Molecular dynamic simulations analysis of ritonavir and lopinavir as SARS-CoV 3CL(pro) inhibitors

Since the emergence of the severe acute respiratory syndrome (SARS) to date, neither an effective antiviral drug nor a vaccine against SARS is available. However, it was found that a mixture of two HIV-1 proteinase inhibitors, lopinavir and ritonavir, exhibited some signs of effectiveness against the SARS virus. To understand the fine details of the molecular interactions between these proteinase inhibitors and the SARS virus via complexation, molecular dynamics simulations were carried out for the SARS-CoV 3CL^pro free enzyme (free SARS) and its complexes with lopinavir (SARS-LPV) and ritonavir (SARS-RTV). The results show that flap closing was clearly observed when the inhibitors bind to the active site of SARS-CoV 3CL^pro. The binding affinities of LPV and RTV to SARS-CoV 3CL^pro do not show any significant difference. In addition, six hydrogen bonds were detected in the SARS-LPV system, while seven hydrogen bonds were found in SARS-RTV complex.     

Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CL(pro): structure-activity relationship studies reveal salient pharmacophore features

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle. In this study, a natural compound called quercetin-3-β-galactoside was identified as an inhibitor of the protease by molecular docking, SPR/FRET-based bioassays, and mutagenesis studies. Both molecular modeling and Q189A mutation revealed that Gln189 plays a key role in the binding. Furthermore, experimental evidence showed that the secondary structure and enzymatic activity of SARS-CoV 3CL^pro were not affected by the Q189A mutation. With the help of molecular modeling, eight new derivatives of the natural product were designed and synthesized. Bioassay results reveal salient features of the structure–activity relationship of the new compounds: (1) removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivatives; (2) acetoxylation of the sugar moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4) replacement of the galactose moiety with other sugars does not affect inhibitor potency. This study not only reveals a new class of compounds as potential drug leads against the SARS virus, but also provides a solid understanding of the mechanism of inhibition against the target enzyme.     

Geranylated flavonoids displaying SARS-CoV papain-like protease inhibition from the fruits of Paulownia tomentosa

SARS-CoV papain-like protease (PLpro) is an important antiviral target due to its key roles in SARS virus replication. The MeOH extracts of the fruits of the Paulownia tree yielded many small molecules capable of targeting PLpro. Five of these compounds were new geranylated flavonoids, tomentin A, tomentin B, tomentin C, tomentin D, tomentin E (1–5). Structure analysis of new compounds (1–5) by NMR showed that they all contain a 3,4-dihydro-2H-pyran moiety. This chemotype is very rare and is derived from cyclization of a geranyl group with a phenol functionality. Most compounds (1–12) inhibited PLpro in a dose dependent manner with IC₅₀’s raging between 5.0 and 14.4 μM. All new compounds having the dihydro-2H-pyran group showed better inhibition than their parent compounds (1 vs 11, 2 vs 9, 4 vs 12, 5 vs 6). In kinetic studies, 1–12 emerged to be reversible, mixed inhibitors.     

18-Oxoferruginol from the leaf of Torreya nucifera

From the leaf of the conifer Torreya nucifera, a new diterpenoid has been isolated and shown to be 18-oxoferruginold (12-hydroxy-8,11,13-abietatrien-18-al) on the basis of its spectroscopic properties and its chemical conversion to the known 18-hydroxyferruginol (8,11,13-abietatriene-12,18-diol).     

