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author:("Yu, kenbo")
1.  Inclusion of multiple fragment types in the Site Identification by Ligand Competitive Saturation (SILCS) approach 
The Site Identification by Ligand Competitive Saturation (SILCS) method identifies the location and approximate affinities of small molecular fragments on a target macromolecular surface by performing Molecular Dynamics (MD) simulations of the target in an aqueous solution of small molecules representative of different chemical functional groups. In this study, we introduce a set of small molecules to map potential interactions made by neutral hydrogen bond donors and acceptors, and charged donor and acceptor fragments in addition to nonpolar fragments. The affinity pattern is obtained in the form of discretized probability or, equivalently, free energy maps, called FragMaps, which can be visualized with the target surface. We performed SILCS simulations for four proteins for which structural and thermodynamic data is available for multiple, diverse ligands. Good overlap is shown between high affinity regions identified by the FragMaps and the crystallographic positions of ligand functional groups with similar chemical functionality, thus demonstrating the validity of the qualitative information obtained from the simulations. To test the ability of FragMaps in providing quantitative predictions, we calculate the previously introduced Ligand Grid Free Energy (LGFE) metric and observe its correspondence with experimentally measured binding affinity. LGFE is computed for different conformational ensembles and improvement in prediction is shown with increasing ligand conformational sampling. Ensemble generation includes a Monte Carlo sampling approach that uses the GFE FragMaps directly as the energy function. The results show some, but not all experimental trends are predicted, and warrant improvements in the scoring methodology. In addition, the potential utility of atom-based free energy contributions to the LGFE scores and the use of multiple ligands in SILCS to identify displaceable water molecules during ligand design are discussed.
PMCID: PMC3947602  PMID: 24245913
2.  N-Acetyl-Serotonin Protects HepG2 Cells from Oxidative Stress Injury Induced by Hydrogen Peroxide 
Oxidative stress plays an important role in the pathogenesis of liver diseases. N-Acetyl-serotonin (NAS) has been reported to protect against oxidative damage, though the mechanisms by which NAS protects hepatocytes from oxidative stress remain unknown. To determine whether pretreatment with NAS could reduce hydrogen peroxide- (H2O2-) induced oxidative stress in HepG2 cells by inhibiting the mitochondrial apoptosis pathway, we investigated the H2O2-induced oxidative damage to HepG2 cells with or without NAS using MTT, Hoechst 33342, rhodamine 123, Terminal dUTP Nick End Labeling Assay (TUNEL), dihydrodichlorofluorescein (H2DCF), Annexin V and propidium iodide (PI) double staining, immunocytochemistry, and western blot. H2O2 produced dramatic injuries in HepG2 cells, represented by classical morphological changes of apoptosis, increased levels of malondialdehyde (MDA) and intracellular reactive oxygen species (ROS), decreased activity of superoxide dismutase (SOD), and increased activities of caspase-9 and caspase-3, release of cytochrome c (Cyt-C) and apoptosis-inducing factor (AIF) from mitochondria, and loss of membrane potential (ΔΨm). NAS significantly inhibited H2O2-induced changes, indicating that it protected against H2O2-induced oxidative damage by reducing MDA levels and increasing SOD activity and that it protected the HepG2 cells from apoptosis through regulating the mitochondrial apoptosis pathway, involving inhibition of mitochondrial hyperpolarization, release of mitochondrial apoptogenic factors, and caspase activity.
PMCID: PMC4074966  PMID: 25013541
3.  A Replicating Modified Vaccinia Tiantan Strain Expressing an Avian-Derived Influenza H5N1 Hemagglutinin Induce Broadly Neutralizing Antibodies and Cross-Clade Protective Immunity in Mice 
PLoS ONE  2013;8(12):e83274.
