The file contains supplemental details related to sequence information of the bicyclic peptides referred to in the text, binding data, organ distribution data, PK parameters, mouse and human plasma stability, in vivo metabolism of BCY-B2 in mice, cell binding and internalization data, additional PET imaging data. It also contains detailed methods related to protein expression, phage selection, peptide synthesis, affinity determination by fluorescence polarization and SPR, radiolabeling, internalization, confocal microscopy, plasma protein binding, plasma stability of radiolabeled compounds, µPET imaging, autoradiography, immunohistochemistry, and 6 references related to these methods. €¢ Supplementary Tables S1 - S14: o Table S1: Affinities and plasma stabilities of BCY-B and stabilized BCY-C. o Table S2: Sequence information and binding data of bicyclic peptides referred to in text. o Table S3: Surface plasmon resonance (SPR) data. o Table S4-6: Organ distribution data, BCY-B3/B4 o Table S7: PK parameters. o Table S8: Selectivity data of BCY-C towards other metalloproteinases. o Table S9-11: Organ distribution data- BCYC2/C4 o Table S12-14: Organ distribution data- BCYD1/D2 o Table S15: Organ distribution data- MAb €¢ Supplementary Figures S1-S6: o Figure S1: Comparative HT1080 cell binding and internalization of non-stabilized BCY-B3 and stabilized BCY-C2 o Figure S2: Organ distribution time course studies with active BCY-B3, and inactive BCY-B4 in HT1080 xenograft mice. o Figure S3: Mouse and human plasma stability of BCY-B5, BCY-C3, BCY-D1; o Figure S4: Pharmacokinetic profile of BCY-B2 in mouse; o Figure S5: In vivo metabolism of BCY-B2 in mice; o Figure S6: Confocal microscopy on HT1080 cells and BCY-C2 o Figure S7: PET imaging. Time-resolved whole-body maximum intensity projections of 68Ga-BCY-C2 in HT1080 xenograft mouse. €¢ Supplementary Methods: o Protein expression, o Phage selection, o Peptide synthesis, o Affinity determination by fluorescence polarization and SPR, o Radiolabeling, o Internalization, o Confocal microscopy, o Plasma protein binding, o Plasma stability of radiolabeled compounds, o µPET imaging, o Autoradiography, o Immunohistochemistry, o 6 references related to these methods.

Molecular imaging of cancers using probes specific for tumor-associated target proteins offers a powerful solution for providing information regarding selection of targeted therapy, patient stratification, and response to therapy. Here we demonstrate the power of bicyclic peptides as targeting probes, exemplified with the tumor-overexpressed matrix metalloproteinase MT1-MMP as a target. A bicyclic peptide with subnanomolar affinity towards MT1-MMP was identified, and its radioconjugate showed selective tumor uptake in an HT1080 xenograft mouse model. Proteolytic stabilization of the peptide by chemical modification significantly enhanced the in vivo tumor signal [from 2.5% ID/g to 12% ID/g at 1 hour post injection (p. i.)]. Studies using mouse xenograft models with different cell lines show a robust correlation between tumor signals and in vivo MT1MMP expression levels. Fatty acid modification of the bicy-clic peptide extended its circulating half-life, resulting in increased tumor signals (36% ID/g at 6 hours p. i.). Comparative work with an equipotent radiolabeled MT1-MMP targeting antibody demonstrated starkly differential biodistribution and tumor accumulation properties, with the tumor signal slowly increasing to 6.2% ID/g within 48 hours. The rapid tumor penetration characteristics of bicyclic peptides, coupled with high potency and chemical versatility, thus offer high-contrast imaging probes for clinical diagnostics with compelling additional potential in targeted therapy. Significance: This work demonstrates the potential of bicyclic peptides as a platform for the development of highcontrast imaging probes for potential use in clinical cancer diagnostics and molecularly targeted therapeutics.

