Haibin Luo

Cinanserin Is an Inhibitor of the 3C-Like Proteinase of Severe Acute Respiratory Syndrome Coronavirus and Strongly Reduces Virus Replication In Vitro

ABSTRACT The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLpro of SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 μM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50 values ranging from 19 to 34 μM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.

Nucleocapsid protein of SARS coronavirus tightly binds to human cyclophilin A

Abstract Severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for SARS infection. Nucleocapsid protein (NP) of SARS-CoV (SARS_NP) functions in enveloping the entire genomic RNA and interacts with viron structural proteins, thus playing important roles in the process of virus particle assembly and release. Protein–protein interaction analysis using bioinformatics tools indicated that SARS_NP may bind to human cyclophilin A (hCypA), and surface plasmon resonance (SPR) technology revealed this binding with the equilibrium dissociation constant ranging from 6 to 160nM. The probable binding sites of these two proteins were detected by modeling the three-dimensional structure of the SARS_NP–hCypA complex, from which the important interaction residue pairs between the proteins were deduced. Mutagenesis experiments were carried out for validating the binding model, whose correctness was assessed by the observed effects on the binding affinities between the proteins. The reliability of the binding sites derived by the molecular modeling was confirmed by the fact that the computationally predicted values of the relative free energies of the binding for SARS_NP (or hCypA) mutants to the wild-type hCypA (or SARS_NP) are in good agreement with the data determined by SPR. Such presently observed SARS_NP–hCypA interaction model might provide a new hint for facilitating the understanding of another possible SARS-CoV infection pathway against human cell.

Ligand-binding regulation of LXR/RXR and LXR/PPAR heterodimerizations: SPR technology-based kinetic analysis correlated with molecular dynamics simulation.

Liver X receptor (LXR) and peroxisome proliferator‐activated receptor (PPAR) are two members of nuclear receptors involved in the nutrient metabolisms of dietary fatty acid and cholesterol. They are found to be of cross‐talk function in that LXR regulates fatty acid synthesis and PPAR controls fatty acid degradation. LXRs (LXRα and LXRβ) function by forming obligate heterodimers with the retinoid X receptor (RXR), and subsequently binding to specific DNA response elements within the regulatory regions of their target genes. In this work, the kinetic features concerning LXR/RXR and LXR/PPAR interactions have been fully investigated using surface plasmon resonance (SPR) technology. It is found that LXRs could bind to all the three PPAR subtypes, PPARα, PPARγ and PPARδ with different binding affinities, and such receptor/receptor interactions could be regulated by ligand binding. Moreover, molecular dynamics (MD) simulations were performed on six typical complex models. The results revealed that ligands may increase the interaction energies between the receptor interfaces of the simulated receptor/receptor complexes. The MD results are in agreement with the SPR data. Further analyses on the MD results indicated that the ligand binding might increase the hydrogen bonds between the interfaces of the receptor/receptor complex.

The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1

The nucleocapsid (N) protein of SARS coronavirus (SARS_CoV) is a major structural component of virions, which appears to be a multifunctional protein involved in viral RNA replication and translation. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is related to the pre‐mRNA splicing in the nucleus and translation regulation in the cytoplasm. In this report, based on the relevant biophysical and biochemical assays, the nucleocapsid protein of SARS_CoV (SARS_N) was discovered to exhibit high binding affinity to human hnRNP A1. GST pull‐down results clearly demonstrated that SARS_N protein could directly and specifically bind to human hnRNP A1 in vitro. Yeast two‐hybrid assays further indicated in vivo that such binding relates to the fragment (aa 161–210) of SARS_N and the Gly‐rich domain (aa 203–320) of hnRNP A1. Moreover, kinetic analyses by surface plasmon resonance (SPR) technology revealed that SARS_N protein has a specific binding affinity against human hnRNP A1 with K D at 0.35 ± 0.02 μM (k on = 5.83 ± 0.42 × 103 M−1 s−1 and k off = 2.06 ± 0.12 × 10−3 s−1). It is suggested that both SARS_N and hnRNP A1 proteins are possibly within the SARS_CoV replication/transcription complex and SARS_N/human hnRNP A1 interaction might function in the regulation of SARS_CoV RNA synthesis. In addition, the determined results showed that SARS_N protein has only one binding domain for interacting with human hnRNP A1, which is different from the mouse hepatitis virus (MHV) binding case where the nucleocapsid protein of MHV (MHV_N) was found to have two binding domains involved in the MHV_N/hnRNP A1 interaction, thereby suggesting that SARS_N protein might carry out a different binding mode to bind to human hnRNP A1 for its further function performance in comparison with MHV_N.