To combat the possibility of a zoonotic H5N1 pandemic in a timely fashion, it is necessary to develop a vaccine that would confer protection against homologous and heterologous human H5N1 influenza viruses. Using a replicating modified vaccinia virus Tian Tan strain (MVTT) as a vaccine vector, we constructed MVTTHA-QH and MVTTHA-AH, which expresses the H5 gene of a goose-derived Qinghai strain A/Bar-headed Goose/Qinghai/1/2005 or human-derived Anhui Strain A/Anhui/1/2005. The immunogenicity profiles of both vaccine candidates were evaluated. Vaccination with MVTTHA-QH induced a significant level of neutralizing antibodies (Nabs) against a homologous strain and a wide range of H5N1 pseudoviruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4). Neutralization tests (NT) and Haemagglutination inhibition (HI) antibodies inhibit the live autologous virus as well as a homologous A/Xingjiang/1/2006 and a heterologous A/Vietnam/1194/2004, representing two human isolates from clade 2.2 and clade 1, respectively. Importantly, mice vaccinated with intranasal MVTTHA-QH were completely protected from challenge with lethal dosages of A/Bar-headed Goose/Qinghai/1/2005 and the A/Viet Nam/1194/2004, respectively, but not control mice that received a mock MVTTS vaccine. However, MVTTHA-AH induced much lower levels of NT against its autologous strain. Our results suggest that it is feasible to use the H5 gene from A/Bar-headed Goose/Qinghai/1/2005 to construct an effective vaccine, when using MVTT as a vector, to prevent infections against homologous and genetically divergent human H5N1 influenza viruses.
PMCID: PMC3866202  PMID: 24358269
4.  Extension of the CHARMM General Force Field to Sulfonyl-Containing Compounds and Its Utility in Biomolecular Simulations 
Journal of computational chemistry  2012;33(31):2451-2468.
Presented is an extension of the CHARMM General force field (CGenFF) to enable the modeling of sulfonyl-containing compounds. Model compounds containing chemical moieties such as sulfone, sulfonamide, sulfonate and sulfamate were used as the basis for the parameter optimization. Targeting high-level quantum mechanical and experimental crystal data, the new parameters were optimized in a hierarchical fashion designed to maintain compatibility with the remainder of the CHARMM additive force field. The optimized parameters satisfactorily reproduced equilibrium geometries, vibrational frequencies, interactions with water, gas phase dipole moments and dihedral potential energy scans. Validation involved both crystalline and liquid phase calculations showing the newly developed parameters to satisfactorily reproduce experimental unit cell geometries, crystal intramolecular geometries and pure solvent densities. The force field was subsequently applied to study conformational preference of a sulfonamide based peptide system. Good agreement with experimental IR/NMR data further validated the newly developed CGenFF parameters as a tool to investigate the dynamic behavior of sulfonyl groups in a biological environment. CGenFF now covers sulfonyl group containing moieties allowing for modeling and simulation of sulfonyl-containing compounds in the context of biomolecular systems including compounds of medicinal interest.
PMCID: PMC3477297  PMID: 22821581
empirical force field; molecular mechanics; molecular dynamics; molecular modeling; potential energy function; sulfonamide; β-strand mimetic; peptidomimetic; medicinal chemistry; drug design
5.  Intranasal LPS-Mediated Parkinson’s Model Challenges the Pathogenesis of Nasal Cavity and Environmental Toxins 
PLoS ONE  2013;8(11):e78418.
Accumulating evidence implicates the relationship between neuroinflammation and pathogenesis in idiopathic Parkinson's disease (iPD). The nose has recently been considered a gate way to the brain which facilitates entry of environmental neurotoxin into the brain. Our study aims to build a PD model by a natural exposure route. In this report, we establish a new endotoxin-based PD model in mice by unilateral intranasal (i.n.) instillation of the lipopolysaccharides (LPS) every other day for 5 months. These mice display a progressive hypokinesia, selective loss of dopaminergic neurons, and reduction in striatal dopamine (DA) content, as well as α-synuclein aggregation in the SN, without systemic inflammatory and immune responses. This new PD model provides a tool for studying the inflammation-mediated chronic pathogenesis and searching for therapeutic intervention in glia-neuron pathway that will slow or halt neurodegeneration in PD.
PMCID: PMC3826714  PMID: 24250796
6.  Turning Defense into Offense: Defensin Mimetics as Novel Antibiotics Targeting Lipid II 
PLoS Pathogens  2013;9(11):e1003732.
We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and in silico analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an in vivo model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections.