Abstract The Bicycle® technology is based on repertoires of short peptides displayed on the surface of bacteriophages which can be modified with homo-trifunctional organochemical scaffolds, thus creating large diverse libraries of constrained, bicyclic peptides. These large combinatorial libraries have been extensively used for iterative selections to identify high affinity binding peptides to a wide array of targets, including receptors, interleukins and proteases. Bicyclic peptides are chemically synthesized macrocyclic entities with drug-like properties that exhibit sub-nanomolar affinities and exquisite selectivity towards targets. Unlike biologics, their synthetic nature allows facile modulation of metabolic and pharmacokinetic properties, as well as site-specific conjugation to effector molecules such as fluorophores, radionuclides, and cytotoxic drugs. In the present work, novel phage display derived bicyclic peptides were identified targeting the matrix metalloproteinase 14 (also known as MT1-MMP), a tumor associated surface protein overexpressed in a variety of cancers (i.e. lung, breast). A prototype bicyclic peptide with high affinity to MT1-MMP (Kd at ~1 nM) was identified, and confocal microscopy using fluoresceinated bicyclic peptide derivatives shows target-dependent internalisation in MT1-MMP+ cells. In the in vivo mouse, selective tumor binding in an MT1-MMP+ xenograft model is demonstrated for a DOTA conjugate loaded with Ga-68 or Lu-177. Upon proteolytic optimization of the prototype bicycle peptide, a striking enhancement in tumor signal is observed in biodistribution studies. Compared to radiolabeled antibodies directed against the same target, the lead compound showed fast background clearance (< 1 %ID/g for all organs apart from kidneys) resulting in high imaging contrast in µPET studies as early as 30 minutes post injection. Importantly, most of the non-tumor associated bicyclic peptide rapidly clears into the bladder. Together, tumor targeting bicyclic peptides can, through their small size and high selectivity, facilitate efficient penetration and visualization of tumors in vivo, demonstrating their potential as diagnostic imaging agents in profiling and therapeutic management of patients. Citation Format: Daniel Teufel, Helen Harrison, Spencer Campbell, Catherine Stace, Edward Walker, Robert J. Lutz, Peter Park, Matthias Eder, Ulrike Bauder-Wüst, Ursula Schierbaum, Karin Leotta, Klaus Kopka, Uwe Haberkorn. Bicyclic peptides for PET imaging of MT1-MMP expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3719. doi:10.1158/1538-7445.AM2017-3719

Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.

The neurotrophin receptor p75NTR is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75NTR antibody or phage scFv library pre-panned against p75NTR are internalized by neurons expressing p75NTR; (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75NTR antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75NTR expression is upregulated in motor neurons in response to injury and in disease, the p75NTR antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.

The cover picture shows a novel bicyclic peptide inhibitor of the serine protease plasma kallikrein identified by phage display. By modulating the loop size of the peptide macrocycles, inhibitors with sub-nanomolar potency and an optimal specificity profile could be generated. For more details, see the Communication by Greg Winter, John Tite, Christian Heinis et al. on p. 1173 ff.

Picky push-bikes! Bicyclic peptides with low to sub-nanomolar inhibitory activities towards the serine protease plasma kallikrein were developed. By modulating the size of the macrocyclic rings, inhibitors with the desired specificity profile could be generated.

Conference Abstract| June 01 2001 Phosphoinositide 3-kinasey signalling: a structural perspective R. L. Williams; R. L. Williams 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB22QH, UK Search for other works by this author on: This Site PubMed Google Scholar M. E. Pacold; M. E. Pacold 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB22QH, UK Search for other works by this author on: This Site PubMed Google Scholar E. H. Walker; E. H. Walker 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB22QH, UK Search for other works by this author on: This Site PubMed Google Scholar O. Perisic; O. Perisic 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB22QH, UK Search for other works by this author on: This Site PubMed Google Scholar S. Suire; S. Suire 2The Babraham Institute, Babraham, Cambridge CB24AT, UK Search for other works by this author on: This Site PubMed Google Scholar L. Stephens; L. Stephens 2The Babraham Institute, Babraham, Cambridge CB24AT, UK Search for other works by this author on: This Site PubMed Google Scholar J. F. Eccleston J. F. Eccleston 3NIMR, The Ridgeway, Mill Hill, London NW7 1AA, UK Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2001) 29 (3): A46. https://doi.org/10.1042/bst029a046c Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation R. L. Williams, M. E. Pacold, E. H. Walker, O. Perisic, S. Suire, L. Stephens, J. F. Eccleston; Phosphoinositide 3-kinasey signalling: a structural perspective. Biochem Soc Trans 1 June 2001; 29 (3): A46. doi: https://doi.org/10.1042/bst029a046c Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2001 Biochemical Society2001 Article PDF first page preview Close Modal You do not currently have access to this content.

