Study on the structure optimization and anti‐hepatitis B virus activity of novel human La protein inhibitor HBSC11

Abstract In our previous study, Methyl pyrazolo[1,5‐a] pyridine‐2‐carboxylate (HBSC11) was shown to combine with La protein, which conferred anti‐hepatitis B virus (HBV) effects. The purpose of this study was to optimize, synthesize, and evaluate the anti‐HBV activity of HBSC11. The methyl group of HBSC11 was substituted with hydrophobic, hydrophilic, and tricyclic groups to generate novel HBV inhibitors with desirable potency. On in vitro evaluation, several derivatives exhibited good anti‐HBV activity compared with control. In particular, compound 5a reduced the level of HBV antigen by approximately 50%, which was similar to the activity of entecavir. In a mouse model, 5a showed 98.9% inhibition rate for HBV DNA, 57.4% for HBsAg, and 46.4% for HBeAg; the corresponding rates in the control group were 90.8, 3.8, and 9.8%, respectively. In addition, prediction of binding modes and physicochemical properties showed that 5a formed hydrogen bonds with La protein and conformed well to the Lipinski's rule of five. Our results suggest that 5a is a potential new anti‐HBV drug.


Chronic hepatitis B virus (HBV) infection is a major global public
health problem with significant morbidity and mortality. 1-3 An estimated 2 billion people have been infected with HBV worldwide till date and approximately 780 000 patients die from HBV-related complications every year (http://www.who.int/en/news-room/factsheets/detail/hepatitis-b.). Currently, there are two main antiviral therapies available for patients with HBV: nucleoside analogs (NAs) and interferon-alpha (IFNα). The NAs are generally safe; however, long-term oral treatment with NAs may lead to the emergence of drug resistance. IFNα is expensive and causes adverse effects. 4 Therefore, development of novel anti-HBV agents with superior efficacy and safety is a key imperative. 5 La protein was first identified as a ribonucleic protein (RNP) targeted by autoantibodies in patients with autoimmune diseases; it is now known to be a multifunctional nucleoprotein involved in diverse aspects of RNA metabolism and translation. La protein binds to nascent RNA polymerase III transcripts, which protects them from exonucleolytic decay; in addition, it plays a role as RNA chaperone to help them correct folding. 6 Studies have shown that combination of La protein to HBV RNA alters its conformation and covers up the RNA cleavage site, which protects the HBV RNA from destruction 7-10 and helps maintain the replication ability of HBV. 11 Mutation and silencing of La protein have been used to confirm its role in HBV. 12 Subsequently, according to the structural characteristics of La protein crystal, Methyl pyrazolo[1,5-a] pyridine-2-carboxylate (HBSC11), a pyrazolo[1,5-a] pyridine compound, was selected as inhibitor of La protein by virtual screening and showed good anti-HBV efficacy. 13 In this study, we aimed towards optimization, synthesis, and evaluation of the anti-HBV activity of HBSC11 as a specific inhibitor of La protein. This study may provide a foundation for developing new non-nucleoside agents for anti-HBV therapy.

| Structural optimization of HBSC11
The derivatives of HBSC11 were designed on the basis of two reduced pressure to afford the desired product as yellow solid. The solid was dissolved in sulfuric acid (15 mL, 50%) and then warmed to 80°C for 3 hours. The reaction mixture was poured into the water at 0°C (50 mL), and then extracted with EA (150 mL × 3); the organic layer was washed with brine (150 mL), and then dried over anhydrous Na 2 SO 4 . After filtration, the filtrate was concentrated under vacuum, the crude product was purified by thin-layer chromatography (TLC; EA/PE = 2:1) to afford the desired Compound 1. Total yield was 46%.
Parallel to this, compound 1 (567 mg, 3.5 mmol) was suspended in dry toluene (20 mL), and three drops DMF and oxalyl chloride (1.0 mL, 11.8 mmol) were added at RT, then warmed to 50°C and stirred for to afford the desired product as shown in supplement (S1).

| Molecular modeling and molecular dynamics simulation
The three-dimensional model of La protein was constructed using validated homology techniques and docking study using the Glide software (Glide, version 5.5, Schrödinger, LLC, New York, NY, 2009). 13,16 Binding affinity between the protein and compounds was estimated using well-established molecular dynamics (MD) experiments. 17,18 S C H E M E 3 derivatives synthetic route

| Prediction of physicochemical properties
Leading compound HBSC11 and its candidate compounds 3a, 5a, and 5b were predicted for their compliance with the Lipinski's rule of five using free online software (http://www.molinspiration.com/). 19

| Ethical statement
The study was reviewed and approved by the Ethics Committee of the Obstetrics & Gynecology Hospital of Fudan University. All efforts were made to minimize animal suffering.