Author Summary
Every year, an increasing number of people are at risk for bacterial infections that cannot be effectively treated. This is because many bacteria are becoming more resistant to antibiotics. Of particular concern is the rise in hospital-acquired infections. Infection caused by the methicillin-resistant Staphylococcus aureus bacterium or MRSA is the cause of many fatalities and puts a burden on health care systems in many countries. The antibiotic of choice for treatment of S. aureus infections is vancomycin, an antimicrobial peptide that kills bacteria by binding to the bacterial cell wall component Lipid II. Here, we have identified for the first time, small synthetic compounds that also bind Lipid II with the aim to develop new antibiotic drugs to fight against bacterial infections.
PMCID: PMC3820767  PMID: 24244161
7.  The novel BH3 α-helix mimetic JY-1-106 induces apoptosis in a subset of cancer cells (lung cancer, colon cancer and mesothelioma) by disrupting Bcl-xL and Mcl-1 protein–protein interactions with Bak 
Molecular Cancer  2013;12:42.
It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-2/Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. We designed the BH3 α-helix mimetic JY-1-106 to engage the hydrophobic BH3-binding grooves on the surfaces of both Bcl-xL and Mcl-1.
JY-1-106–protein complexes were studied using molecular dynamics (MD) simulations and the SILCS methodology. We have evaluated the in vitro effects of JY-1-106 by using a fluorescence polarization (FP) assay, an XTT assay, apoptosis assays, and immunoprecipitation and western-blot assays. A preclinical human cancer xenograft model was used to test the efficacy of JY-1-106 in vivo.
MD and SILCS simulations of the JY-1-106–protein complexes indicated the importance of the aliphatic side chains of JY-1-106 to binding and successfully predicted the improved affinity of the ligand for Bcl-xL over Mcl-1. Ligand binding affinities were measured via an FP assay using a fluorescently labeled Bak-BH3 peptide in vitro. Apoptosis induction via JY-1-106 was evidenced by TUNEL assay and PARP cleavage as well as by Bax–Bax dimerization. Release of multi-domain Bak from its inhibitory binding to Bcl-2/Bcl-xL and Mcl-1 using JY-1-106 was detected via immunoprecipitation (IP) western blotting.
At the cellular level, we compared the growth proliferation IC50s of JY-1-106 and ABT-737 in multiple cancer cell lines with various Bcl-xL and Mcl-1 expression levels. JY-1-106 effectively induced cell death regardless of the Mcl-1 expression level in ABT-737 resistant solid tumor cells, whilst toxicity toward normal human endothelial cells was limited. Furthermore, synergistic effects were observed in A549 cells using a combination of JY-1-106 and multiple chemotherapeutic agents. We also observed that JY-1-106 was a very effective agent in inducing apoptosis in metabolically stressed tumors. Finally, JY-1-106 was evaluated in a tumor-bearing nude mouse model, and was found to effectively repress tumor growth. Strong TUNEL signals in the tumor cells demonstrated the effectiveness of JY-1-106 in this animal model. No significant side effects were observed in mouse organs after multiple injections.
Taken together, these observations demonstrate that JY-1-106 is an effective pan-Bcl-2 inhibitor with very promising clinical potential.
PMCID: PMC3663763  PMID: 23680104
Mcl-1; Bcl-xL; Small molecule inhibitor; Cancer; BH3 mimetic
8.  PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice  
The Journal of Clinical Investigation  2013;123(6):2629-2642.
Viral vector–based vaccines that induce protective CD8+ T cell immunity can prevent or control pathogenic SIV infections, but issues of preexisting immunity and safety have impeded their implementation in HIV-1. Here, we report the development of what we believe to be a novel antigen-targeting DNA vaccine strategy that exploits the binding of programmed death-1 (PD1) to its ligands expressed on dendritic cells (DCs) by fusing soluble PD1 with HIV-1 GAG p24 antigen. As compared with non–DC-targeting vaccines, intramuscular immunization via electroporation (EP) of the fusion DNA in mice elicited consistently high frequencies of GAG-specific, broadly reactive, polyfunctional, long-lived, and cytotoxic CD8+ T cells and robust anti-GAG antibody titers. Vaccination conferred remarkable protection against mucosal challenge with vaccinia GAG viruses. Soluble PD1–based vaccination potentiated CD8+ T cell responses by enhancing antigen binding and uptake in DCs and activation in the draining lymph node. It also increased IL-12–producing DCs and engaged antigen cross-presentation when compared with anti-DEC205 antibody-mediated DC targeting. The high frequency of durable and protective GAG-specific CD8+ T cell immunity induced by soluble PD1–based vaccination suggests that PD1-based DNA vaccines could potentially be used against HIV-1 and other pathogens.