Inhibition of SARS-CoV 3CL protease by flavonoids

Abstract

There were severe panics caused by Severe Acute Respiratory Syndrome (SARS) and Middle-East Respiratory Syndrome-Coronavirus. Therefore, researches targeting these viruses have been required. Coronaviruses (CoVs) have been rising targets of some flavonoids. The antiviral activity of some flavonoids against CoVs is presumed directly caused by inhibiting 3C-like protease (3CLpro). Here, we applied a flavonoid library to systematically probe inhibitory compounds against SARS-CoV 3CLpro. Herbacetin, rhoifolin and pectolinarin were found to efficiently block the enzymatic activity of SARS-CoV 3CLpro. The interaction of the three flavonoids was confirmed using a tryptophan-based fluorescence method, too. An induced-fit docking analysis indicated that S1, S2 and S3′ sites are involved in binding with flavonoids. The comparison with previous studies showed that Triton X-100 played a critical role in objecting false positive or overestimated inhibitory activity of flavonoids. With the systematic analysis, the three flavonoids are suggested to be templates to design functionally improved inhibitors.     

Structure-based virtual screening against SARS-3CL(pro) to identify novel non-peptidic hits

Severe acute respiratory syndrome is a highly infectious upper respiratory tract disease caused by SARS-CoV, a previously unidentified human coronavirus. SARS-3CL(pro) is a viral cysteine protease critical to the pathogen's life cycle and hence a therapeutic target of importance. The recently elucidated crystal structures of this enzyme provide an opportunity for the discovery of inhibitors through rational drug design. In the current study, Gold docking program was utilized to conduct extensive docking studies against the target crystal structure to develop a robust and predictive docking protocol. The validated docking protocol was used to conduct a structure-based virtual screening of the Asinex Platinum collection. Biological evaluation of a screened selection of compounds was carried out to identify novel inhibitors of the viral protease.     

Mechanism of the maturation process of SARS-CoV 3CL protease

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CL(pro)) to cleave the virus-encoded polyproteins. We report here that the 3CL(pro) containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln(-1)-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (K(d) = 0.35 nm) than for the mutant (C145A) containing 10 extra N-terminal (K(d) = 17.2 nM) or C-terminal amino acids (K(d) = 5.6 nM). The data indicate that immature 3CL(pro) can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CL(pro) from the polyprotein and design of new structure-based inhibitors.     

Effects of Japanese Torreya (Torreya nucifera) seed oil on the activities and mRNA expression of lipid metabolism-related enzymes in rats

The mechanism for the plasma and liver triacylglycerol-reducing effects of Japanese torreya (Torreya nucifera) seed oil containing sciadonic acid (all-cis-5, 11, 14-eicosatrienoic acid) is reported. Male SD rats were fed experimental diets containing 10% torreya, corn, or soybean oil for 4 weeks, and the activities and mRNA expression of the enzymes involved in lipid metabolism were measured in the liver. The activities of some hepatic enzymes involved in fatty acid synthesis were lower in the rats fed torreya oil.     

Abietane diterpenoids from suspension cultured cells of Torreya nucifera var. radicans

Three abietane diterpenoids were isolated from the suspension cultured cells of Torreya nucifera var. radicans along with four known abietane diterpenoids. Based on spectroscopic evidence, the structures of the three were elucidated as (3S,5R,10S)-7-oxo-12-methoxyabieta-8,11,13-triene-3,11-diol, (3S,5R,10S)-7-oxo-12-methoxyabieta-8,11,13-triene-3,11,14-triol and (5R,10S)-3-oxo-7R,12-dimethoxyabieta-8,11,13-trien-11-ol, respectively.     

Binding interaction of SARS coronavirus 3CL(pro) protease with vacuolar-H+ ATPase G1 subunit

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important issue for treatment and prevention of SARS. Recently, SARS-CoV 3CL(pro) protease has been implied to be possible relevance to SARS-CoV pathogenesis. In this study, we intended to identify potential 3CL(pro)-interacting cellular protein(s) using the phage-displayed human lung cDNA library. The vacuolar-H+ ATPase (V-ATPase) G1 subunit that contained a 3CL(pro) cleavage site-like motif was identified as a 3CL(pro)-interacting protein, as confirmed using the co-immunoprecipitation assay and the relative affinity assay. In addition, our result also demonstrated the cleavage of the V-ATPase G1 fusion protein and the immunoprecipitation of cellular V-ATPase G1 by the 3CL(pro). Moreover, loading cells with SNARF-1 pH-sensitive dye showed that the intracellular pH in 3CL(pro)-expressing cells was significantly lower as compared to mock cells.     

