Discovery of ZFD-10 of a pyridazino[4,5-b]indol-4(5H)-one derivative as an anti-ZIKV agent and a ZIKV NS5 RdRp inhibitor.

Zika virus (ZIKV) infection is associated with the birth defect microcephaly and Guillain-Barré syndrome in adults. There is no approved vaccine or specific antiviral agent against ZIKV. ZFD-10, a novel structural skeleton of 1H-pyridazino[4,5-b]indol-4(5H)-one, was firstly synthesized and discovered to be a potent anti-ZIKV inhibitor with very low cytotoxicity. ZFD-10's anti-ZIKV potency is independent of cell lines and ZFD-10 mainly targets the post-entry stages of ZIKV life cycle. Time-of-addition and time-of-withdrawal assays showed that 10 µM ZFD-10 displayed the ability to decrease mainly at the RNA level and weakly the viral progeny particle load. Furthermore, ZFD-10 could protect ZIKV NS5 from thermal unfolding and aggregation and increase the Tagg value of ZIKV NS5 protein from 44.6 to 49.3 °C, while ZFD-10 dose-dependently inhibits ZIKV NS5 RdRp activity using in vitro RNA polymerase assays. Molecular docking study suggests that ZFD-10 affects RdRp enzymatic function through interfering with the fingers and thumb subdomains. These results supported that ZFD-10's cell-based anti-ZIKV activity is related to its anti-RdRp activity of ZIKV NS5. The in vivo anti-ZIKV study shows that the middle-dose (4.77 mg/kg/d) of ZFD-10 protected mice from ZIKV infection and the viral loads of the blood, liver, kidney and brain in the middle-dose and high-dose (9.54 mg/kg/d) were significantly reduced compared to those of the ZIKV control. These results confirm that ZFD-10 has a certain antiviral effect against ZIKV infection in vivo.

Antiviral effects of the fused tricyclic derivatives of indoline and imidazolidinone on ZIKV infection and RdRp activities of ZIKV and DENV.

The prevalence and ravages of Zika virus (ZIKV) seriously endanger human health, especially causing significant neurological defects in both neonates as pediatric microcephaly and adults as Guillain-Barré syndrome. In this work, we studied anti-ZIKV effects of the fused tricyclic derivatives of indoline and imidazolidinone and discovered that some of them are valuable leads for drug discovery of anti-ZIKV agents. The current results show that certain compounds are broad-spectrum inhibitors of ZIKV- and dengue virus (DENV)-infection while distinctive compounds are selective ZIKV inhibitors or selective DENV inhibitors. Compounds of 12, 17 and 28 are more active against Asian ZIKV SZ-VIV01 strain than African ZIKV MR766 strain. It is valued that silylation makes six TBS compounds of 4-nitrophenyl hydrazine series and phenyl hydrazine series more active against ZIKV infection than their phenols. Time-of-addition and withdrawal studies indicate that compound 12 majorly acts on post-infection of RNA synthesis stage of ZIKV life cycle. Moreover, compounds of 12, 17 and 18 are anti-ZIKV agents with the inhibitory activities to ZIKV NS5 RdRp while 12 doesn't inhibit DENV infection even though it is a DENV RdRp inhibitor, 17 is an active agent against DENV infection but is only a weak DENV NS5 RdRp inhibitor, and 28 is inactive against DENV infection and not a DENV NS5 RdRp inhibitor. As a result, a compound's antiviral difference between ZIKV and DENV is not always related to anti-RdRp difference between ZIKV RdRp and DENV RdRp, and structural features of a compound play important roles in executing antiviral and anti-RdRp functions. Further discovery of highly potent broad-spectrum or selective agents against infection by ZIKV and DENV will be facilitated.

Inhibiting cardiac autophagy suppresses Zika virus replication.

Zika Virus (ZIKV) infection is a global threat. Other than the congenital neurological disorders it causes, ZIKV infection has been reported to induce cardiac complications. However, the precise treatment plans are unclear. Thus, illustrating the pathogenic mechanism of ZIKV in the heart is critical to providing effective prevention and treatment of ZIKV infection. The mechanism of autophagy has been reported recently in Dengue virus infection. Whether or not autophagy participates in ZIKV infection and its role remains unrevealed. This study successfully established the in vitro cardiomyocytes and in vivo mouse models of ZIKV infection to investigate the involvement of autophagy in ZIKV infection. The results showed that ZIKV infection is both time and gradient-dependent. The key autophagy protein, LC3B, increased remarkably after ZIKV infection. Meanwhile, autophagic flux was detected by immunofluorescence. Applying autophagy inhibitors decreased the LC3B levels. Furthermore, the number of viral copies was quantified to evaluate the influence of autophagy during infection. We found that autophagy was actively involved in the ZIKV infection and the inhibition of autophagy could effectively reduce the viral copies, suggesting a potential intervention strategy for reducing ZIKV infection and the undesired complications caused by ZIKV.