The Dipeptide H-Trp-Glu-OH Shows Highly Antagonistic Activity against PPARγ: Bioassay with Molecular Modeling Simulation

The peroxisome proliferator‐activated receptor γ (PPARγ) is an important therapeutic drug target for several conditions, including diabetes, inflammation, dyslipidemia, hypertension, and cancer. It is shown that an antagonist or partial agonist of PPARγ has attractive potential applications in the discovery of novel antidiabetic agents that may retain efficacious insulin‐sensitizing properties and minimize potential side effects. In this work, the dipeptide H‐Trp‐Glu‐OH (G3335) was discovered to be a novel PPARγ antagonist. Biacore 3000 results based on the surface plasmon resonance (SPR) technique showed that G3335 exhibits a highly specific binding affinity against PPARγ (KD=8.34 μM) and is able to block rosiglitazone, a potent PPARγ agonist, in the stimulation of the interaction between the PPARγ ligand‐binding domain (LBD) and RXRα‐LBD. Yeast two‐hybrid assays demonstrated that G3335 exhibits strong antagonistic activity (IC50=8.67 μM) in perturbing rosiglitazone in the promotion of the PPARγ‐LBD–CBP interaction. Moreover, in transactivation assays, G3335 was further confirmed as an antagonist of PPARγ in that G3335 could competitively bind to PPARγ against 0.1 μM rosiglitazone to repress reporter‐gene expression with an IC50 value of 31.9 μM. In addition, homology modeling and molecular‐docking analyses were performed to investigate the binding mode of PPARγ‐LBD with G3335 at the atomic level. The results suggested that residues Cys285, Arg288, Ser289, and His449 in PPARγ play vital roles in PPARγ‐LBD–G3335 binding. The significance of Cys285 for PPARγ‐LBD–G3335 interaction was further demonstrated by PPARγ point mutation (PPARγ‐LBD‐Cys285Ala). It is hoped our current work will provide a powerful approach for the discovery of PPARγ antagonists, and that G3335 might be developed as a possible lead compound in diabetes research.

Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus.

Abstract Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CLpro) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CLpro, in this report the synthetic genes encoding 3CLpro of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded (∼15mg/L) expressed protease was purified by use of NTA-Ni2+ affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.

Severe acute respiratory syndrome coronavirus membrane protein interacts with nucleocapsid protein mostly through their carboxyl termini by electrostatic attraction

Abstract The severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein is an abundant virion protein, and its interaction with the nucleocapsid protein is crucial for viral assembly and morphogenesis. Although the interacting region in the nucleocapsid protein was mapped to residues 168–208, the interacting region in the membrane protein and the interaction nature are still unclear. In this work, by using yeast two-hybrid and surface plasmon resonance techniques, the residues 197–221 of the membrane protein and the residues 351–422 of the nucleocapsid protein were determined to be involved in their interaction. Sequence analysis revealed that these two fragments are highly charged at neutral pH, suggesting that their interaction may be of ionic nature. Kinetic assays indicated that the endodomain (aa102–221) of the membrane protein interacts with the nucleocapsid protein with high affinity (K D =0.55±0.04μM), however, this interaction could be weakened greatly by acidification, higher salt concentration (400mM NaCl) and divalent cation (50mM Ca2+), which suggests that electrostatic attraction might play an important role in this interaction. In addition, it is noted that two highly conserved amino acids (L218 and L219) in the membrane protein are not involved in this interaction. Here, we show that electrostatic interactions between the carboxyl termini of SARS-CoV membrane protein and nucleocapsid protein largely mediate the interaction of these two proteins. These results might facilitate therapeutic strategies aiming at the disruption of the association between SARS-CoV membrane and nucleocapsid proteins.