Synthesis and evaluation of phenylisoserine derivatives for the SARS-CoV 3CL protease inhibitor

Synthesis and evaluation of new scaffold phenylisoserine derivatives connected with the essential functional groups against SARS CoV 3CL protease are described. The phenylisoserine backbone was found by simulation on GOLD software and the structure activity relationship study of phenylisoserine derivatives gave SK80 with an IC₅₀ value of 43 μM against SARS CoV 3CL R188I mutant protease.     

Phenols displaying tyrosinase inhibition from Humulus lupulus

Tyrosinase is the rate-limiting enzyme for the production of melanin and other pigments via the oxidation of l-tyrosine. The methanol extract from Humulus lupulus showed potent inhibition against mushroom tyrosinase. The bioactivity-guided fractionation of this methanol extract resulted in the isolation of seven flavonoids (1–7), identified as xanthohumol (1), 4′-O-methylxanthohumol (2), xanthohumol C (3), flavokawain C (4), xanthoumol B (5), 6-prenylnaringenin (6) and isoxanthohumol (7). All isolated flavonoids (1–7) effectively inhibited the monophenolase (IC₅₀s?=?15.4–58.4?µM) and diphenolase (IC₅₀s?=?27.1–117.4?µM) activities of tyrosinase. Kinetic studies using Lineweaver–Burk and Dixon-plots revealed that chalcones (1–5) were competitive inhibitors, whereas flavanones (6 and 7) exhibited both mixed and non-competitive inhibitory characteristics. In conclusion, this study is the first to demonstrate that the phenolic phytochemicals of H. lupulus display potent inhibitory activities against tyrosinase.     

Biflavonoids from the Wollemi Pine,Wollemia nobilis (Araucariaceae)

Biflavonoids from the leaves of Wollemia nobilis (Wollemi Pine) have been investigated and compared with data on other members of the family Araucariaceae. Based on ¹H and ¹³C NMR as well as High Resolution Mass Spectrometry (HRMS) the structures of seven isolated biflavonoids were fully determined. Although all of them were previously characterised especially at Agathis and in lesser degree at Araucaria genera this is the first report on their occurrence at Wollemi Pine, one of the world's oldest and rarest plants.     

Biflavonoids from the bark roots of Poincianella pyramidalis (Fabaceae)

Poincianella pyramidalis (Fabaceae) is an endemic tree that grows in semiarid regions of Brazil. Phytochemical investigations on the bark roots of this plant led to the isolation of four new biflavonoids named (+)-5-hydroxy-7,4′-dimethoxyflavone-3α-2′′′-hydroxy-4′′′,4′′-dimethoxydihydrochalcone (1), (+)-5,7-dihydroxy-4′-methoxyflavone-3α-2′′′-hydroxy-4′′′,4′′-dimethoxydihydrochalcone (2), (−)-7-hydroxy-4′-methoxyflavone-3α-2′′′,4′′′-dihydroxy-4′′-methoxydihydrochalcone (3), (−)-7,4′-dihydroxy-flavanone-3,8-5′′,6′′,4′′-trihydroxy-flavone (4), and the previously identified compound 4,2′,4′,4′′,2′′′,4′′′-hexahydroxy-3,5′′′-bichalcone (rhuschalcone VI, 5). Their structures were determined by HR-ESI-MS and extensive analyses of NMR spectroscopic data.     

