Yu-Ching Lee

Discovery of Highly Potent Tyrosinase Inhibitor, T1, with Significant Anti-Melanogenesis Ability by zebrafish in vivo Assay and Computational Molecular Modeling

Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53 μM, Ki = 58 ± 6 nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50 μM of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.

Rationalization and Design of the Complementarity Determining Region Sequences in an Antibody-Antigen Recognition Interface

Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes.

A comparison of different electrostatic potentials on prediction accuracy in CoMFA and CoMSIA studies

Computational chemistry is playing an increasingly important role in drug design and discovery, structural biology, and quantitative structure-activity relationship (QSAR) studies. For QSAR work, selecting an appropriate and accurate method to assign the electrostatic potentials of each atom in a molecule is a critical first step. So far several commonly used methods are available to assign charges. However, no systematic comparison of the effects of electrostatic potentials on QSAR quality has been made. In this study, twelve semi-empirical and empirical charge-assigning methods, AM1, AM1-BCC, CFF, Del-Re, Formal, Gasteiger, Gasteiger-Hückel, Hückel, MMFF, PRODRG, Pullman, and VC2003 charges, have been compared for their performances in CoMFA and CoMSIA modeling using several standard datasets. Some charge assignment models, such as Del-Re, PRODRG, and Pullman, are limited to specific atom and bond types, and, therefore, were excluded from this study. Among the remaining nine methods, the Gasteiger-Hückel charge, though commonly used, performed poorly in prediction accuracy. The AM1-BCC method was better than most charge-assigning methods based on prediction accuracy, though it was not successful in yielding overall higher cross-validation correlation coefficient (q(2)) values than others. The CFF charge model worked the best in prediction accuracy when q(2) was used as the evaluation criterion. The results presented should help the selection of electrostatic potential models in CoMFA and CoMSIA studies.

Identification of salt-inducible kinase 3 as a novel tumor antigen associated with tumorigenesis of ovarian cancer

Existence of humoral immunity has been previously demonstrated in malignant ascitic fluids. However, only a limited number of immunogenic tumor-associated antigens (TAAs) were identified, and few of which are associated with ovarian cancer. Here, we identified salt-inducible kinase 3 (SIK3) as a TAA through screening of a random peptide library in the phage display system. Overexpression of SIK3 markedly promoted cell proliferation, attenuated p21Waf/Cip1 and p27Kip expressions in low-grade OVCAR3 cells, and permitted the cells to grow in mice. Decrease in SIK3 expression in high-grade SK-OV3 cells consistently demonstrated its tumorigenic potency by modulating the protein levels of cell cycle regulators. When the expressions of SIK3 and CA125 were compared in cancer tissues, immunohistochemical (IHC) studies indicated that cytoplasm-localized SIK3 was highly expressed in 55% of the ovarian cancer samples. In contrast, it was rarely detected in adenomyosis, leiomyoma and normal ovary tissues, showing its higher specificity (97%) to CA125 (65%) in ovarian cancer. Moreover, experiments using pharmacological inhibitors to block SIK3-induced p21Waf/Cip1 expression revealed that activation of c-Src and phosphoinositide-3-kinase were critically required for its biological activity, suggesting that they are the downstream signaling mediators of SIK3. These data were further supported by IHC studies, showing coexpression of c-Src with SIK3 in 85% of the ovarian tumor samples stained positive for SIK3. Collectively, our findings indicate that SIK3 is a novel ovarian TAA. Overexpression of SIK3 promotes G1/S cell cycle progression, bestows survival advantages to cancer cells for growth and correlates the clinicopathological conditions of patients with ovarian cancer.

Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and sc-dsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv.

Loop-sequence features and stability determinants in antibody variable domains by high-throughput experiments.

Protein loops are frequently considered as critical determinants in protein structure and function. Recent advances in high-throughput methods for DNA sequencing and thermal stability measurement have enabled effective exploration of sequence-structure-function relationships in local protein regions. Using these data-intensive technologies, we investigated the sequence-structure-function relationships of six complementarity-determining regions (CDRs) and ten non-CDR loops in the variable domains of a model vascular endothelial growth factor (VEGF)-binding single-chain antibody variable fragment (scFv) whose sequence had been optimized via a consensus-sequence approach. The results show that only a handful of residues involving long-range tertiary interactions distant from the antigen-binding site are strongly coupled with antigen binding. This implies that the loops are passive regions in protein folding; the essential sequences of these regions are dictated by conserved tertiary interactions and the consensus local loop-sequence features contribute little to protein stability and function.