PMCID: PMC3668817  PMID: 23635778
9.  Comprehensive Conformational Studies of Five Tripeptides and a Deduced Method for Efficient Determinations of Peptide Structures 
The Journal of Physical Chemistry. B  2012;116(7):2269-2283.
Thorough searches on the potential energy surfaces of five tripeptides, GGG, GYG, GWG, TGG and MGG, were performed by considering all possible combinations of the bond rotational degrees of freedom with a semi-empirical and ab initio combined computational approach. Structural characteristics of the obtained stable tripeptide conformers were carefully analyzed. Conformers of the five tripeptides were found to be closely connected with conformers of their constituting dipeptides and amino acids. A method for finding all important tripeptide conformers by optimizing a small number of trial structures generated by suitable superposition of the parent amino acid and dipeptide conformers is thus proposed. Applying the method to another five tripeptides, YGG, FGG, WGG, GFA and GGF, studied before shows that the new approach is both efficient and reliable by providing the most complete ensembles of tripeptide conformers. The method is further generalized for application to larger peptides by introducing the breeding and mutation concepts in a genetic algorithm way. The generalized method is verified to be capable of finding tetrapeptide conformers with secondary structures of strands, helices and turns which are highly populated in larger peptides. This show some promise for the proposed method to be applied for the structural determination of larger peptides.
PMCID: PMC3288331  PMID: 22260814
amino acids; dipeptides; tripeptides; structure determination; genetic algorithm
10.  Targeting zymogen activation to control the matriptase-prostasin proteolytic cascade 
Journal of medicinal chemistry  2011;54(21):7567-7578.
Membrane-associated serine protease matriptase has been implicated in human diseases, and might be a drug target. In the present study, a novel class of matriptase inhibitors targeting zymogen activation is developed by a combination of the screening of compound library using a cell-based matriptase activation assay and a computer-aided search of commercially available analogs of a selected compound. Four structurally related compounds are identified that can inhibit matriptase activation with IC50 at low μM in both intact-cell and cell-free systems, suggesting that these inhibitors target the matriptase autoactivation machinery rather than the intracellular signaling pathways. These activation inhibitors can also inhibit prostasin activation, a downstream event that occurs in lockstep with matriptase activation. In contrast, the matriptase catalytic inhibitor CVS-3983 at a concentration 300-fold higher than its Ki fails to inhibit activation of either protease. Our results suggest that inhibiting matriptase activation is an efficient way to control matriptase function.
PMCID: PMC3214968  PMID: 21966950
11.  Reproducing Crystal Binding Modes of Ligand Functional Groups using Site-Identification by Ligand Competitive Saturation (SILCS) Simulations 
The applicability of a computational method, Site Identification by Ligand Competitive Saturation (SILCS), to identify regions on a protein surface with which different types of functional groups on low-molecular weight inhibitors interact is demonstrated. The method involves molecular dynamics (MD) simulations of a protein in an aqueous solution of chemically diverse small molecules from which probability distributions of fragments types, termed FragMaps, are obtained. In the present application, SILCS simulations are performed with an aqueous solution of 1 M benzene and propane to map the affinity pattern of the protein for aromatic and aliphatic functional groups. In addition, water hydrogen and oxygen atoms serve as probes for hydrogen bond donor and acceptor affinity, respectively. The method is tested using a set of 7 proteins for which crystal structures of complexes with several high affinity inhibitors are known. Good agreement is obtained between FragMaps and the positions of chemically similar functional groups in inhibitors as observed in the X-ray crystallographic structures. Quantitative capabilities of the SILCS approach are demonstrated by converting FragMaps to free energies, termed Grid Free Energies (GFE), and showing correlation between the GFE values and experimental binding affinities. For proteins for which ligand decoy sets are available, GFE values are shown to typically score the crystal conformation and conformations similar to it more favorable than decoys. Additionally, SILCS is tested for its ability to capture the subtle differences in ligand affinity across homologous proteins, information which may be of utility towards specificity-guided drug design. Taken together, our results show that SILCS can recapitulate the known location of functional groups of bound inhibitors for a number of proteins, suggesting that the method may be of utility for rational drug design.
