Ching-Yao Su

High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms are useful. In this study, we developed a screening procedure to search for new antiinfluenza inhibitors from 1,200,000 compounds and identified previously reported as well as new antiinfluenza compounds. Several antiinfluenza compounds were inhibitory to the influenza RNA-dependent RNA polymerase (RdRP), including nucleozin and its analogs. The most potent nucleozin analog, 3061 (FA-2), inhibited the replication of the influenza A/WSN/33 (H1N1) virus in MDCK cells at submicromolar concentrations and protected the lethal H1N1 infection of mice. Influenza variants resistant to 3061 (FA-2) were isolated and shown to have the mutation on nucleoprotein (NP) that is distinct from the recently reported resistant mutation of Y289H [Kao R, et al. (2010) Nat Biotechnol 28:600]. Recombinant influenza carrying the Y52H NP is also resistant to 3061 (FA-2), and NP aggregation induced by 3061 (FA-2) was identified as the most likely cause for inhibition. In addition, we identified another antiinfluenza RdRP inhibitor 367 which targets PB1 protein but not NP. A mutant resistant to 367 has H456P mutation at the PB1 protein and both the recombinant influenza and the RdRP expressing the PB1 H456P mutation have elevated resistance to 367. Our high-throughput screening (HTS) campaign thus resulted in the identification of antiinfluenza compounds targeting RdRP activity.

Differential Receptor Binding Affinities of Influenza Hemagglutinins on Glycan Arrays

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.

A Broadly Neutralizing Antibody Targets the Dynamic HIV Envelope Trimer Apex via a Long, Rigidified, and Anionic β-Hairpin Structure

&NA; Broadly neutralizing antibodies (bnAbs) to HIV delineate vaccine targets and are prophylactic and therapeutic agents. Some of the most potent bnAbs target a quaternary epitope at the apex of the surface HIV envelope (Env) trimer. Using cryo‐electron microscopy, we solved the atomic structure of an apex bnAb, PGT145, in complex with Env. We showed that the long anionic HCDR3 of PGT145 penetrated between glycans at the trimer 3‐fold axis, to contact peptide residues from all three Env protomers, and thus explains its highly trimer‐specific nature. Somatic hypermutation in the other CDRs of PGT145 were crucially involved in stabilizing the structure of the HCDR3, similar to bovine antibodies, to aid in recognition of a cluster of conserved basic residues hypothesized to facilitate trimer disassembly during viral entry. Overall, the findings exemplify the creative solutions that the human immune system can evolve to recognize a conserved motif buried under a canopy of glycans. HighlightsApex binding antibody PGT145 engages all three gp120 protomers simultaneouslyEpitope recognition is chemical‐feature specificPGT145‐class antibodies exhibit structural features that reflect bovine antibodiesPGT145‐class antibody maturation is dependent on structural stabilization of HCDR3 &NA; Broadly neutralizing antibodies of the PGT145‐family target the HIV‐1 Env trimer apex via a long &bgr;‐hairpin HCDR3, but the molecular basis of recognition is unknown. Using cryoEM, Lee et al. (2017) reveal how PGT145 binds its quaternary epitope and the importance of HCDR2 evolution despite its lack of contacts with Env.

In Vivo Protection Provided by a Synthetic New Alpha-Galactosyl Ceramide Analog against Bacterial and Viral Infections in Murine Models

ABSTRACT Alpha-galactosyl ceramide (α-GalCer) has been known to bind to the CD1d receptor on dendritic cells and activate invariant natural killer T (iNKT) cells, which subsequently secrete T-helper-cell 1 (Th1) and Th2 cytokines, which correlate with anti-infection activity and the prevention of autoimmune diseases, respectively. α-GalCer elicits the secretion of these two cytokines nonselectively, and thus, its effectiveness is limited by the opposing effects of the Th1 and Th2 cytokines. Reported here is the synthesis of a new α-GalCer analog (compound C34), based on the structure of CD1d, with a 4-(4-fluorophenoxy) phenyl undecanoyl modification of the N-acyl moiety of α-GalCer. Using several murine bacterial and viral infection models, we demonstrated that C34 has superior antibacterial and antiviral activities in comparison with those of several other Th1-selective glycolipids and that it is most effective by administering it to mice in a prophylactic manner before or shortly after infection.

