Synthesis and in vitro DMPK profiling of a 1,2-dioxolane-based library with activity against Plasmodium falciparum

A 43-member 1,2-dioxolane library was synthesized by coupling a 1,2-dioxolane-3-acetic acid derivative to a range of amines. Ten compounds had EC₅₀s ? 30 nM against Plasmodium falciparum 3D7 and Dd2 strains, and another 15 compounds had EC₅₀s ? 50 nM against both 3D7 and Dd2. The library was then subjected to a range of in vitro DMPK assays, which revealed that side chains with a heteroatom were required for favorable solubility, Log D and membrane permeability. CYP450 inhibition was isoform dependent, with 2C19 and 3A4 particularly susceptible, and the majority of compounds tested against rat and human microsomes were metabolized rapidly.     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses

RNA viruses are a major source of respiratory diseases worldwide. The lack of effective therapeutical treatment underlines the importance of research for new antiviral compounds. Raoulic acid is a principal ingredient of the plant Raoulia australis Hook. F. Antiviral assay using cytopathic effect (CPE) reduction method showed that raoulic acid possessed strong antiviral activity against human rhinovirus 2 (HRV2) with a 50% inhibition concentration (IC₅₀) value of less than 0.1 μg/ml, human rhinovirus 3 (HRV3) with a IC₅₀ value of 0.19 μg/ml, coxsackie B3 (CB3) virus with IC₅₀ values of 0.33 μg/ml, coxsackie B4 (CB4) virus with IC₅₀ values of 0.40 μg/ml, and enterovirus 71 (EV71) virus with IC₅₀ values of less than 0.1 μg/ml. However, the compound did not possess antiviral activity against influenza A (Flu A/PR, Flu A/WS, H1N1) and B viruses at four concentrations ranging from 0.1 to 100 μg/ml.     

C ring may be dispensable for β-carboline: Design,synthesis, and bioactivities evaluation of tryptophan analog derivatives based on the biosynthesis of β-carboline alkaloids

According to our previous work and the latest research on the biosynthesis of β-carboline, and using the reverse thinking strategy, tryptophan, the biosynthesis precursor of β-carboline alkaloids, and their derivatives were synthesized, and their biological activities and structure–activity relationships were studied. This bioassay showed that these compounds exhibited good inhibitory activities against tobacco mosaic virus (TMV); especially (S)-2-amino-3-(1H-indol-3-yl)-N-octylpropanamide (4) (63.3 ± 2.1%, 67.1 ± 1.9%, 68.7 ± 1.3%, and 64.5 ± 3.1%, 500 μg/mL) exhibited the best antiviral activity both in vitro and in vivo. Compound 4 was chosen for the field trials and the acute oral toxicity test, the results showed that the compound exhibited good anti-TMV activity in the field and low acute oral toxicity. We also found that these compounds showed antifungal activities and insecticidal activities.     

Protection of Procambarus clarkii against white spot syndrome virus using inactivated WSSV

White spot syndrome virus (WSSV) is a highly pathogenic and prevalent virus infecting shrimp and other crustaceans. The potentiality of binary ethylenimine (BEI)-inactivated WSSV against WSSV in crayfish, Procambarus clarkii, was investigated in this study. Efficacy of BEI-inactivated WSSV was tested by vaccination trials followed by challenge of crayfish with WSSV. The crayfish injected with BEI-inactivated WSSV showed a better survival (P < 0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared to the control. Calculated relative percent survival (RPS) values were 77% and 60% on the 7th and 21st dpv for 2 mM BEI-inactivated WSSV, and 63%, 30% on 7th and 21st dpv for 3 mM BEI-inactivated WSSV. However, heat-inactivated WSSV did not provide protection from WSSV even on 7th dpv. In the inactivation process WSSV especially their envelope proteins maybe changed as happened to 3 mM BEI and heat-inactivated WSSV particles. These results indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect P. clarkii from WSSV.     

