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James H Cochran - One of the best experts on this subject based on the ideXlab platform.
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Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1.
Structure (London England : 1993), 2019Co-Authors: Johannes F. Van Agthoven, James R. Kintzing, Frank V. Cochran, Hengameh Shams, José Luis Alonso, Kiavash Garakani, Brian D. Adair, Jian-ping Xiong, Mohammad R. K. Mofrad, James H CochranAbstract:Summary Targeting both integrins αVβ3 and α5β1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing Knottin 2.5F binds selectively to αVβ3 and α5β1, whereas Knottin 2.5D is αVβ3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with αVβ3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages αVβ3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.
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Structural basis of the differential binding of engineered Knottins to integrins αVβ3 and α5β1
2019Co-Authors: Johannes F. Van Agthoven, James R. Kintzing, Frank V. Cochran, Hengameh Shams, José Luis Alonso, Kiavash Garakani, Brian D. Adair, Jian-ping Xiong, Mohammad R. K. Mofrad, James H CochranAbstract:Targeting both integrins αVβ3 and α5β1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins has not been fully elucidated. RGD-containing Knottin 2.5F binds selectively to αVβ3 and α5β1, whereas Knottin 2.5D is αVβ3-specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo-proteins and in complex with αVβ3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages αVβ3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low affinity linear RGD peptides to multiple integrins, suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.
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Heterochiral Knottin Protein: Folding and Solution Structure
Biochemistry, 2017Co-Authors: Surin K. Mong, James H Cochran, Frank V. Cochran, Zachary Graziano, Yu-shan Lin, Bradley L. PenteluteAbstract:Homochirality is a general feature of biological macromolecules, and Nature includes few examples of heterochiral proteins. Herein, we report on the design, chemical synthesis, and structural characterization of heterochiral proteins possessing loops of amino acids of chirality opposite to that of the rest of a protein scaffold. Using the protein Ecballium elaterium trypsin inhibitor II, we discover that selective β-alanine substitution favors the efficient folding of our heterochiral constructs. Solution nuclear magnetic resonance spectroscopy of one such heterochiral protein reveals a homogeneous global fold. Additionally, steered molecular dynamics simulation indicate β-alanine reduces the free energy required to fold the protein. We also find these heterochiral proteins to be more resistant to proteolysis than homochiral l-proteins. This work informs the design of heterochiral protein architectures containing stretches of both d- and l-amino acids.
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Engineered Knottin peptides as diagnostics, therapeutics, and drug delivery vehicles
Current opinion in chemical biology, 2016Co-Authors: James R. Kintzing, James H CochranAbstract:Inhibitor cystine-knots, also known as Knottins, are a structural family of ultra-stable peptides with diverse functions. Knottins and related backbone-cyclized peptides called cyclotides contain three disulfide bonds connected in a particular arrangement that endows these peptides with high thermal, proteolytic, and chemical stability. Knottins have gained interest as candidates for non-invasive molecular imaging and for drug development as they can possess the pharmacological properties of small molecules and the target affinity and selectively of protein biologics. Naturally occurring Knottins are clinically approved for treating chronic pain and GI disorders. Combinatorial methods are being used to engineer Knottins that can bind to other clinically relevant targets in cancer, and inflammatory and cardiac disease. This review details recent examples of engineered Knottin peptides; their use as molecular imaging agents, therapeutics, and drug delivery vehicles; modifications that can be introduced to improve peptide folding and bioactivity; and future perspectives and challenges in the field.
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Integrin‐Targeting Knottin Peptide–Drug Conjugates Are Potent Inhibitors of Tumor Cell Proliferation
Angewandte Chemie (International ed. in English), 2016Co-Authors: Nick Cox, James R. Kintzing, Mark Smith, Gerald A. Grant, James H CochranAbstract:Antibody-drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide-drug delivery, which has the potential for increased tumor penetration and facile synthesis. We report a Knottin peptide-drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor-associated integrins. This KDC binds to tumor cells with low-nanomolar affinity, is internalized by an integrin-mediated process, releases its payload intracellularly, and is a highly potent inhibitor of brain, breast, ovarian, and pancreatic cancer cell lines. Notably, these features enable this KDC to bypass a gemcitabine-resistance mechanism found in pancreatic cancer cells. This work expands the therapeutic relevance of Knottin peptides to include targeted drug delivery, and further motivates efforts to expand the drug-conjugate toolkit to include non-antibody protein scaffolds.
Glenn F. King - One of the best experts on this subject based on the ideXlab platform.
