Toby Passioura
University of Tokyo
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Featured researches published by Toby Passioura.
Annual Review of Biochemistry | 2014
Toby Passioura; Takayuki Katoh; Yuki Goto; Hiroaki Suga
Macrocyclic peptides are an emerging class of therapeutics that can modulate protein-protein interactions. In contrast to the heavily automated high-throughput screening systems traditionally used for the identification of chemically synthesized small-molecule drugs, peptide-based macrocycles can be synthesized by ribosomal translation and identified using in vitro selection techniques, allowing for extremely rapid (hours to days) screening of compound libraries comprising more than 10(13) different species. Furthermore, chemical modification of translated peptides and engineering of the genetic code have greatly expanded the structural diversity of the available peptide libraries. In this review, we discuss the use of these technologies for the identification of bioactive macrocyclic peptides, emphasizing recent developments.
BMC Immunology | 2009
Amber Goodchild; Nicole Nopper; Andrew King; Tram Bich Doan; Marcel Tanudji; Greg M. Arndt; Michael Poidinger; Laurent P. Rivory; Toby Passioura
BackgroundShort interfering RNAs (siRNAs) have been shown to induce immune stimulation through a number of different receptors in a range of cell types. In primary cells, both TLR7 and TLR8 have been shown to recognise siRNAs however, despite the identification of a number of TLR7/8 stimulatory RNA motifs, the complete and definitive sequence determinants of TLR7 and TLR8 are yet to be elucidated.ResultsA total of 207 siRNA sequences were screened for TLR7/8 stimulation in human PBMCs. There was a significant correlation between the U count of the U-rich strand and the immunostimulatory activity of the duplex. Using siRNAs specifically designed to analyse the effect of base substitutions and hybridisation of the two strands, we found that sequence motifs and the thermodynamic properties of the duplexes appeared to be the major determinants of siRNA immunogenicity and that the strength of the hybridisation interaction between the two strands correlated negatively with immunostimulatory activity.ConclusionThe data presented favour a model of TLR7/8 activation by siRNAs, in which the two strands are denatured in the endosome, and single-stranded, U-rich RNA species activate TLR7/8. These findings have relevance to the design of siRNAs, particularly for in vivo or clinical applications.
Molecules | 2013
Kenichiro Ito; Toby Passioura; Hiroaki Suga
In this review, we discuss emerging technologies for drug discovery, which yields novel molecular scaffolds based on natural product-inspired non-traditional peptides expressed using the translation machinery. Unlike natural products, these technologies allow for constructing mRNA-encoding libraries of macrocyclic peptides containing non-canonical sidechains and N-methyl-modified backbones. The complexity of sequence space in such libraries reaches as high as a trillion (>1012), affording initial hits of high affinity ligands against protein targets. Although this article comprehensively covers several related technologies, we discuss in greater detail the technical development and advantages of the Random non-standard Peptide Integration Discovery (RaPID) system, including the recent identification of inhibitors against various therapeutic targets.
Nature Communications | 2017
Akane Kawamura; Martin Münzel; Tatsuya Kojima; Clarence Yapp; Bhaskar Bhushan; Yuki Goto; Anthony Tumber; Takayuki Katoh; Oliver N. King; Toby Passioura; Louise J. Walport; Stephanie B. Hatch; Sarah Madden; Susanne Müller; Paul E. Brennan; Rasheduzzaman Chowdhury; Richard J. Hopkinson; Hiroaki Suga; Christopher J. Schofield
The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.
Chemistry: A European Journal | 2013
Toby Passioura; Hiroaki Suga
Noncanonical peptides occur frequently in Nature, and often display high bioactivity. However, the lack of tractable systems for the synthesis of diverse libraries of such peptides has thus far hampered their development as drugs. Genetic reprogramming techniques, in which noncanonical amino acids may be incorporated into peptides, have largely removed this limitation. This Concept article outlines the development of these techniques with an emphasis on drug discovery.
Journal of Biomolecular Screening | 2009
Amber Goodchild; Nicole Nopper; Alexis Craddock; Tamara Law; Andrew King; Gregory Fanning; Laurent P. Rivory; Toby Passioura
The innate immune system of mammals is a key defense mechanism against invading foreign pathogens. Innate immune stimulants may have applications as vaccine adjuvants as well as in the treatment of cancer and some viral diseases, and clinical studies have been performed using agonists of Toll-like receptors (TLRs) 7, 8, and 9. The high-throughput screens for such agonists have typically relied on the overexpression of a single TLR gene in an immortalized cell line and are inherently artificial systems that are restricted to the identification of agonists for a single receptor. The authors describe 2 assays for the identification of immunostimulants that employ primary human leukocytes cocultured with hepatitis C virus (HCV) replicon-expressing cells. In these assays, stimulation of innate immune pathways in leukocytes induces interferon (IFN) expression, which acts to inhibit HCV replication, providing a high-throughput and low-cost readout for leukocyte activation. These assays are highly sensitive and provide a physiologically relevant system for the identification of a broad range of immunostimulant agents. (Journal of Biomolecular Screening 2009:723-730)
Nucleic Acids Research | 2007
Amber Goodchild; Andrew King; Mary Margaret Gozar; Toby Passioura; Carly Tucker; Laurent P. Rivory
It has recently been shown that certain oligodeoxynucleotides (ODNs) designed as catalytic DNA molecules (DNAzymes) exhibit potent cytotoxicity independent of RNA-cleavage activity in a number of cell lines. These cytotoxic ODNs all featured a 5′ G-rich sequence and induced cell death by a TLR9-independent mechanism. In this study, we examined the sequence and length dependence of ODNs for cytotoxicity. A G-rich sequence at the 5′ terminus of the molecule was necessary for cytotoxicity and the potency of ODNs with active 5′ sequences was length dependent. Cytotoxicity appeared to be generally independent of 3′ sequence composition, although 3′ sequences totally lacking G-nucleotides were mostly inactive. Nucleolin, elongation factor 1-alpha (eEF1A) and vimentin were identified as binding to a cytotoxic ODN (Dz13) using protein pull-down assays and LC-MS/MS. Although these proteins have previously been described to bind G-rich ODNs, the binding of eEF1A correlated with cytotoxicity, whereas binding of nucleolin and vimentin did not. Quiescent non-proliferating cells were resistant to cytotoxicity, indicating cytotoxicity may be cell cycle dependent. Although the exact mechanism of cytotoxicity remains unknown, marked potency of the longer (⩾25 nt) ODNs in particular, indicates the potential of these molecules for treatment of diseases associated with abnormal cell proliferation.
