH2 inclines to the ethanol formation because the coordinatively unsaturated Cu+ site could maintain the C-O bond of *OCHCH2, and the weak binding between *O and Cu+/Cu0 sites helps inhibit the pathway toward ethylene. These findings may provide guidelines for the design of CO and CO2 reduction active sites with enhanced ethanol selectivity.

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. Reductions of HBsAg were a result of the compound's effect on HBsAg mRNA levels. However, commercial development by Roche of a DHQ lead compound, RG-7834, was stopped due to undisclosed toxicity issues. Herein we report our effort to convert the systemic RG7834 compound to a hepatoselective DHQ analog to limit its distribution to the bloodstream and thus to other body tissues.

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. Further biochemical analyses showed that the putative amphipathic alpha helix was not required for the disulfide-linked S protein oligomerization, but was essential for SVP morphogenesis. Pharmacological disruption of vesicle trafficking between the ER and Golgi complex in SVP producing cells supported the hypothesis that S protein-directed SVP morphogenesis takes place at the ER-Golgi intermediate compartment (ERGIC). Moreover, it was demonstrated that S protein is degraded in hepatocytes via a 20S proteasome-dependent, but ubiquitination-independent non-classic ER-associated degradation (ERAD) pathway. Taken together, the results reported herein favor a model in which the amphipathic alpha helix at the antigenic loop of S protein attaches to the lumen leaflet to facilitate SVP budding from the ERGIC compartment, whereas the failure of budding process may result in S protein degradation by 20S proteasome in an ubiquitination-independent manner.Importance Subviral particles are the predominant viral product produced by HBV-infected hepatocytes. Their levels exceed the virion particles by 10,000 to 100,000-fold in the blood of HBV infected individuals. The high levels of SVPs, or HBV surface antigen (HBsAg), in the circulation induces immune tolerance and contributes to the establishment of persistent HBV infection. The loss of HBsAg, often accompanied by appearance of anti-HBs antibodies, is the hallmark of durable immune control of HBV infection. Therapeutic induction of HBsAg loss is, therefore, considered to be essential for the restoration of host antiviral immune response and functional cure of chronic hepatitis B. Our findings on the mechanism of SVP morphogenesis and S protein metabolism will facilitate the rational discovery and development of antiviral drugs to achieve this therapeutic goal.

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. RG7834 is a small-molecule compound that binds PAPD5/7 and inhibits HBV gene production in both tissue culture and animal study. We reported that RG7834 was able to destabilize multiple HBV mRNA species, ranging from the 3.5-kb pregenomic/precore mRNAs to the 2.4/2.1-kb hepatitis B virus surface protein (HBs) mRNAs, except for the smallest 0.7-kb X protein (HBx) mRNA. Compound-induced HBV mRNA destabilization was initiated by a shortening of the poly(A) tail, followed by an accelerated degradation process in both the nucleus and cytoplasm. In cells expressing HBV mRNA, both PAPD5/7 were found to be physically associated with the viral RNA, and the polyadenylating activities of PAPD5/7 were susceptible to RG7834 repression in a biochemical assay. Moreover, in PAPD5/7 double-knockout cells, viral transcripts with a regular length of the poly(A) sequence could be initially synthesized but became shortened in hours, suggesting that participation of PAPD5/7 in RNA 3' end processing, either during adenosine oligomerization or afterward, is crucial for RNA stabilization.

HBsAg mRNA degradation induced by a dihydroquinolizinone compound depends on the HBV posttranscriptional regulatory element.

In pursuit of novel therapeutics targeting the hepatitis B virus (HBV) infection, we evaluated a dihydroquinolizinone compound (DHQ-1) that in the nanomolar range reduced the production of virion and surface protein (HBsAg) in tissue culture. This compound also showed broad HBV genotype coverage, but was inactive against a panel of DNA and RNA viruses of other species. Oral administration of DHQ-1 in the AAV-HBV mouse model resulted in a significant reduction of serum HBsAg as soon as 4 days following the commencement of treatment. Reduction of HBV markers in both in vitro and in vivo experiments was related to the reduced amount of viral RNA including pre-genomic RNA (pgRNA) and 2.4/2.1 kb HBsAg mRNA. Nuclear run-on and subcellular fractionation experiments indicated that DHQ-1 mediated HBV RNA reduction was the result of accelerated viral RNA degradation in the nucleus, rather than the consequence of inhibition of transcription initiation. Through mutagenesis of HBsAg gene sequences, we found induction of HBsAg mRNA decay by DHQ-1 required the presence of the HBV posttranscriptional regulatory element (HPRE), with a 109 nucleotides sequence within the central region of the HPRE alpha sub-element being the most critical. Taken together, the current study shows that a small molecule can reduce the overall levels of HBV RNA, especially the HBsAg mRNA, and viral surface proteins. This may shed light on the development of a new class of HBV therapeutics.

A Long-Term Prediction Model of Beijing Haze Episodes Using Time Series Analysis.

The rapid industrial development has led to the intermittent outbreak of pm2.5 or haze in developing countries, which has brought about great environmental issues, especially in big cities such as Beijing and New Delhi. We investigated the factors and mechanisms of haze change and present a long-term prediction model of Beijing haze episodes using time series analysis. We construct a dynamic structural measurement model of daily haze increment and reduce the model to a vector autoregressive model. Typical case studies on 886 continuous days indicate that our model performs very well on next day's Air Quality Index (AQI) prediction, and in severely polluted cases (AQI ≥ 300) the accuracy rate of AQI prediction even reaches up to 87.8%. The experiment of one-week prediction shows that our model has excellent sensitivity when a sudden haze burst or dissipation happens, which results in good long-term stability on the accuracy of the next 3-7 days' AQI prediction.