Antibody Variable Domain Interface and Framework Sequence Requirements for Stability and Function by High-Throughput Experiments

Protein structural stability and biological functionality are dictated by the formation of intradomain cores and interdomain interfaces, but the intricate sequence-structure-function interrelationships in the packing of protein cores and interfaces remain difficult to elucidate due to the intractability of enumerating all packing possibilities and assessing the consequences of all the variations. In this work, groups of β strand residues of model antibody variable domains were randomized with saturated mutagenesis and the functional variants were selected for high-throughput sequencing and high-throughput thermal stability measurements. The results show that the sequence preferences of the intradomain hydrophobic core residues are strikingly flexible among hydrophobic residues, implying that these residues are coupled indirectly with antigen binding through energetic stabilization of the protein structures. By contrast, the interdomain interface residues are directly coupled with antigen binding. The interdomain interface should be treated as an integral part of the antigen-binding site.

Serendipitous Discovery of Short Peptides from Natural Products as Tyrosinase Inhibitors

Tyrosinase, which is the crucial copper-containing enzyme involved in melanin synthesis, is strongly associated with hyperpigmentation disorders, cancer, and neurodegenerative disease; thus, it has attracted considerable interest in the fields of medicine and cosmetics. The known tyrosinase inhibitors show numerous adverse side effects, and there is a lack of safety regulations governing their use. As a result, there is a need to develop novel inhibitors with no toxicity and long-term stability. In this study, we use molecular docking and pharmacophore modeling to construct a reasonable and reliable pharmacophore model, called Hypo 1, that could be used for identifying potent natural products with crucial complementary functional groups for mushroom tyrosinase inhibition. It was observed that, out of 47,263 natural compounds, A5 structurally resembles a dipeptide (WY) and natural compound B16 is the equivalent of a tripeptide (KFY), revealing that the C-terminus tyrosine residues play a key role in tyrosinase inhibition. Tripeptides RCY and CRY, which show high tyrosinase inhibitory potency, revealed a positional and functional preference for the cysteine residue at the N-terminus of the tripeptides, essentially determining the capacity of tyrosinase inhibition. CRY and RCY used the thiol group of cysteine residues to coordinate with the Cu ions in the active site of tyrosinase and showed reduced tyrosinase activity. We discovered the novel tripeptide CRY that shows the most striking inhibitory potency against mushroom tyrosinase (IC50 = 6.16 μM); this tripeptide is more potent than the known oligopeptides and comparable with kojic acid-tripeptides. Our study provides an insight into the structural and functional roles of key amino acids of tripeptides derived from the natural compound B16, and the results are expected to be useful for the development of tyrosinase inhibitors.

Phage Display-Mediated Discovery of Novel Tyrosinase-Targeting Tetrapeptide Inhibitors Reveals the Significance of N-Terminal Preference of Cysteine Residues and Their Functional Sulfur Atom

Tyrosinase, a key copper-containing enzyme involved in melanin biosynthesis, is closely associated with hyperpigmentation disorders, cancer, and neurodegenerative diseases, and as such, it is an essential target in medicine and cosmetics. Known tyrosinase inhibitors possess adverse side effects, and there are no safety regulations; therefore, it is necessary to develop new inhibitors with fewer side effects and less toxicity. Peptides are exquisitely specific to their in vivo targets, with high potencies and relatively few off-target side effects. Thus, we systematically and comprehensively investigated the tyrosinase-inhibitory abilities of N- and C-terminal cysteine/tyrosine-containing tetrapeptides by constructing a phage-display random tetrapeptide library and conducting computational molecular docking studies on novel tyrosinase tetrapeptide inhibitors. We found that N-terminal cysteine-containing tetrapeptides exhibited the most potent tyrosinase-inhibitory abilities. The positional preference of cysteine residues at the N terminus in the tetrapeptides significantly contributed to their tyrosinase-inhibitory function. The sulfur atom in cysteine moieties of N- and C-terminal cysteine-containing tetrapeptides coordinated with copper ions, which then tightly blocked substrate-binding sites. N- and C-terminal tyrosine-containing tetrapeptides functioned as competitive inhibitors against mushroom tyrosinase by using the phenol ring of tyrosine to stack with the imidazole ring of His263, thus competing for the substrate-binding site. The N-terminal cysteine-containing tetrapeptide CRVI exhibited the strongest tyrosinase-inhibitory potency (with an IC50 of 2.7 ± 0.5 μM), which was superior to those of the known tyrosinase inhibitors (arbutin and kojic acid) and outperformed kojic acid–tripeptides, mimosine-FFY, and short-sequence oligopeptides at inhibiting mushroom tyrosinase.

Chicken single-chain variable fragments against the SARS-CoV spike protein

Abstract The major concern for severe acute respiratory syndrome (SARS), caused by the SARS-associated coronavirus (SARS-CoV), is the lack of diagnostic and therapeutic agents. Using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the SARS-CoV spike protein were characterized. Ten truncated spike protein gene fragments were expressed in Escherichia coli cells. Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5×107 and 9×106 transformants, respectively. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were demonstrated by Coomassie blue staining, and verified by western blot analysis. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750–1000. In conclusion, the results suggest that the chicken scFv phage display system can be a potential model for mass production of high-affinity antibodies against the SARS-CoV spike protein.