Synthesis and in vitro activities of a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via an octadecylglycerol residue

To prepare a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via a lipophilic octadecylglycerol residue we condensed 1-O-4-monomethoxytrityl-3-O-octadecyl-sn-glycerol-2-hydrogenphosphonate obtained from 3-O-octadecyl-sn-glycerol with AZT by the phosphonate method. The purified condensation product was de-tritylated resulting in 3′-azido-3′-deoxythymidylyl-(5′ → 2-O)-3-O-octadecyl-sn-glycerol, followed by treatment with (ethoxycarbonyl)phosphoric dichloride. The resulting 3′-azido-3′-deoxy-thymidylyl-(5′ → 2)-3-O-octadecyl-sn-glycerol-1-O-(ethoxycarbonyl)phosphonate was purified by preparative RP-18 column chromatography. The antiviral duplex drug 3′-azido-3′-deoxythymidylyl-(5′ → 2-O)-3-O-octadecyl-sn-glycerol-1-O-phosphonoformate trisodium salt (AZT–lipid–PFA) was obtained after alkaline cleavage of the phosphonoformate ethylester residue. The overall yield of the five step synthesis performed at gram scale was about 30%. According to a supposed pathway AZT–lipid–PFA could be cleaved to yield a mixture of different antiviral compounds such as AZT, AZT-5′-monophosphate, octadecylglycerol–AZT, PFA and octadecylglycerol–PFA, possibly producing additive and/or synergistic antiviral effects. In vitro studies showed that the duplex drug exhibits antiviral activities against HIV and especially against drug-resistant strains and clinical isolates of HSV and HCMV. The E₅₀ values of AZT–lipid–PFA against HIV ranged between 170 and 200 nM. The half-maximal inhibitory doses (IC₅₀) against highly acyclovir (ACV)-resistant HSV isolates determined by a plaque reduction assay ranged between 1.87 and 4.59 μM. Using ganciclovir (GCV)-sensitive, GCV resistant and drug cross-resistant HCMV strains the IC₅₀-values of AZT–lipid–PFA were between 2.78 and 1.18 μM. With regard to PFA, the IC₅₀-value of AZT–lipid–PFA determined on a multi-drug-resistant HCMV strain was about 90-fold lower than that of PFA, demonstrating the superior antiviral effect of the duplex-drug.     

Synthesis and biological evaluation of fluoro analogues of antimitotic phenstatin

With the aim of investigating the influence of fluorine, in particular on the A-ring, a new series of fluoro analogues (7a–l) of phenstatin (3) was synthesized and tested for interactions with tubulin polymerization and evaluated for cytotoxicity on an NCI-60 human cancer cell lines panel. We have shown that the replacement of 3,4,5-trimethoxyphenyl A-ring of phenstatin with 2,4,5-trifluoro-3-methoxyphenyl unit, results in the conservation of both antitubulin and cytotoxic effect. Fluoro isocombretastatin 7k was the most effective anticancer agent in the present study and demonstrated the highest antiproliferative potential on leukemia cell lines SR (GI₅₀ = 15 nM) and HL-60(TB) (GI₅₀ = 23 nM) and on melanoma cell line MDA-MB-435 (GI₅₀ = 19 nM).     

Design,synthesis, antiviral and cytostatic activity of ω-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues

The efficient synthesis of a new series of polyhydroxylated dibenzyl ω-(1H-1,2,3-triazol-1-yl)alkylphosphonates as acyclic nucleotide analogues is described starting from dibenzyl ω-azido(polyhydroxy)alkylphosphonates and selected alkynes under microwave irradiation. Selected O,O-dibenzylphosphonate acyclonucleotides were transformed into the respective phosphonic acids. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses and for cytostatic activity against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells. Compound (1S,2S)-16b exhibited antiviral activity against Influenza A H3N2 subtype (EC₅₀ = 20 μM—visual CPE score; EC₅₀ = 18 μM—MTS method; MCC >100 μM, CC₅₀ >100 μM) in Madin Darby canine kidney cell cultures (MDCK), and (1S,2S)-16k was active against vesicular stomatitis virus and respiratory syncytial virus in HeLa cells (EC₅₀ = 9 and 12 μM, respectively). Moreover, compound (1R,2S)-16l showed activity against both herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC₅₀ = 2.9 and 4 μM, respectively) and feline herpes virus in CRFK cells (EC₅₀ = 4 μM) but at the same time it exhibited cytotoxicity toward uninfected cell (MCC ⩾ 4 μM). Several other compounds have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC₅₀ in the 4–50 μM range. Among them compounds (1S,2S)- and (1R,2S)-16l were the most active (IC₅₀ in the 4–7 μM range).     