Xanthones from Cudrania tricuspidata displaying potent alpha-glucosidase inhibition

We have proven that xanthones 1-8 isolated from the root of C. tricuspidata possess highly potent alphaalpha-glucosidase inhibition properties. Compound 1 was identified as a new isoprenylated tetrahydroxy xanthone, 1,3,6,7-tetrahydroxy-2-(3-methylbut-2-enyl)-8-(2-methylbut-3-en-2-yl)-9H-xanthen-9-one (1). These are the first natural xanthones documented to exhibit such inhibition. The IC(50) values of compounds 1-8 inhibiting alpha-glucosidase activity were determined to be up to 16.2microM. Mechanistic analysis showed the xanthones 1-8 exhibited full mixed inhibition.     

Isolation of 6-Hydroxydehydroabietinol and Hinokiol from the Leaves of Torreya nucifera Sieb,et Zucc

6-Hydroxydehydroabietinol and hinokiol were isolated from the non-steam-volatile fraction of the leaves of Torreya nucifera Sieb, et Zucc. (Taxaceae) (Japanese name “Kaya”). The former was also isolated from the immature fruits of the same plant. This is the first report of the isolation of 6-hydroxydehydroabietinol from a natural source. This compound was prepared previously and reported to show marked estrogenic activity.     

Biflavonoids from Fumana procumbens (Dunal) Gren. & Godr

The first phytochemical investigation on the aerial parts of Fumana procumbens (Dunal) Gren. & Godr. led to the isolation and identification of six compounds, including two biflavonoids, i.e. dihydrodaphnodorin B (1) and daphnodorin B (2); three flavonoids, i.e. quercitrin (3), myricitrin (4), and quercetin (5); and a flavan derivative, i.e. epigallocatechin (6). The structures of the compounds were elucidated by extensive 1D- and 2D-NMR spectroscopic analysis in combination with MS experiments. This is the first report on the isolation of biflavonoids from the genus Fumana and from the family Cistaceae.     

Caged xanthones displaying protein tyrosine phosphatase 1B (PTP1B) inhibition from Cratoxylum cochinchinense

Four new caged xanthones (1–4) and two known compounds (5, 6) were isolated from the roots of Cratoxylum cochinchinense, a polyphenol rich plant, collected in China. The structures of the isolated compounds (1–6) were characterized by obtaining their detailed spectroscopic data. In particular, compounds 1 and 6 were fully identified by X-ray crystallographic data. The isolated compounds (1–6) were evaluated against protein tyrosine phosphatase 1B (PTP1B), which plays an important role in diabetes, obesity, and cancer. Among these compounds, 3, 4, and 6 displayed significant inhibition with IC₅₀ values of 76.3, 43.2, and 6.6 µM, respectively. A detailed kinetic study was conducted by determining K_m, V_max, and the ratio of K_ik and K_iv, which revealed that all the compounds behaved as competitive inhibitors.     

Increase of SARS-CoV 3CL peptidase activity due to macromolecular crowding effects in the milieu composition

The 3C-like peptidase of the severe acute respiratory syndrome virus (SARS-CoV) is strictly required for viral replication, thus being a potential target for the development of antiviral agents. In contrast to monomeric picornavirus 3C peptidases, SARS-CoV 3CLpro exists in equilibrium between the monomer and dimer forms in solution, and only the dimer is proteolytically active in dilute buffer solutions. In this study, the increase of SARS-CoV 3CLpro peptidase activity in presence of kosmotropic salts and crowding agents is described. The activation followed the Hofmeister series of anions, with two orders of magnitude enhancement in the presence of Na?SO?, whereas the crowding agents polyethylene glycol and bovine serum albumin increased the hydrolytic rate up to 3 times. Kinetic determinations of the monomer dimer dissociation constant (K(d)) indicated that activation was a result of a more active dimer, without significant changes in K(d) values. The activation was found to be independent of substrate length and was derived from both k(cat) increase and K(m) decrease. The viral peptidase activation described here could be related to the crowded intracellular environment and indicates a further fine-tuning mechanism for biological control, particularly in the microenvironment of the vesicles that are induced in host cells during positive strand RNA virus infection.