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Tying pest insects in knots: the deployment of spider‐venom‐derived Knottins as bioinsecticides
Pest Management Science, 2019Co-Authors: Glenn F. KingAbstract:Spider venoms are complex chemical arsenals that contain a rich variety of insecticidal toxins. However, the major toxin class in many spider venoms are disulfide-rich peptides known as Knottins. The knotted three-dimensional fold of these mini-proteins provides them with exceptional chemical and thermal stability as well as resistance to proteases. In contrast with other bioinsecticides, which are often slow-acting, spider Knottins are fast-acting neurotoxins. In addition to being potently insecticidal, some Knottins have exceptional taxonomic selectivity, being lethal to key agricultural pests but innocuous to vertebrates and beneficial insects such as bees. The intrinsic oral activity of these peptides, combined with the ability of aerosolized Knottins to penetrate insect spiracles, has enabled them to be developed commercially as eco-friendly bioinsecticides. Moreover, it has been demonstrated that spider-Knottin transgenes can be used to engineer faster-acting entomopathogens and insect-resistant crops. This article is protected by copyright. All rights reserved.
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Tying pest insects in knots: the deployment of spider‐venom‐derived Knottins as bioinsecticides
Pest management science, 2019Co-Authors: Glenn F. KingAbstract:Spider venoms are complex chemical arsenals that contain a rich variety of insecticidal toxins. However, the major toxin class in many spider venoms is disulfide-rich peptides known as Knottins. The knotted three-dimensional fold of these mini-proteins provides them with exceptional chemical and thermal stability as well as resistance to proteases. In contrast with other bioinsecticides, which are often slow-acting, spider Knottins are fast-acting neurotoxins. In addition to being potently insecticidal, some Knottins have exceptional taxonomic selectivity, being lethal to key agricultural pests but innocuous to vertebrates and beneficial insects such as bees. The intrinsic oral activity of these peptides, combined with the ability of aerosolized Knottins to penetrate insect spiracles, has enabled them to be developed commercially as eco-friendly bioinsecticides. Moreover, it has been demonstrated that spider-Knottin transgenes can be used to engineer faster-acting entomopathogens and insect-resistant crops. © 2019 Society of Chemical Industry.
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Efficient enzymatic ligation of inhibitor cystine knot spider venom peptides: using sortase a to form double-Knottins that probe voltage-gated sodium channel NaV1.7
Bioconjugate chemistry, 2018Co-Authors: Akello J. Agwa, Glenn F. King, Linda V. Blomster, David J. Craik, Christina I. SchroederAbstract:Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These Knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-Knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-Knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent Knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-Knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent Knottins can cooperatively modulate NaV1.7. We anticipate that double-Knottins will provid...
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the insecticidal spider toxin sfi1 is a Knottin peptide that blocks the pore of insect voltage gated sodium channels via a large β hairpin loop
FEBS Journal, 2015Co-Authors: Niraj S Bende, Glenn F. King, Volker Herzig, Graham M. Nicholson, Slawomir Dziemborowicz, Venkatraman Ramanujam, Geoffrey W Brown, Frank Bosmans, Mehdi MobliAbstract:Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a Knottin peptide with an unusually large β-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in Knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker.
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A proteomics and transcriptomics investigation of the venom from the barychelid spider Trittame loki (brush-foot trapdoor).
Toxins, 2013Co-Authors: Eivind A. B. Undheim, Glenn F. King, Volker Herzig, Kartik Sunagar, Laurence Kely, Dolyce H.w. Low, Timothy N. W. Jackson, Alun Jones, Nyoman D. Kurniawan, Syed Abid AliAbstract:Although known for their potent venom and ability to prey upon both invertebrate and vertebrate species, the Barychelidae spider family has been entirely neglected by toxinologists. In striking contrast, the sister family Theraphosidae (commonly known as tarantulas), which last shared a most recent common ancestor with Barychelidae over 200 million years ago, has received much attention, accounting for 25% of all the described spider toxins while representing only 2% of all spider species. In this study, we evaluated for the first time the venom arsenal of a barychelid spider, Trittame loki, using transcriptomic, proteomic, and bioinformatic methods. The venom was revealed to be dominated by extremely diverse inhibitor cystine knot (ICK)/Knottin peptides, accounting for 42 of the 46 full-length toxin precursors recovered in the transcriptomic sequencing. In addition to documenting differential rates of evolution adopted by different ICK/Knottin toxin lineages, we discovered homologues with completely novel cysteine skeletal architecture. Moreover, acetylcholinesterase and neprilysin were revealed for the first time as part of the spider-venom arsenal and CAP (CRiSP/Allergen/PR-1) were identified for the first time in mygalomorph spider venoms. These results not only highlight the extent of venom diversification in this neglected ancient spider lineage, but also reinforce the idea that unique venomous lineages are rich pools of novel biomolecules that may have significant applied uses as therapeutics and/or insecticides.