A-ring modified betulinic acid derivatives as potent cancer preventive agents

Ten new 3,4-seco betulinic acid (BA) derivatives were designed and synthesized. Among them, compounds 7–15 exhibited enhanced chemopreventive ability in an in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein–Barr virus early antigen (EBV-EA) activation assay in Raji cells. Specifically, analogs with a free C-28 carboxylic acid, including 7, 8, 11, and 13, inhibited EBV-EA activation significantly. The most potent compound 8 displayed 100% inhibition at 1 × 10³ mol ratio/TPA and 73.4%, 35.9%, and 8.4% inhibition at 5 × 10², 1 × 10², and 1 × 10 mol ratio/TPA, respectively, comparable with curcumin at high concentration and better than curcumin at low concentration. The potent chemopreventive activity of novel seco A-ring BAs (8 and 11) was further confirmed in an in vivo mouse skin carcinogenesis assay.     

Antiviral efficacy of Limonium densiflorum against HSV-1 and influenza viruses

Viral infections remain a major threat to humans and animals and there is a crucial need for new antiviral agents especially with the development of resistant viruses. Several Limonium genus members (Plumbaginacea) have been widely used in traditional medicine for the treatment of infections. In this study, we investigated the antiviral activities of different fractions after successive extraction (hexane, dichloromethane, ethanol and methanol) of the halophyte Limonium densiflorum against H1N1 influenza and HSV-1 herpes viruses. In addition, TLC phytochemicals of the shoot extracts were analyzed. All extracts were tested for their cytotoxicity using a fluorometric resazurin assay. The antiviral activity of extracts was tested using four modes of action: virucidal test, pretreatment of cells with samples before infection, attachment assay and plaque reduction test. A good antiviral activity was found with ethanol and methanol extracts. They were most potent in HSV-1 inhibition than H1N1 influenza virus. The most potent inhibition was observed with ethanol extract, and it exhibited high levels of virucidal activity against HSV-1 (IC₅₀ = 6 μg/mL). It inhibits the replication of the virus by 75% when added after penetration of the virus, and by 100% when added during the viral attachment. It protects MDCK cells against influenza virus by abolishing virus to entry into the host cell (IC₅₀ = 55 μg/mL). After attachment of influenza virus, the ethanol extract displayed an appreciable inhibition of virus replication (IC₅₀ = 193 μg/mL). Methanol extract showed a moderate antiviral capacity against both viruses. While dichloromethane has excellent antiherpes potential, results were inappropriate because it was toxic to Vero cells, hexane extract has no effect. TLC analysis of these extracts showed that flavonoids and saponins were the major classes of natural products found in the shoot extracts that may be responsible for these antiviral activities.     

Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue

NS5 is the largest and most conserved protein among the four dengue virus (DENV) serotypes. It has been the target of interest for antiviral drug development due to its major role in replication. NS5 consists of two domains, the N-terminal methyltransferase domain and C-terminal catalytic RNA-dependent RNA polymerase (RdRp) domain. It is an unstable protein and is prone to inactivation upon prolonged incubation at room temperature, thus affecting the inhibitor screening assays. In the current study, we expressed and purified DENV RdRp alone in Esherichia coli (E. coli) cells. The N-terminally His-tagged construct of DENV RdRp was transformed into E. coli expression strain BL-21 (DE3) pLysS cells. Protein expression was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM. The induced cultures were then grown for 20 h at 18 °C and cells were harvested by centrifugation at 6000 x g for 15 min at 4 °C. The recombinant protein was purified using HisTrap affinity column (Ni-NTA) and then the sample was subjected to size exclusion chromatography, which successfully removed the degradation product obtained during the previous purification step. The in vitro polymerase activity of RdRp was successfully demonstrated using homopolymeric polycytidylic acid (poly(rC)) RNA template. This study describes the high level production of enzymatically active DENV RdRp protein which can be used to develop assays for testing large number of compounds in a high-throughput manner. RdRp has the de novo initiation activity and the in vitro polymerase assays for the protein provide a platform for highly robust and efficient antiviral compound screening systems.     

