Discovery of Allopregnanolone Against Inuenza Virus With Broad Spectrum in Vitro

Background: Inuenza virus infection with seasonal or occasional but devastating morbidity and mortality, is a severe threat to public health. The frequent emergence of resistant viral strains limited application of current antivirals and posing an urgent need for novel antiviral therapies. Natural products offered a broad prospect in the screening and development of new inuenza inhibitors. Methods: In this research, a high-throughput antiviral screening for 891 natural products was performed based on a recombinant reporter inuenza A virus. According to the cytotoxicity assay and dose-response relationship, alloprogesterone (ALLO), as the positive hit was selected, and veried by viral titer reduction assay and immunouorescence using a wild-type virus. Followingly, we explored its antiviral potency of counteracting with IAV and IBV, and preliminary investigated the mechanism of ALLO through time-of-addition assay and mini-replicon system. Results: Under the criteria of 80% inhibition and 70% cell viability, ALLO was screened out and conrmed antiviral activity in varied cells. The inhibitory effect of ALLO against inuenza virus with a dose-dependent manner and signicantly reduced viral yield of ve different inuenza viruses in the presence of 40 µM ALLO, including oseltamivir-resistant virus. Moreover, ALLO exhibited no inuence on IAV entry or release during the viral replication cycle, but obviously interfered with the genome replication regarding post-infection 2 hrs to 6 hrs, which is consistent with the evidence of decreased polymerase activity. Conclusions: In summary, we rstly identied a new pharmacological activity of ALLO, as a broad spectrum inhibitor for treatment inuenza infections, targeting viral replication stage and possessing great value of further development.


Background
In uenza virus is the main pathogen causing human acute respiratory tract diseases, which is prefer to induce seasonal infections and pandemic outbreaks with signi cant morbidity and mortality globally [1][2][3]. Clinical diagnosis analyses suggest that groups with low immunity including pregnant women, infants and the elderly, are susceptible to the in uenza virus, and patients with chronic basic diseases have a higher risk of suffering more serious problems and even death [4][5][6][7]. Currently, the prevention and treatment therapy of in uenza infection mainly focuses on vaccines and antivirals [8]. However, the application of vaccines is restricted by high costs, long cycle and the limited effectiveness caused by mismatching with circulating viral strains [9,10]. Besides, drug resistance and adverse reactions in the clinical usage of existing listed antivirals also urge us to nd potential novel in uenza viral inhibitors [11].
For instance, the ion channel blocker, amantadine, is no longer recommended for the treatment of in uenza virus since its side effects of vertigo, insomnia, vomiting and others and no more than effective for most in uenza viruses [12,13]. As the preferred drug for combating in uenza diseases recommended by WHO, oseltamivir (Tami u®, NA inhibitor) also presented a declined e cacy to combat more than 20 relevant resistant viral strains in 2009, such as H274Y, D151G, K136Q, N386K, P431S, D334N, A193T and so on [13][14][15][16][17]. Therefore, the urgent need for novel in uenza-speci c antiviral inhibitors is emphasized to control infection and reduce incidences of resistance.
The natural products with unique pharmacological activity and chemical structure remain popular in potential therapeutic value, of which the important source of new drug research and development [18]. For instance, 49% agents applied in clinically cancer therapy are derived from natural compounds [19].
And a series of traditional Chinese medicines or extracted active ingredients, not only in vitro antiviral experiments, but also in the clinical treatment of in uenza, all displayed vital value and broad development prospects [20][21][22]. In this study, we performed a high-throughput antiviral screening of 891 natural products using a recombinant replication-competent reporter in uenza virus, and Allopregnanolone (ALLO) was identi ed possessing the anti-in uenza e cacy with broad spectrum, acting inhibition on the viral replication stage.
The compound library containing 891 natural products dissolved in 100 µL DMSO at a stored concentration of 10 mM, was purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA).
Samples were arranged in 96-well plates and diluted with Opti-MEM containing 1.5 µg/mL TPCK-trypsin to 20 µM screened concentration.