Identification of 6ω-cyclohexyl-2-(phenylamino carbonylmethylthio)pyrimidin-4(3H)-ones targeting the ZIKV NS5 RNA dependent RNA polymerase.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a global health concern because of its association with severe neurological disorders such as neonatal microcephaly and adult Guillain-Barre syndrome. Although many efforts have been made to combat ZIKV infection, there is currently no approved vaccines or antiviral drugs available and there is an urgent need to develop effective anti-ZIKV agents. In this study, 26 acetylarylamine-S-DACOs derivatives were prepared, and eight of them were found to have inhibitory activity against Zika virus. Among these substances, 2-[(4-cyclohexyl-5-ethyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(3,5-difluorophenyl)acetamide (4w) with the best anti-ZIKV activity was selected for in-depth study of antiviral activity and mechanism of action. Here, we discovered 4w targeted on the ZIKV NS5 RNA -dependent RNA polymerase (RdRp), which exhibited good in vitro antiviral activity without cell species specificity, both at the protein level and at the RNA level can significantly inhibit ZIKV replication. Preliminary molecular docking studies showed that 4w preferentially binds to the palm region of NS5A RdRp through hydrogen bonding with residues such as LYS468, PHE466, GLU465, and GLY467. ZIKV NS5 RdRp enzyme activity experiment showed that 4w could directly inhibit ZIKV RdRp activity with EC50 = 11.38 ± 0.51 µM. In antiviral activity studies, 4w was found to inhibit ZIKV RNA replication with EC50 = 6.87 ± 1.21 µM. ZIKV-induced plaque formation was inhibited with EC50 = 7.65 ± 0.31 µM. In conclusion, our study disclosed that acetylarylamine-S-DACOs is a new active scaffolds against ZIKV, among which compound 4w was proved to be a potent novel anti-ZIKV compound target ZIKV RdRp protein. These promising results provide a future prospective for the development of ZIKV RdRp inhibitors.

Discovery of dehydroandrographolide derivatives with C19 hindered ether as potent anti-ZIKV agents with inhibitory activities to MTase of ZIKV NS5.

Infection by Zika virus (ZIKV), a mosquito-transmitted arbovirus and a member of Flavivirus, could make pediatric microcephaly and Guillain-Barré syndrome, which remains an ongoing global threat. The efficient antivirals to ZIKV infection are of great medical need. Andrographolide and its analogues were discovered to be active against flaviviral infection. In this study, we discovered some dehydroandrographolide derivatives of 3-oximido- or 3-alcohol-19-hindered ether to be potent anti-ZIKV agents with low cytotoxicities (CC50 > 200 µM). Time of addition assay suggests that compound 5a and its analogues act on inhibition of post-entry stage of ZIKV life cycle. It is discovered by experimental and molecular docking studies that active anti-ZIKV compounds of 3a, 5a, 5b and 5c possess inhibitory activities of ZIKV NS5 MTase (methyl transferase) enzymatic activity. Preliminary SAR reveals that C19-modification with bulky groups is necessary for anti-ZIKV infection and replication, anti-ZIKV activity of 5a comes from itself bearing hindered trityl ether but not from its instability, the backbone of dehydroandrographolide is more effective against ZIKV infection than that of andrographolide, and 3-oxime derivatives are more active against ZIKV infection than 3-alcohol derivatives. To our knowledge, 5a is the first reported MTase inhibitor of andrographolide derivatives. More importantly, discovery of active compound 5b with acid-stable 19-OCHPh2 against ZIKV infection is valued and gives us a clue to design and discover generally acid-stable anti-ZIKV agents.

Design, synthesis, discovery and SAR of the fused tricyclic derivatives of indoline and imidazolidinone against DENV replication and infection.

Dengue virus, belonging to a genus Flavivirus, caused public health problem in recent years. One controversial vaccine of DENV was approved and there is no antiviral for the clinic treatment of DENV, therefore, efficient antivirals to DENV are of great medical significance. In this study, we conducted the design, synthesis, cell-based and target-based activity evaluation of 28 compounds based on indoline structural skeleton against DENV infection. Among them, 13 active compounds against DENV infection were discovered and their structure-activity relationship (SAR) was summarized. In this study, indoline carbohydrazine has derived more active compounds than indoline carboamide. It is discovered that TBS group exhibits a good pharmacophore to enhance anti-DENV activity. Further exploration indicated that post-treatment acts as effective time of addition and compound 15 targeting the post-entry stages of DENV2 viral life cycle. SPR imaging results support there are strong interaction of 13 and 15 with RdRp and compounds 13 and 15 reduce RdRp enzymatic activity, revealing that RdRp of DENV NS5 is the drug target for these series of compounds. Molecular docking deciphered the relationship of the structural feature with the putative binding mode by 13 and 15 with RdRp domain of DENV2 NS5 by hydrogen bonds and hydrophobic interactions to establish the fitted low energy conformation. Future studies will focus on designing more potent inhibitors for the treatment and prevention of dengue virus replication and infection, and understanding the more profound underlying structural features of inhibitors and drug action of the mechanism.

[A practical method for the calculation of wound water loss in burn patients].

OBJECTIVE: To evaluate a calculating method of wound water loss (WWL) in hospitalized burn patients. METHODS: Eighty-five adult burn patients with total burn area over 50% were enrolled in the study. The WWL formula after shock stage (after 3rd postburn day) was given as: WWL (ml/%/kg/24 h) = [input (ml/24 h)-urine output (ml/24 h)-500 ml] / actual burn wound area(%) / body weight (kg). The result was compared with that determined by other formulas such as Davies, S.M.S.P and Lamke and palm rule. RESULTS: The calculated daily WWL in burn patients after shock stage was found out to be 0.9 +/- 0.1 ml/%/kg/24 h by this method. This figure is comparable with that calculated by several different methods. Palm rule could be simply applied in the patients with body weight around 60 kg. CONCLUSION: This method and palm rule were proved to be simple and practical for the calculation of WWL in burn patients, which could be applied in clinical work.