Kaori Takakusagi

Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

Background Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Principal Findings Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. Conclusions We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

Use of phage display technology for the determination of the targets for small-molecule therapeutics

Importance of the field: Target discovery of drug-like small-molecules contributes to our understanding of biological phenomena at the molecular level as well as elucidating the mode of action of bioactive compounds. Research in this field is of high value because, in addition to basic observations, the data can be used to directly identify molecular targets or investigate pharmacokinetic characteristics of drugs in clinical use. Areas covered in this review: In addition to providing a brief overview of phage display (PD) technology, we discuss screening platforms, different types of phage libraries and the application of this method to the determination of targets for small-molecule therapeutics over the past decade. What the reader will gain: Readers will gain an understanding of the basis of PD technology through successful examples of the use of this method for the determination of targets for small-molecule therapeutics. They will learn what kinds of small-molecules were used to identify their binding partner, what characteristics and drawbacks are present in the use of small-molecule as bait, and what kinds of approaches were introduced in order to improve the technique to overcome the limitations of conventional strategies. Take home message: A suitable combination of diverse technologies from various different fields can act synergistically to increase throughput and enhance the efficiency of PD technology for the determination of targets for small-molecule therapeutics. The most suitable method for successful target identification of small-molecules of interest using PD technology can often be determined by referring to past examples.

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool.

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.

Camptothecin (CPT) directly binds to human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and inhibits the hnRNP A1/topoisomerase I interaction.

Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a K(D) value of 82.7 nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (K(D): 260 nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5-50 μM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster.

Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site.

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix.

A sulfoglycolipid beta-sulfoquinovosyldiacylglycerol (βSQDG) binds to Met1-Arg95 region of murine DNA polymerase lambda (Mmpol λ) and inhibits its nuclear transit

Beta-sulfoquinovosyldiacylglycerol (betaSQDG) is a synthetic sulfoglycolipid that shows inhibitory activity of DNA polymerase lambda (pol lambda). Here we identified a betaSQDG binding region within murine pol lambda (Mmpol lambda) using T7 phage display technology. We compared the binding intensity of betaSQDG with recombinant phages (phages lambda1-6) that displayed different segments of Mmpol lambda. The binding assay clearly showed that phage lambda1, which displayed the non-structural Met1-Arg95 region including the nuclear localization signal (NLS) and part of the BRCT domain, bound more strongly to betaSQDG than the other recombinant phages. Binding assays using recombinant proteins gave similar results, showing specific betaSQDG binding to Met1-Arg95 with a K(D) value of 9.9 nM. Furthermore, in a cell-based assay, nuclear localization of EGFP-pollambda was inhibited in the presence of betaSQDG possibly due to binding of betaSQDG to NLS. These experiments clearly show that the binding region of betaSQDG within Mmpol lambda could be successfully identified using T7 phage display technology. We suggest that the strategy we describe here will be of value for identifying the binding site within a protein for small ligands, and will provide information that cannot be obtained using other experimental techniques due to their inherent technical limitations.

Mapping a Disordered Portion of the Brz2001-Binding Site on a Plant Monooxygenase, DWARF4, Using a Quartz-Crystal Microbalance Biosensor-Based T7 Phage Display

In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor-ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp450) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage-displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the amino-acid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule-recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.

Exploration of the binding proteins of perfluorooctane sulfonate by a T7 phage display screen.

Perfluorooctane sulfonate (PFOS) is a pollutant widely found throughout nature and is toxic to animals. We created a PFOS analogue on a polyethylene glycol polyacrylamide copolymer and isolated peptides that preferentially bound the PFOS analogue using a T7 phage display system. Bioinformatic analysis using the FASTAskan program on the RELIC bioinformatics server showed several human proteins that likely bound PFOS. Among them, we confirmed binding between PFOS and a recombinant soluble form of monocyte differentiation antigen CD14 (sCD14) by a surface plasmon biosensor. Furthermore, PFOS inhibited TNF-α production induced by the sCD14 in mouse macrophage RAW264.7 cells.

Multimodal biopanning of T7 phage-displayed peptides reveals angiomotin as a potential receptor of the anti-angiogenic macrolide Roxithromycin

Roxithromycin (RXM) is a semi-synthetic fourteen-membered macrolide antibiotic that shows anti-angiogenic activity in solid tumors. In the present study, we conducted biopanning of T7 phage-displayed peptides either on a 96-well formatted microplate, a flow injection-type quartz-crystal microbalance (QCM) biosensor, or a cuvette-type QCM. RXM-selected peptides of different sequence, length and number were obtained from each mode of screening. Subsequent bioinformatics analysis of the RXM-selected peptides consistently gave positive scores for the extracellular domain (E458-T596) of angiomotin (Amot), indicating that this may comprise a binding region for RXM. Bead pull down assay and QCM analysis confirmed that RXM directly interacts with Amot via the screen-guided region, which also corresponds to the binding site for the endogenous anti-angiogenic inhibitor angiostatin (Anst). Thus, multimodal biopanning of T7PD revealed that RXM binds to the extracellular domain on Amot as a common binding site with Anst, leading to inhibition of angiogenesis-dependent tumor growth and metastasis. These data might explain the molecular basis underlying the mechanism of action for the anti-angiogenic activity of RXM.

Binding region and interaction properties of sulfoquinovosylacylglycerol (SQAG) with human vascular endothelial growth factor 165 revealed by biosensor-based assays

Sulfoquinovosylacylglycerol (SQAG) is a sulfoglycolipid showing anti-angiogenic and radiosensitizing effects for treatment of solid tumors both in vitro and in vivo. Here we elucidated the interaction of SQAG with various growth factors and their cognate receptors for vascular formation using biosensor-based assays. The structure–binding relationship was also determined. Our results show that βSQDG selectively recognizes heparin binding domain (HBD) in human vascular endothelial growth factor 165 (hVEGF165) with an affinity in the order of 10−11 M. The presence of both a sulfate moiety and at least one C18 length fatty acid chain is essential for binding. Conversion of anomeric configurations in SQAG did not alter the affinity with hVEGF165. This SQAG association inhibited T7 phage-displayed HBD binding to neuropilin-1 (NRP1), a VEGF receptor on the endothelial cell surface of blood vessels that specifically recognizes HBD in hVEGF165.