Cell-Based High-throughput antiviral screening
The replication-competent reporter IAV carrying Gaussia luciferase gene (PR8-PB2-Gluc) has been reported as a sensitive and convenient tool to characterize the virus titer [23]. A cell-based phenotypic high-throughput screening (HTS) that allows for multicycle viral replication to identify potential inhibitors was carried out as previously described [24,25]. Brie y, the adherent growth of monolayer MDCK cells growing in white, at-bottom, 96-well culture plates (PerkinElmer, Waltham, MA) were infected with PR8-PB2-Gluc virus at 0.01 multiplicities of infection (MOI) in presence of test samples of 20 µM. After incubation 36 hours (hrs), infections were determined through the Guassia luciferase activity which was measured using Pierce Guassia luciferase glow assay kit (Thermo scienti c, Rockford, IL, USA) following the manufacturer's instructions. Mock infected cells were used as blank control. DMSO and baloxavir acid (BA, diluted to 20 nM concentration) were set as negative and positive control, respectively. The compounds with inhibition rate above 80% were selected for detection of cytotoxicity.

Cytotoxicity assay
The cytotoxicity analysis was performed according to the manufacturer's instructions of CCK-8 kit (MCE).
As described previously, MDCK cells at a density of 10000 cells/well grown in 96-well assay plates were treated with speci ed screening concentration and incubated for 36 hrs. Cell viability was assessed by measuring the absorbance at a wavelength of 450 nm of a microplate reader.

Dose-response assay
The dose-effect relationship was veri ed for the selected active natural product. Serial diluted samples were added into infected cells and mock cells, separately to obtain IC 50 (50% inhibiting concentration) and CC 50 (50% cytotoxic concentration) by tting dose-response curves with a four-parameter logistic regression to the data in GraphPad Prism software (version 5.02, La Jolla, CA, United States), and the selectivity index (SI) was further analyzed.

Viral Titer Reduction Assay
Viral yield reduction assay was performed as previously described 22 . Brie y, MDCK cells growing in 24well plates were infected with A/H1N1/PR8, oseltamivirresistant in uenza A/H1N1/pdm(09), A/H3N2/Brisbane, B-Yamagata-like, and B-Victoria-like strains at MOI of 0.01 with or without various concentrations of ALLO, respectively. The culture supernatants were harvested at 36 hrs post-infection (p.i.), and virus titers (TCID 50 /mL) were determined using cytopathic effect (CPE) experiment. DMSO (0.2%) and uninfected MDCK cells were used as the negative control and the blank control, BA at different concentrations was designed as an antiviral positive drug control group.

Time of Addition Assay
A classic experiment to further investigate which stage of viral life cycle ALLO disturbed was performed as previously reported [27]. MDCK cells seeded in 24-well plates at a density of 1×10 5 cells/well were infected with 0.1 MOI PR8-wt virus, and interfered with 20 µM compounds at indicated time (-2, 0, 2, 4, 6, 8) to investigate the period of the viral life cycle targeted by ALLO. At 12 hrs pi, the expression of newly produced virion was analyzed by viral titer test.

Mini-replicon System
The polymerase activity assay was determined by using viral ribonucleoprotein complex (vRNP) minigenome system in A549 cells as previously described [26,28,29]. In a brief, A549 cells grown in 6-well plates were transfected with viral polymerase plasmids (PB2/pCAGGS, PB1/pCAGGS, PA/pCAGGS, and NP/pCAGGS) and pPolI-Fluc (a re y luciferase reporter plasmid) together with hRluc-TK (Renilla luciferase plasmid) using Lipofectamine 2000 reagent. At 5 hrs co-transfection, the cells were washed with PBS, re-suspended with fresh DMEM (phenol red free) and seeded into white, 96-well plates at a density of 10,000 cells per well, with various concentrations of ALLO or 0.5% DMSO. After incubation at 37 ℃ for 24 hrs, luciferase activity was measured by dual-luciferase reporter system (Promega, Madison, WI USA) according to manufacturer's instruction.