| IHC staining for LA protein
The liver tissues of mice were collected from mice killed at 12 days.
La protein and cell nucleus were visualized by IHC staining of tissues embedded in OCT by rabbit anti-La (Abcam, UK) and Hoechst (Hoechst AG, Germany), respectively.

| Statistics
The data are presented as mean ± standard deviation (n = 3). Oneway ANOVA test was performed using the SAS system (SAS Institute Inc) or GraphPad Prism version 5 (GraphPad Software Inc, San Diego) software. P value < .05 vs control was considered statistically significant.
Ten derivatives of HBSC11 (Table 1)  this was followed by quantification of the HBV DNA level by qPCR.
As shown in Figure 1, some derivatives such as 3c, 3d, 3f, and 5a showed better inhibitory activity (38, 82, 62, and 34%, respectively) compared with lamivudine at the indicated time-points. HBSC11 inhibited HBV DNA up to 54% and 71% at 24 hours and 48 hours To further confirm the anti-HBV activity of compounds 5a, 5b, The extracellular HBV DNA was collected and quantified by qPCR ( Figure 4A). Compounds 5b, 5a, and HBSC11 inhibited HBV DNA by up to 50%, although ETV showed better inhibitory activity. However, 3a and ETV were found preferable to 5a, 5b, and HBSC11 with respect to the effect on intracellular cccDNA ( Figure 4B). On the contrary, the intracellular viral RNA level was increased upon ETV treatment ( Figure   4C). Similarly, HBV DNA synthesis was significantly inhibited, whereas viral RNA was increased. 21 The antigen inhibitory efficiency of compounds was assessed by ELISA. As shown in Figures 4D,E, compound 5a and HBSC11 showed similar effect compared with ETV in reducing the antigen levels (approximately 50%). The results indicated that the effects of our compounds on HBV were not mediated via direct inhibition of the replication of HBV DNA; however, these did show some effect on HBsAg and HBeAg. However, because the compounds did not show any effect on inhibition of La protein, we presumed that HBSC11 and its derivatives do not act directly on La protein synthesis, but on cellular localization or function ( Figure 4F).

| In vivo antiviral activity and cytotoxicity of candidate derivatives
On the basis of the results of in vitro experiments, HBSC11, 5a, and 5b were selected for assessment of antiviral activity and toxicity in vivo. The Interestingly, HBSC11, 5a, and 5b reduced HBeAg up to 53.0%, 46.4%, and 40.2%, respectively, on Day 12 (only 9.8% in control group), which was consistent with the in vitro results.
In addition, we sought to assess whether the expression of La protein in liver tissue or cell localization could be affected by the compounds. Immunofluorescence assay showed a decreasing trend of La protein in the hepatocytes after administration, especially in the nucleus; besides, there may be a tendency of protein exchange between hepatocytes and immune cells ( Figure 6). Further verification of specific results and the underlying mechanisms is required in future research.

| Molecular modeling and MD simulation study
To gain insights into the mechanism by which 3a-3f and 5a-5d compounds affect the biological function of La protein, we carried out docking simulation for binding modes of compounds to human La protein by Schrodinger's GLIDE components (

| Prediction of physicochemical properties
As shown in Table 3, some physicochemical properties of HBSC11, 5a, 5b, and 3a were calculated using free online software. The results showed good compliance with the Lipinski's rule of five, which suggests their potential use as a drug.

| DISCUSSION
La protein is a multifunctional nucleoprotein that is involved in diverse aspects of RNA metabolism and translation. Heise T and colleagues have focused on the relationship between La protein and HBV for many years; they found that La protein binds to the 5′ end of HBV RNA, which protects the HBV RNA against the nuclear RNase activity. 9 Subsequently, other researchers used specific siRNA or La mutation to prove that La protein is involved in the life cycle of HBV RNA. 12,22,23 These results indicate that La protein is a potential target for novel anti-HBV strategies. In our previous study, we used virtual high-throughput screening and biological evaluation and discovered a new La inhibitor with anti-HBV activity. 13 In this study, we aimed at optimization, synthesis, and evaluation of HBSC11 as a specific inhibitor of La protein.
Firstly, we identified and retained active sites between HBSC11 and La, and obtained 10 derivatives by introducing hydrophobic, hydrophilic and tricyclic groups containing aromatic rings on the side chain. Secondly, we screened three promising candidates with good anti-HBV activity in HepG2.2.15 cells (compounds 3a, 5a, and 5b).

CONFLICTS OF INTEREST
The authors declare that there is no conflict of interest.