Zhen Cheng - One of the best experts on this subject based on the ideXlab platform.
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Tumor imaging with a radiofluorinated divalent Knottin
The Journal of Nuclear Medicine, 2014Co-Authors: Lei Jiang, Zheng Miao, Richard H. Kimura, Sanjiv S. Gambhir, Bin Shen, Frederick T. Chin, Hongcheng Shi, Zhen ChengAbstract:1084 Objectives A divalent Knottin 3-4A containing two separate integrin binding epitopes (RGD) in the adjacent loops was recently discovered in our previous report.Here,3-4A was radiofluorinated with a 18F-fluoropropionate (NFP) group,and was evaluated as a novel imaging probe to detect integrin αvβ3 positive tumors in living animals. Methods Knottin 3-4A was synthesized by solid phase peptide synthesis,folded and site-specifically conjugated with 4-nitrophenyl 2-18/19F-NFP to produce the fluorinated peptide 18/19F-FP-3-4A.The stability of 18F-FP-3-4A was tested in PBS buffer and mouse serum.Cell uptake assay was performed using U87MG cell lines.microPET imaging and biodistribution studies were performed in U87MG tumor-bearing mice. Results 18F-FP-3-4A exhibited excellent stability in PBS buffer and mouse serum.microPET imaging and biodistribution data revealed that 18F-FP-3-4A exhibited rapid and good tumor uptake (3.76±0.59%ID/g at 0.5h),and rapidly clear from normal tissues(liver:0.68±0.12%ID/g at 0.5h),resulting in excellent tumor-to-normal tissue contrasts.Moreover,co-injection of 18F-FP-3-4A with c(RGDyK) significantly inhibited tumor uptake,demonstrating binding specificity. Conclusions 18F-FP-3-4A is characterized by rapid and high tumor uptake,and excellent tumor-to-normal tissue ratios. It is a highly promising Knottin based PET probe for translating into clinical imaging of tumor angiogenesis. Research Support Office of Science (BER), U.S. Department of Energy (DE-SC0008397), NCI In Vivo Cellular Molecular Imaging Center (ICMIC) grant P50 CA114747, National Science Foundation for Young Scholars of China (Grant No. 81101072).
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A radiofluorinated divalent cystine knot peptide for tumor PET imaging.
Molecular pharmaceutics, 2014Co-Authors: Lei Jiang, Zheng Miao, Richard H. Kimura, Sanjiv S. Gambhir, Bin Shen, Frederick T. Chin, Hongcheng Shi, Zhen ChengAbstract:A divalent Knottin containing two separate integrin binding epitopes (RGD) in the adjacent loops, 3-4A, was recently developed and reported in our previous publication. In the current study, 3-4A was radiofluorinated with a 4-nitrophenyl 2-18F-fluoropropinate (18F-NFP) group and the resulting divalent positron emission tomography (PET) probe, 18F-FP–3-4A, was evaluated as a novel imaging probe to detect integrin αvβ3 positive tumors in living animals. Knottin 3-4A was synthesized by solid phase peptide synthesis, folded, and site-specifically conjugated with 18/19F-NFP to produce the fluorinated peptide 18/19F-fluoropropinate-3-4A (18/19F-FP–3-4A). The stability of 18F-FP–3-4A was tested in both phosphate buffered saline (PBS) buffer and mouse serum. Cell uptake assays of the radiolabeled peptides were performed using U87MG cells. In addition, small animal PET imaging and biodistribution studies of 18F-FP–3-4A were performed in U87MG tumor-bearing mice. The receptor targeting specificity of the radiolabel...