Statistical Analysis
In order to quantify the robustness of the screen, Z' factor was calculated from the normalized signals from positive and negative control wells on each plate as follows: Z' = 1 3 × (SD of positive control + SD of negative control) / (mean of negative control mean of positive control). SD represents the standard deviation. Z' value between 0.5 and 1.0 is regarded as appropriate for an HTS assay [30].
The antiviral inhibition rate was calculated according measured luciferase expression with the following equation: inhibition rate = (signal of negative control -signal of tested compound) / (signal of negative control -signal of positive control) × 100%. In this study, 891 samples were included in the primary screen, 96 hits that met the cutoff of 80% inhibition were then proceeded in the toxicity screen to exclude cytotoxicity caused false positive ones.

In uenza Virus Inhibitors Identi cation Screen Assay
Under the screening threshold of 80% inhibition and 30% cytotoxicity dual authentication, it nally yielded one selected hit which continued to carry out the dose-response assay to con rm antiviral activity of candidate inhibitor. The stability and quality of primary HTS system was evaluated by the Z' values of each screening plate, which ranges from 0.54 to 0.91, indicating our preliminary screen was robust and reliable.
ALLO inhibits viral replication in a dose -dependent manner Throughout antiviral screening and validation assay, allopregnanolone (ALLO, structure indicated in Fig. 2a), as a progesterone metabolite, was selected out from natural product library. We tested a 6-point diluted concentrations of ALLO in the presence of reporter virus or in the absence of virus for inhibition and cytotoxicity dose-response assay in varied cells. The results in MDCK, A549 and 293T cells, all showed that the antiviral activity was regulated by ALLO amount with a positive correlation re ected in the tted dose-response curves (Fig. 2b). ALLO signi cantly inhibited IAV replication in a dose-dependent manner with IC 50 ranged from 3.01 to 10.64 µM among three cell lines, and all exhibited inconsiderable cytotoxicity besides relatively sensitive 293T. Calculated selective index (SI) of ALLO against in uenza virus in MDCK, A549 and 293T cells were 77.4, more than 47 and 21.7 respectively (Table 1). The signi cant antiviral effect of ALLO was further reassessed in wild-type IAV (A/H1N1/PR8) replication by monitoring the reduction of viral titer and immuno uorescence phenomenon of viral protein expression after compounds treatment. As a result, ALLO at 40 µM concentration greatly decreased viral generation over 5 log compared to the viral load of negative group, which had a comparable antiviral effect with 40 nM baloxavir acid (viral titer reductions of > 6 log). In the meanwhile, IF observed the expression of viral protein stained 8 hrs p.i. was inversely proportional to the drug concentration, that is, the higher ALLO dose, the less uorescence amount of NP protein particle and the weaker uorescence intensity (Fig. 4a, MDCK cells). When treated with a concentration of 40 µM, ALLO also almost completely suppressed viral replication in wt-PR8 virus (MOI = 1) infected A549 cells (Fig. 4b).
ALLO is effective to inhibit in uenza A and B virus To further explore the broad-spectrum antiviral potency of ALLO, IAV-pdm09 (H1N1), which was resistant to oseltamivir with NA/H274Y mutations, IAV-Brisbane (H3N2), IBV-Yamagate and IBV-Victoria strains were carried out for viral yield reduction assay. Each viral strain was inoculated with different concentrations of ALLO for 36 hrs, the supernatants were harvested and the virus titers were determined. All groups with triplicate measurements. As indicated in Fig. 5, ALLO at 40 µM concentration could inhibit the replication of in uenza A and B virus, especially H3N2 virus (viral yield decreased > 6 log) and oseltamivir-resistant virus (viral yield decreased > 5 log), showing more sensitivity to ALLO, which compared to IBV two strains (> 3 log decreased in Yamagate, about 2 log decreased in Victoria). Thus, we concluded that ALLO exhibits a broad spectrum of anti-in uenza virus activity.