The specific phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kgamma bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.

Morphine dehydrogenase (MDH) of Pseudomonas putida M10 catalyses the NADP(+)-dependent oxidation of morphine and codeine to morphinone and codeinone. This enzyme forms the basis of a sensitive detection and assay method for heroin metabolites and a biotransformation process for production of hydromorphone and hydrocodone. To improve these processes we have undertaken a thorough examination of the kinetic mechanism of MDH. Sequence comparisons indicated that MDH belongs within the aldose reductase enzyme family. MDH was shown to be specific for the pro-R hydrogen of NADPH. In steady-state kinetic studies, product inhibition patterns suggested that MDH follows a Theorell-Chance mechanism for codeinone reduction at pH 7, and a non-Theorell-Chance sequential ordered mechanism for codeine oxidation at pH 9.5. Residues corresponding to the catalytically important Tyr-48, Lys-77 and Asp-43 of aldose reductase were modified by site-directed mutagenesis, resulting in substantial loss of activity consistent with a catalytic role for these residues. Loss of activity of MDH in the presence of the reaction product morphinone was found to be due to the formation of a covalent adduct with Cys-80; alteration of Cys-80 to serine resulted in an enzyme with greatly enhanced stability.

ABSTRACT We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP + -dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.

Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that function both as signal transducers downstream of cell-surface receptors and in constitutive intracellular membrane and protein trafficking pathways. All PI3Ks are dual-specificity enzymes with a lipid kinase activity which phosphorylates phosphoinositides at the 3-hydroxyl, and a protein kinase activity. The products of PI3K-catalysed reactions, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), PtdIns(3,4)P2 and PtdIns(3)P, are second messengers in a variety of signal transduction pathways, including those essential to cell proliferation, adhesion, survival, cytoskeletal rearrangement and vesicle trafficking1,2. Here we report the 2.2 Å X-ray crystallographic structure of the catalytic subunit of PI3Kγ, the class I enzyme that is activated by heterotrimeric G-protein βγ subunits and Ras. PI3Kγ has a modular organization centred around a helical-domain spine, with C2 and catalytic domains positioned to interact with phospholipid membranes, and a Ras-binding domain placed against the catalytic domain where it could drive allosteric activation of the enzyme.

Recent discoveries of plant retinoblastoma (Rb) protein homologues and D-type cyclins suggest that control of the onset of cell division in plants may have stronger parallels with mammalian G1/S controls than with yeasts. In mammals, the Rb protein interacts specifically with D-type cyclins and regulates cell proliferation by binding and inhibiting E2F transcription factors. However, the developmental role of Rb in plants and its potential interaction with cell cycle regulators is unknown. We show that the maize Rb homologue ZmRb-1 is temporally and spatially regulated during maize leaf development. ZmRb-1 is highly expressed in differentiating cells, but almost undetectable in proliferating cells. In vitro, both ZmRb-1 and human Rb bind all classes of plant D-type cyclins with the involvement of a conserved N-terminal Leu-x-Cys-x-Glu (LxCxE) Rb-interaction motif. This binding is strongly reduced by mutation of the conserved Cys-470 of ZmRb-1. ZmRb-1 binds human and Drosophila E2F, and inhibits transcriptional activation of human E2F. We also show that ZmRb-1 is a good in vitro substrate for all human G1/S protein kinases. The functional conservation of proteins that control the G1/S transition in mammals and plants points to the existence of plant E2F homologues. We conclude that evolution of Rb and cyclin D proteins occurred after separation of the fungi from the higher eukaryotic lineage, but preceded the divergence of plant and animal kingdoms.