PMCID: PMC3090225  PMID: 21456594
12.  Mucosal Immunization Induces a Higher Level of Lasting Neutralizing Antibody Response in Mice by a Replication-Competent Smallpox Vaccine: Vaccinia Tiantan Strain 
The possible bioterrorism threat using the variola virus, the causative agent of smallpox, has promoted us to further investigate the immunogenicity profiles of existing vaccines. Here, we study for the first time the immunogenicity profile of a replication-competent smallpox vaccine (vaccinia Tiantan, VTT strain) for inducing neutralizing antibodies (Nabs) through mucosal vaccination, which is noninvasive and has a critical implication for massive vaccination programs. Four different routes of vaccination were tested in parallel including intramuscular (i.m.), intranasal (i.n.), oral (i.o.), and subcutaneous (s.c.) inoculations in mice. We found that one time vaccination with an optimal dose of VTT was able to induce anti-VTT Nabs via each of the four routes. Higher levels of antiviral Nabs, however, were induced via the i.n. and i.o. inoculations when compared with the i.m. and s.c. routes. Moreover, the i.n. and i.o. vaccinations also induced higher sustained levels of Nabs overtime, which conferred better protections against homologous or alternating mucosal routes of viral challenges six months post vaccination. The VTT-induced immunity via all four routes, however, was partially effective against the intramuscular viral challenge. Our data have implications for understanding the potential application of mucosal smallpox vaccination and for developing VTT-based vaccines to overcome preexisting antivaccinia immunity.
PMCID: PMC3134386  PMID: 21765641
13.  A Novel Replication-Competent Vaccinia Vector MVTT Is Superior to MVA for Inducing High Levels of Neutralizing Antibody via Mucosal Vaccination 
PLoS ONE  2009;4(1):e4180.
Mucosal vaccination offers great advantage for inducing protective immune response to prevent viral transmission and dissemination. Here, we report our findings of a head-to-head comparison of two viral vectors modified vaccinia Ankara (MVA) and a novel replication-competent modified vaccinia Tian Tan (MVTT) for inducing neutralizing antibodies (Nabs) via intramuscular and mucosal vaccinations in mice. MVTT is an attenuated variant of the wild-type VTT, which was historically used as a smallpox vaccine for millions of Chinese people. The spike glycoprotein (S) of SARS-CoV was used as the test antigen after the S gene was constructed in the identical genomic location of two vectors to generate vaccine candidates MVTT-S and MVA-S. Using identical doses, MVTT-S induced lower levels (∼2-3-fold) of anti- SARS-CoV neutralizing antibodies (Nabs) than MVA-S through intramuscular inoculation. MVTT-S, however, was capable of inducing consistently 20-to-100-fold higher levels of Nabs than MVA-S when inoculated via either intranasal or intraoral routes. These levels of MVTT-S-induced Nab responses were substantially (∼10-fold) higher than that induced via the intramuscular route in the same experiments. Moreover, pre-exposure to the wild-type VTT via intranasal or intraoral route impaired the Nab response via the same routes of MVTT-S vaccination probably due to the pre-existing anti-VTT Nab response. The efficacy of intranasal or intraoral vaccination, however, was still 20-to-50-fold better than intramuscular inoculation despite the subcutaneous pre-exposure to wild-type VTT. Our data have implications for people who maintain low levels of anti-VTT Nabs after historical smallpox vaccination. MVTT is therefore an attractive live viral vector for mucosal vaccination.
PMCID: PMC2613559  PMID: 19159014

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