ALLO exhibits inhibitory effect on the stage of the viral replication
We further investigated which stage of viral life cycle was disturbed by ALLO according classic time-ofaddition assay as previously reported [27]. Over the single viral replication cycle, viral yield did not appear signi cant reduction when cells were treated with 20 µM ALLO at -2-0h and 8-12h intervals. Instead, infected cells administrated with ALLO at 0h, 2h, 4h and 6h, during viral replication proceeding, the viral titer was inhibited. It is indicated that the antiviral effect of ALLO is associated with viral genome replication rather than virus entry and release periods. In the meanwhile, polymerase, the vital replicated machine, was disturbed by ALLO at active concentrations, with a decline of the uorescence value ranging from 50-90% using the mini-replicon system.
Taken together, although the more detailed antiviral mechanism is in progress, these results already suggested that the inhibitory effect of ALLO through acting on the stage of viral replication, and in uenced the expression of polymerase.

Discussion
In uenza virus is a member of the Orthomyxoviridae family, being segmented, single stranded, negative sense RNA viruses [31]. It mainly adheres to respiratory tract epithelium leading viral pneumonia with typically clinical manifestations of cellular in ltration, diffuse in ammation and ground-glass opacities, following highly morbidity not only in immunocompromised individuals and patients with underlying comorbid conditions, but also in young healthy adults [32]. Natural products occupy an important position in the development of new antiviral inhibitors to cope with the widespread occurrence of drug resistance events in existing listed agents.
In this research, we employed a recombinant reporter virus expressing Gaussia luciferase to carry out the antiviral HTS and eventually obtained one potent compound named ALLO (Allopregnanolone, the metabolite of progesterone). It could dose-dependently decrease the viral luciferase expression among MDCK, A549 and 293T cells, indicating that ALLO exerted an inhibitory effect on in uenza virus without cell speci city. Additionally, immuno uorescence of viral NP protein con rmed that ALLO inhibited viral replication in a dose-dependent manner. We further explored the inhibitory effect of ALLO on various subtypes of in uenza viruses in a titer reduction assay. The viral yields in the infected MDCK cells could be remarkably reduced by 40 µM ALLO, implying a broad spectrum of anti-IAV and IBV activity. Moreover, it was preliminarily concluded that the mode of action of ALLO by blocking viral replication stage according a single-cycle replication assay and a mini-replicon system.
It has been reported that ALLO is an allosteric regulator of GABA receptors playing a role in depression and anxiety [33], as well as a variety of neurodegenerative diseases such as diabetic neuropathy [34], Alzheimer's disease [35] and Parkinson's disease [36]. It has been investigated for the treatment of Severe Postpartum Depression as the main ingredient of Zulresso (the rst FDA-authorized PPD inhibitor) [37]. Eminently, our nding is the rst time revealing the novel pharmacological activity of ALLO against in uenza virus with broad-spectrum and identi ed ALLO playing an antiviral role in the replication period in vitro. Interestingly, another reported anti-in uenza ingredient acting on downregulating NF-κB expression, cirsimaritin, derived from a avone of A. scoparia, can bind weakly with GABAA receptors with antidepressant and anxiolytic activity [38,39]. The commonality of these two in uenza inhibitors' pharmacoactive suggests whether ALLO can also play an indirect antiviral role by stimulating the immune function. The speci c of treating PPD of ALLO makes us suppose that the antiviral activity of ALLO whether or not be associated with clinical changes in progesterone levels in patients with in uenza during pregnancy and postpartum. Also, the further mechanism research and in vivo antiviral study of ALLO are under way, with respect to the activity of its analogues and synergy effect in combination with current inhibitors are also deserved to be explored. In conclusion, this article provided a new perspective on potential activity of ALLO.

Conclusions
In this study, we provided a novel insight about anti-in uenza virus potency of ALLO. The promising prospect of ALLO with broad-spectrum antiviral activity in vitro, rendering us further in vivo study and detailed mechanism research are necessary. And it is of great signi cance to be an alternative antiviral agent against in uenza viruses in the future.  Figure 1 The