Oncogene | 2005
Toby Passioura; Sylvie Shen; Geoff Symonds; Alla Dolnikov
Activating mutations of the N-ras gene occur at relatively high frequency in acute myeloid leukemia and myelodysplastic syndrome. Somewhat paradoxically, ectopic expression of activated N-ras in primary hematopoietic cells and myeloid cell lines (in some cases) can lead to inhibition of proliferation. Expression of mutant N-ras in murine hematopoietic stem/progenitor cells is sufficient to induce myeloid malignancies, but these pathologies occur with long latency. This suggests that mutations that disable the growth suppressive properties of N-ras in hematopoietic cells are required for the development of frank malignancy. In the present work, the growth suppression induced by a mutant N-ras gene in U937 myeloid cells was used as the basis to screen a retroviral cDNA library for genes that prevent mutant N-ras-induced growth suppression (i.e., putative cooperating oncogenes). This screen identified the gene for the transcription factor interferon regulatory factor-2 (IRF-2), and as confirmation of the screen, overexpression of this gene in U937 cells was shown to inhibit mutant N-ras-induced growth suppression. Also recovered from the screen were two truncated clones of an uncharacterized gene (interim official symbol: PP2135). Overexpression of this truncated PP2135 gene in U937 cells did not appear to abrogate mutant N-ras-induced growth suppression, but rather appeared to confer an increased sensitivity of U937 cells to retroviral infection, accounting for the recovery of this gene from the genetic screen.
Current Gene Therapy | 2006
Ailyn Choo; Patricia Palladinetti; Toby Passioura; Sylvie Shen; Richard B. Lock; Geoff Symonds; Alla Dolnikov
Acute myeloid leukaemia (AML) is the most common form of leukaemia in adults. Although of the order of 75-85% of patients will achieve complete remission after induction chemotherapy, long-term survival is still relatively low. Despite the progress in the rational design of drugs in disorders such as chronic myeloid leukaemia, AML lacks a single specific pathogenomic event to act as a drug target. Interferon regulatory factor 1 (IRF1) is a member of a family of related proteins that act as transcriptional activators or repressors. IRF1 and its functional antagonist IRF2 originally discovered as transcription factors regulating the interferon-beta (IFN-beta) gene, are involved in the regulation of normal haematopoiesis and leukaemogenesis. IRF1 appears to act as a tumour suppressor gene and IRF2 as an oncogene. IRF1 acts to repress IRF2 function through the repression of cyclin-dependent kinase (CDK) inhibitor p21WAF1 critical for cell growth control. It appears that the tumour suppression function of IRF1 is abolished by IRF2. This review focuses on the interaction between IRF1 and IRF2 in myeloid development and leukaemogenesis, particularly in relation to the Ras signalling pathway. IRF2 may be a viable and specific therapeutic target in human leukaemia.
Oligonucleotides | 2008
Mary Margaret Gozar; Amber Goodchild; Toby Passioura; Andrew King; Angela Lai; Craig Witherington; Laurent P. Rivory
We have previously shown that Dz13, a catalytic DNA molecule (DNAzyme) designed against c-jun, is cytotoxic to nonquiescent cells by a mechanism independent of c-jun mRNA cleavage. In this report, we evaluated programmed cell death (PCD) pathways in order to gain further insight into the mechanism of action of Dz13. Using human dermal microvascular endothelial cells (HMEC-1), we found that Dz13-mediated cell death is characterized by mitochondrial depolarization, caspase-8 activation, lysosomal increase, and autophagosome formation. Classical DNA laddering and translocation of mitochondrial proteins were not observed. An array of inhibitors, including those targeting caspases, failed to abrogate cytotoxicity and mitochondrial depolarization. Cytotoxicity did not proceed from endoplasmic reticulum (ER) stress. The possible involvement of PARP-1 in Dz13-mediated cytotoxicity was indicated by its differential release as gauged by protein extraction data and its apparent binding to Dz13, as evidenced by protein pull-down experiments. This study on Dz13-mediated cytotoxicity presents a detailed investigation into the interplay of cell death effectors involved in apoptosis, autophagy, and necrosis, and demonstrates a novel form of oligonucleotide-mediated cytotoxicity with features of PCD.