Effect of bone area on spine density in Chinese men and women in Taiwan

Areal bone mineral density (BMD), the quotient of bone mineral content (BMC) divided by the projectional bone area (BA), measured with dual-energy X-ray absorptiometers (DXA), is the most common parameter used today to evaluate spinal osteoporosis. To evaluate whether gender, age, weight, and height can determine spinal BA, and to compare BA and analyze its effects on spinal density in the two genders, we measured BA and BMC, and calculated areal BMD, and the bone mineral apparent density (BMAD = BMD/the square root of BA) of the L-2 to L-4 vertebrae of 604 female and 223 male Chinese volunteers from 20 to 70 years of age using a Norland XR-26 DXA. Standardized for height and weight, BA showed a relatively large variation and a significant increase with increasing age in both genders. On the other hand, BMC stayed unchanged in men > 50 years of age and decreased with aging in postmenopausal women. Younger men (< 51 years) had a much larger mean BA (by 15.5%) and larger mean BMC (only 10%) than that of age-matched women. As a result, younger men had a slightly and significantly lower areal BMD (by 7.1%) and a much lower BMAD (by 16%) (p < 0.0001 for both) than premenopausal women of similar age. Men had higher areal BMD and BMAD values than age-matched women only after age 50 years. Although taller body height, heavier weight, and increasing age were associated with a larger BA, these factors could not explain most of the interindividual variations in BA in both genders. Thus anteroposterior BA of lumbar vertebrae measured with DXA seems to affect the areal BMD and BMAD readings in the two genders. The larger BA caused a low BMAD and probably underestimated the true volumetric spine density in men.

HA-Pseudotyped Retroviral Vectors for Influenza Antagonist Screening

Influenza infections are initiated by the binding of the influenza hemagglutinin (HA) and the cellular receptor sialic acids. The binding is followed by internalization, endocytosis, and uncoating to release the influenza genome to the cytoplasm. It is conceivable that specific inhibitors that antagonize any one of these events could prevent the replication of influenza infections. The authors made HA pseudotyped retroviral vectors that express luciferase reporter activities upon transduction to several recipient cells. The transduction of the HA-pseudotype virus particles (HApp) was mediated through the specific interactions between an avian HA and the terminal disaccharides of sialic acid (SA) and galactose (Gal) in α-2,3 linkage. The HApp-mediated transduction method was used to develop a high-throughput screening assay and to screen for hits from a fermentation extract library. Specific hits that inhibited the HA-mediated but were noninhibitory to the vesicular stomatitis virus—mediated pseudoviral transductions were identified. A few of these hits have anti-influenza activities that prevent the replication of both H1N1 (WSN) and H5N1 (RG14) influenza viruses. ( Journal of Biomolecular Screening 2009:294-302)

Elicitation of Neutralizing Antibodies Targeting the V2 Apex of the HIV Envelope Trimer in a Wild-Type Animal Model

Summary Recent efforts toward HIV vaccine development include the design of immunogens that can engage B cell receptors with the potential to affinity mature into broadly neutralizing antibodies (bnAbs). V2-apex bnAbs, which bind a protein-glycan region on HIV envelope glycoprotein (Env) trimer, are among the most broad and potent described. We show here that a rare “glycan hole” at the V2 apex is enriched in HIV isolates neutralized by inferred precursors of prototype V2-apex bnAbs. To investigate whether this feature could focus neutralizing responses onto the apex bnAb region, we immunized wild-type rabbits with soluble trimers adapted from these Envs. Potent autologous tier 2 neutralizing responses targeting basic residues in strand C of the V2 region, which forms the core epitope for V2-apex bnAbs, were observed. Neutralizing monoclonal antibodies (mAbs) derived from these animals display features promising for subsequent broadening of the response.

Chemical Probes for Drug-Resistance Assessment by Binding Competition (RABC): Oseltamivir Susceptibility Evaluation†

The wizard of OS (resistance): the binding difference of neuraminidase inhibitors (zanamivir versus oseltamivir (OS)) was used to establish an assay to identify the influenza subtypes that are resistant to OS but still sensitive to zanamivir. This assay used a zanamivir-biotin conjugate to determine the OS susceptibility of a wide range of influenza viruses and over 200 clinical isolates.