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PET Imaging of Integrin Positive Tumors Using 18F Labeled Knottin Peptides
Theranostics, 2011Co-Authors: Shuanglong Liu, James H Cochran, Gang Ren, Hongguang Liu, Richard H. Kimura, Zhen ChengAbstract:Cystine knot (Knottin) peptides, engineered to bind with high affinity to integrin receptors, have shown promise as molecular imaging agents in living subjects. The aim of the current study was to evaluate tumor uptake and in vivo biodistribution of (18)F-labeled Knottins in a U87MG glioblastoma model. Engineered Knottin mutants 2.5D and 2.5F were synthesized using solid phase peptide synthesis and were folded in vitro, followed by radiolabeling with 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP). The resulting probes, (18)F-FP-2.5D and (18)F-FP-2.5F, were evaluated in nude mice bearing U87MG tumor xenografts using microPET and biodistribution studies. MicroPET imaging studies with (18)F-FP-2.5D and (18)F-FP-2.5F demonstrated high tumor uptake in U87MG xenograft mouse models. The probes exhibited rapid clearance from the blood and kidneys, thus leading to excellent tumor-to-normal tissue contrast. Specificity studies confirmed that (18)F-FP-2.5D and (18)F-FP-2.5F had reduced tumor uptake when co-injected with a large excess of the peptidomimetic c(RGDyK) as a blocking agent. (18)F-FP-2.5D and (18)F-FP-2.5F showed reduced gallbladder uptake compared with previously published (18)F-FB-2.5D. (18)F-FP-2.5D and (18)F-FP-2.5F enabled integrin-specific PET imaging of U87MG tumors with good imaging contrasts. (18)F-FP-2.5D demonstrated more desirable pharmacokinetics compared to (18)F-FP-2.5F, and thus has greater potential for clinical translation.
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Functional Mutation of Multiple Solvent-Exposed Loops in the Ecballium elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein
PloS one, 2011Co-Authors: Richard H. Kimura, Zheng Miao, Lei Jiang, Zhen Cheng, Douglas Jones, James H CochranAbstract:BACKGROUND The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the Knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops. METHODOLOGY/PRINCIPAL FINDINGS Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting Knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This Knottin peptide bound to α(v)β(3) and α(v)β(5) integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α(5)β(1) and α(iib)β(3). In addition, the engineered Knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α(v)β(3) and α(v)β(5) integrins. A (64)Cu radiolabeled version of this Knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3-5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys. CONCLUSIONS/SIGNIFICANCE We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting Knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this Knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II Knottin peptide containing only one mutated loop.
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pet imaging of integrin positive tumors using 18f labeled Knottin peptides
Theranostics, 2011Co-Authors: Shuanglong Liu, J Cochran, Gang Ren, Hongguang Liu, Richard H. Kimura, Zhen ChengAbstract:Purpose: Cystine knot (Knottin) peptides, engineered to bind with high affinity to integrin receptors, have shown promise as molecular imaging agents in living subjects. The aim of the current study was to evaluate tumor uptake and in vivo biodistribution of 18F-labeled Knottins in a U87MG glioblastoma model. Procedures: Engineered Knottin mutants 2.5D and 2.5F were synthesized using solid phase peptide synthesis and were folded in vitro, followed by radiolabeling with 4-nitrophenyl 2-18F-fluoropropionate (18F-NFP). The resulting probes, 18F-FP-2.5D and 18F-FP-2.5F, were evaluated in nude mice bearing U87MG tumor xenografts using microPET and biodistribution studies. Results: MicroPET imaging studies with 18F-FP-2.5D and 18F-FP-2.5F demonstrated high tumor uptake in U87MG xenograft mouse models. The probes exhibited rapid clearance from the blood and kidneys, thus leading to excellent tumor-to-normal tissue contrast. Specificity studies confirmed that 18F-FP-2.5D and 18F-FP-2.5F had reduced tumor uptake when co-injected with a large excess of the peptidomimetic c(RGDyK) as a blocking agent. Conclusions: 18F-FP-2.5D and 18F-FP-2.5F showed reduced gallbladder uptake compared with previously published 18F-FB-2.5D. 18F-FP-2.5D and 18F-FP-2.5F enabled integrin-specific PET imaging of U87MG tumors with good imaging contrasts. 18F-FP-2.5D demonstrated more desirable pharmacokinetics compared to 18F-FP-2.5F, and thus has greater potential for clinical translation.
Laurent Chiche - One of the best experts on this subject based on the ideXlab platform.
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Knottin: the database of inhibitor cystine knot scaffold after 10 years, toward a systematic structure modeling.