In vitro biochemical and thermodynamic characterization of nucleocapsid protein of SARS

Abstract The major biochemical and thermodynamic features of nucelocapsid protein of SARS coronavirus (SARS_NP) were characterized by use of non-denatured gel electrophoresis, size-exclusion chromatographic and surface plasmon resonance (SPR) techniques. The results showed that SARS_NP existed in vitro as oligomer, more probably dimer, as the basic functional unit. This protein shows its maximum conformational stability near pH 9.0, and it seems that its oligomer dissociation and protein unfolding occur simultaneously. Thermal-induced unfolding for SARS_NP was totally irreversible. Both the thermal and chemical denaturant-induced denaturation analyses showed that oligomeric SARS_NP unfolds and refolds through a two-state model, and the electrostatic interactions among the charge groups of SARS_NP made a significant contribution to its conformational stability.

Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique 1

AbstractAim:To characterize enzymatic activity of severe acute respiratory syndrome (SARS) coronavirus (CoV) 3C-like protease (3CLpro) and its four site-directed mutants.Methods:Based on the fluorescence resonance energy transfer (FRET) principle using 5-[(2′-aminoethyl)-amino] naphthelenesulfonic acid (EDANS) and 4-[[4-(dimethylamino) phenyl] azo] benzoic acid (Dabcyl) as the energy transfer pair, one fluorogenic substrate was designed for the evaluation of SARS-CoV 3CLpro proteolytic activity.Results:The kinetic parameters of the fluorogenic substrate have been determined as Km=404 μmol·L−1, kcat=1.08 min−1, and kcat/Km=2.7 mmol−1·L·min−1. SARS-CoV 3CLpro showed substantial pH and temperature-triggered activity switches, and site-directed mutagenesis analysis of SARS-CoV 3CLpro revealed that substitutions of His41, Cys145, and His163 resulted in complete loss of enzymatic activity, while replacement of Met162 with Ala caused strongly increased activity.Conclusion:This present work has provided valuable information for understanding the catalytic mechanism of SARS-CoV 3CLpro. This FRET-based assay might supply an ideal approach for the exploration SARS-CoV 3CLpro putative inhibitors.

Ferroelectric, dielectric and pyroelectric properties of Sr and Sn codoped BCZT lead free ceramics

The 1 mol.-%Sr and 1 mol.-%Sn codoped (Ba0·84Ca0·15Sr0·01)(Ti0·90Zr0·09Sn0·01)O3 (BCSTZS) ceramics were synthesised by the normal solid state sintering method. The electric field and temperature dependence of the ferroelectric properties of the BCSTZS ceramics were investigated. Their energy storage density depending on electric field and temperature was determined from the polarisation–electric field (P–E ) hysteresis loops. According to the dielectric analysis, the BCSTZS ceramics experience three-phase transitions upon cooling. At room temperature, the pyroelectric coefficient p calculated from the remnant polarisation–temperature (Pr–T ) curve is 1116·7 μC K− 1 m− 2, and the figures of merit Fd is 18·1 μPa− 1/2, Fv is 0·013 m2 C− 1 and Fi is 479·3 pm V− 1 respectively. The pyroelectric figures of merit exhibit high frequency stability over a wide range from 100 to 2000 Hz, whereas these values vary gradually with the increase in temperature, which deserves further research to improve their stability. The excellent pyroelectric property of the BCSTZS ceramics is considered as correlating with a polymorphic phase transition occurring around room temperature. The present study demonstrates that the lead free BCSTZS ceramics are promising candidate for replacing the lead zirconate titanate based ceramics.