Caffeic acid derivatives: A new type of influenza neuraminidase inhibitors

Recently, many natural products, especially some plant-derived polyphenols have been found to exert antiviral effects against influenza virus and show inhibitory activities on neuraminidases (NAs). In our research, we took caffeic acid which contained two phenolic hydroxyl groups as the basic fragment to build a small compound library with various structures. The enzyme inhibition result indicated that some compounds exhibited moderate activities against NA and compound 15d was the best with IC₅₀ = 7.2 μM and 8.5 μM against N2 and N1 NAs, respectively. The 3,4-dihydroxyphenyl group from caffeic acid was important for the activity according to the docking analysis. Besides, compound 15d was found to be a non-competitive inhibitor with K_i = 11.5 ± 0.25 μM by the kinetic study and also presented anti-influenza virus activity in chicken embryo fibroblast cells. It seemed promising to discover more potent NA inhibitors from caffeic acid derivatives to cope with influenza virus.     

In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2

Human Cathelicidin antimicrobial peptide LL-37 is known to have antiviral activity against many viruses. In the present study, we investigated the in-vitro effect of LL-37 on dengue virus type 2 (DENV-2) infection and replication in Vero E6 cells. To study the effect of pretreatment of virus or cells with LL-37, the virus was pretreated with different concentrations of LL-37 (2.5 μM–15 μM) or scrambled (Scr) LL-37(5 μM–15 μM) and used for infection or the cells were first treated with LL-37 and infected. To study the effect of LL-37 post infection (PI), the cells were infected first followed by addition of LL-37 to the culture medium 24 h after infection. In all conditions, after the incubation, the culture supernatant was assessed for viral RNA copy number by real time RT-PCR, infectious virus particles by focus forming unit assay (FFU) and non structural protein 1 (NS1) antigen levels by ELISA. Percentage of infection was assessed using immunoflourescence assay (IFA). The results revealed that pretreatment of virus with 10–15 μM LL-37 significantly reduced its infectivity as compared to virus control (P < 0.0001). Moreover, pretreatment of virus with 10–15 μM LL-37 significantly reduced the levels of viral genomic RNA and NS1 antigen (P < 0.0001). Treatment of virus with 10–15 μM LL-37 resulted in two to three log reduction of mean log₁₀ FFU/ml as compared to virus control (P < 0.0001). Treatment of the virus with scrambled LL-37 had no effect on percentage of infection and viral load as compared to virus control cultures (P > 0.05). Pretreatment of cells before infection or addition of LL-37 to the culture 24 h PI had no effect on viral load. Molecular docking studies revealed possible binding of LL-37 to both the units of DENV envelope (E) protein dimer. Together, the in-vitro experiments and in-silico analyses suggest that LL-37 inhibits DENV-2 at the stage of entry into the cells by binding to the E protein. The results might have implications for prophylaxis against DENV infections and need further in-vivo studies.     

In silico and in vitro methods to identify ebola virus VP35-dsRNA inhibitors

Ebola virus continues to be problematic as sporadic outbreaks in Africa continue to arise, and as terrorist organizations have considered the virus for bioterrorism use. Several proteins within the virus have been targeted for antiviral chemotherapy, including VP35, a dsRNA binding protein that promotes viral replication, protects dsRNA from degradation, and prevents detection of the viral genome by immune complexes. To augment the scope of our antiviral research, we have now employed molecular modeling techniques to enrich the population of compounds for further testing in vitro. In the initial docking of a static VP35 structure with an 80,000 compound library, 40 compounds were selected, of which four compounds inhibited VP35 with IC₅₀ <200 μM, with the best compounds having an IC₅₀ of 20 μM. By superimposing 26 VP35 structures, we determined four aspartic acid residues were highly flexible and the docking was repeated under flexible parameters. Of 14 compounds chosen for testing, five compounds inhibited VP35 with IC₅₀ <200 μM and one compound with an IC₅₀ of 4 μM. These studies demonstrate the value of docking in silico for enriching compounds for testing in vitro, and specifically using multiple structures as a guide for detecting flexibility and provide a foundation for further development of small molecule inhibitors directed towards VP35.     