Nucleic acids research, 2017Co-Authors: Guillaume Postic, Laurent Chiche, Jérôme Gracy, Charlotte Périn, Jean-christophe GellyAbstract:Knottins, or inhibitor cystine knots (ICKs), are ultra-stable miniproteins with multiple applications in drug design and medical imaging. These widespread and functionally diverse proteins are characterized by the presence of three interwoven disulfide bridges in their structure, which form a unique pseudoknot. Since 2004, the Knottin database (www.dsimb.inserm.fr/Knottin/) has been gathering standardized information about Knottin sequences, structures, functions and evolution. The website also provides access to bibliographic data and to computational tools that have been specifically developed for ICKs. Here, we present a major upgrade of our database, both in terms of data content and user interface. In addition to the new features, this article describes how Knottin has seen its size multiplied over the past ten years (since its last publication), notably with the recent inclusion of predicted ICKs structures. Finally, we report how our web resource has proved usefulness for the researchers working on ICKs, and how the new version of the Knottin website will continue to serve this active community.
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Structure and modeling of Knottins, a promising molecular scaffold for drug discovery.
Current pharmaceutical design, 2011Co-Authors: Jérôme Gracy, Laurent ChicheAbstract:The Knottins are extremely stable miniproteins present in many species and are able to perform various tasks. Owing to its small size and its amazing stability, the Knottin structural domain is considered as an excellent scaffold for drug development. Several recent databases and web servers dedicated to or aware of Knottins have appeared and are shortly described. Altogether they provide a valuable ensemble of data and of specific tools that greatly facilitate Knottin-based studies. The essential structural features of the Knottin scaffold, which heavily rest on the three knotted disulfide bridges for its stability, are reviewed. These include small but well-conserved secondary structures and hydrogen bonding networks, but also several further interactions that have been shown to be essential for stability and/or activity. Examples are supplementary disulfide bridges, side chain hydrogen bonds, or circularization. General structure prediction and modeling tools are not well fitted to Knottins, and several specific tools have been developed. Specifically, methods to assign a disulfide connectivity pattern to small disulfide-rich sequences or to build accurate 3D models of Knottins are available and are discussed in the review. Although more works are still needed to better understand sequence-structure-function relationships, recent studies strongly suggest that existing applications of Knottins as drugs (i.e. painkillers), molecules for diagnosis, or insecticidal crop treatment should rapidly generalize and extend to other fields as well, e.g. as antimicrobials.
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Optimizing structural modeling for a specific protein scaffold: Knottins or inhibitor cystine knots
BMC bioinformatics, 2010Co-Authors: Jérôme Gracy, Laurent ChicheAbstract:Background Knottins are small, diverse and stable proteins with important drug design potential. They can be classified in 30 families which cover a wide range of sequences (1621 sequenced), three-dimensional structures (155 solved) and functions (> 10). Inter Knottin similarity lies mainly between 15% and 40% sequence identity and 1.5 to 4.5 A backbone deviations although they all share a tightly knotted disulfide core. This important variability is likely to arise from the highly diverse loops which connect the successive knotted cysteines. The prediction of structural models for all Knottin sequences would open new directions for the analysis of interaction sites and to provide a better understanding of the structural and functional organization of proteins sharing this scaffold.
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Optimizing structural modeling for a specific protein scaffold: Knottins or inhibitor cystine knots
BMC Bioinformatics, 2010Co-Authors: Jérôme Gracy, Laurent ChicheAbstract:Background: Knottins are small, diverse and stable proteins with important drug design potential. They can be classified in 30 families which cover a wide range of sequences (1621 sequenced), three-dimensional structures (155 solved) and functions (> 10). Inter Knottin similarity lies mainly between 15% and 40% sequence identity and 1.5 to 4.5 Å backbone deviations although they all share a tightly knotted disulfide core. This important variability is likely to arise from the highly diverse loops which connect the successive knotted cysteines. The prediction of structural models for all Knottin sequences would open new directions for the analysis of interaction sites and to provide a better understanding of the structural and functional organization of proteins sharing this scaffold. Results: We have designed an automated modeling procedure for predicting the three-dimensionnal structure of Knottins. The different steps of the homology modeling pipeline were carefully optimized relatively to a test set of Knottins with known structures: template selection and alignment, extraction of structural constraints and model building, model evaluation and refinement. After optimization, the accuracy of predicted models was shown to lie between 1.50 and 1.96 Å from native structures at 50% and 10% maximum sequence identity levels, respectively. These average model deviations represent an improvement varying between 0.74 and 1.17 Å over a basic homology modeling derived from a unique template. A database of 1621 structural models for all known Knottin sequences was generated and is freely accessible from our web server at http://Knottin.cbs.cnrs.fr. Models can also be interactively constructed from any Knottin sequence using the structure prediction module Knoter1D3D available from our protein analysis toolkit PAT at http://pat.cbs.cnrs.fr.
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Knottin cyclization: impact on structure and dynamics.