Highly potent tyrosinase inhibitor,neorauflavane from Campylotropis hirtella and inhibitory mechanism with molecular docking

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson’s disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20 μg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC₅₀ of 30 nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC₅₀ = 500 nM), significantly. Another potent inhibitor 1 (IC₅₀ = 2.9 μM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: K_i^app = 1.48 nM, k₃ = 0.0033 nM⁻¹ min⁻¹ and k₄ = 0.0049 min⁻¹. Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95 μM of IC₅₀. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.     

Design,synthesis, assessment,and molecular docking of novel pyrrolopyrimidine (7-deazapurine) derivatives as non-nucleoside hepatitis C virus NS5B polymerase inhibitors

Hepatitis C virus (HCV) infection is highly persistent and presents an unmet medical need requiring more effective treatment options. This has spurred intensive efforts to discover novel anti-HCV agents. The RNA-dependent RNA polymerase (RdRp), NS5B of HCV, constitutes a selective target for drug discovery due to its absence in human cells; also, it is the centerpiece for viral replication. Here, we synthesized novel pyrrole, pyrrolo[2,3-d]pyrimidine and pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives. The non-toxic doses of these compounds on Huh 7.5 cell line were determined and their antiviral activity against HCVcc genotype 4a was examined. Compounds 7j, 7f, 5c, 12i and 12f showed significant anti HCV activity. The percent of reduction for the non-toxic doses of 7j, 7f, 5c, 12i and 12f were 90%, 76.7 ± 5.8%, 73.3 ± 5.8%, 70% and 63.3 ± 5.8%, respectively. The activity of these compounds was interpreted by molecular docking against HCV NS5B polymerase enzyme.     

The first activation study of a bacterial carbonic anhydrase (CA). The thermostable α-CA from Sulfurihydrogenibium yellowstonense YO3AOP1 is highly activated by amino acids and amines

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) was investigated for its activation with a series of amino acids and amines. d-His, l-Phe, l-Tyr, l- and d-Trp were the most effective SspCA activators, with activation constants in the range of 1–12 nM, whereas l-His, l/d-DOPA, d-Tyr, and several biogenic amines/catecholamines were slightly less effective activators (K_A in the range of 37 nM–0.97 μM). The least effective SspCA activator was d-Phe (K_A of 5.13 μM). The thermal stability, robustness and very high catalytic activity of SspCA make this enzyme an ideal candidate for biomimetic CO₂ capture processes.     

Antiplasmodial activity of synthetic ellipticine derivatives and an isolated analog

Ellipticine has been shown previously to exhibit excellent in vitro antiplasmodial activity and in vivo antimalarial properties that are comparable to those of the control drug chloroquine in a mouse malaria model. Ellipticine derivatives and analogs exhibit antimalarial potential however only a few have been studied to date. Herein, ellipticine and a structural analog were isolated from Aspidosperma vargasii bark. A-ring brominated and nitrated ellipticine derivatives exhibit good in vitro inhibition of Plasmodium falciparum K1 and 3D7 strains. Several of the compounds were found not to be toxic to human fetal lung fibroblasts. 9-Nitroellipticine (IC₅₀ = 0.55 μM) exhibits greater antiplasmodial activity than ellipticine. These results are further evidence of the antimalarial potential of ellipticine derivatives.     

Green synthesis,inhibition studies of yeast α-glucosidase and molecular docking of pyrazolylpyridazine amines

An efficient and environmentally benign simple fusion reaction of 3-chloro-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine (1a) or 3-chloro-6-(3,5-dimethyl-4-nitro-1H-pyrazol-1-yl)pyridazine (2a) with different aliphatic/aromatic amines have produced a series of novel pyrazolylpyridazine amines (4a–4c & 5a–5m). All compounds exhibited moderate in vitro yeast α-glucosidase inhibition except m-chloro derivative 5g, which was found potent inhibitor of this enzyme with IC₅₀ value of 19.27 ± 0.005 µM. The molecular docking further helped in understanding the structure activity relationship of these compounds including 5g.