BMC structural biology, 2008Co-Authors: Annie Heitz, Jérôme Gracy, Dung Le-nguyen, Olga Avrutina, Ulf Diederichsen, Jean-françois Hernandez, Harald Kolmar, Laurent ChicheAbstract:Background Present in various species, the Knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, Knottins are considered as excellent leads or scaffolds in drug design. Two Knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack.
Richard H. Kimura - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of integrin αvβ6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.
Nature communications, 2019Co-Authors: Richard H. Kimura, Ling Wang, Bin Shen, Li Huo, Willemieke S. Tummers, Fabian V. Filipp, Haiwei H. Guo, Thomas Haywood, Lotfi Abou-elkacem, Lucia BarattoAbstract:Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (Knottin) that selectively recognizes human integrin αvβ6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the Knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvβ6.
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Tumor imaging with a radiofluorinated divalent Knottin
The Journal of Nuclear Medicine, 2014Co-Authors: Lei Jiang, Zheng Miao, Richard H. Kimura, Sanjiv S. Gambhir, Bin Shen, Frederick T. Chin, Hongcheng Shi, Zhen ChengAbstract:1084 Objectives A divalent Knottin 3-4A containing two separate integrin binding epitopes (RGD) in the adjacent loops was recently discovered in our previous report.Here,3-4A was radiofluorinated with a 18F-fluoropropionate (NFP) group,and was evaluated as a novel imaging probe to detect integrin αvβ3 positive tumors in living animals. Methods Knottin 3-4A was synthesized by solid phase peptide synthesis,folded and site-specifically conjugated with 4-nitrophenyl 2-18/19F-NFP to produce the fluorinated peptide 18/19F-FP-3-4A.The stability of 18F-FP-3-4A was tested in PBS buffer and mouse serum.Cell uptake assay was performed using U87MG cell lines.microPET imaging and biodistribution studies were performed in U87MG tumor-bearing mice. Results 18F-FP-3-4A exhibited excellent stability in PBS buffer and mouse serum.microPET imaging and biodistribution data revealed that 18F-FP-3-4A exhibited rapid and good tumor uptake (3.76±0.59%ID/g at 0.5h),and rapidly clear from normal tissues(liver:0.68±0.12%ID/g at 0.5h),resulting in excellent tumor-to-normal tissue contrasts.Moreover,co-injection of 18F-FP-3-4A with c(RGDyK) significantly inhibited tumor uptake,demonstrating binding specificity. Conclusions 18F-FP-3-4A is characterized by rapid and high tumor uptake,and excellent tumor-to-normal tissue ratios. It is a highly promising Knottin based PET probe for translating into clinical imaging of tumor angiogenesis. Research Support Office of Science (BER), U.S. Department of Energy (DE-SC0008397), NCI In Vivo Cellular Molecular Imaging Center (ICMIC) grant P50 CA114747, National Science Foundation for Young Scholars of China (Grant No. 81101072).
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A radiofluorinated divalent cystine knot peptide for tumor PET imaging.
Molecular pharmaceutics, 2014Co-Authors: Lei Jiang, Zheng Miao, Richard H. Kimura, Sanjiv S. Gambhir, Bin Shen, Frederick T. Chin, Hongcheng Shi, Zhen ChengAbstract:A divalent Knottin containing two separate integrin binding epitopes (RGD) in the adjacent loops, 3-4A, was recently developed and reported in our previous publication. In the current study, 3-4A was radiofluorinated with a 4-nitrophenyl 2-18F-fluoropropinate (18F-NFP) group and the resulting divalent positron emission tomography (PET) probe, 18F-FP–3-4A, was evaluated as a novel imaging probe to detect integrin αvβ3 positive tumors in living animals. Knottin 3-4A was synthesized by solid phase peptide synthesis, folded, and site-specifically conjugated with 18/19F-NFP to produce the fluorinated peptide 18/19F-fluoropropinate-3-4A (18/19F-FP–3-4A). The stability of 18F-FP–3-4A was tested in both phosphate buffered saline (PBS) buffer and mouse serum. Cell uptake assays of the radiolabeled peptides were performed using U87MG cells. In addition, small animal PET imaging and biodistribution studies of 18F-FP–3-4A were performed in U87MG tumor-bearing mice. The receptor targeting specificity of the radiolabel...
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PET Imaging of Integrin Positive Tumors Using 18F Labeled Knottin Peptides
Theranostics, 2011Co-Authors: Shuanglong Liu, James H Cochran, Gang Ren, Hongguang Liu, Richard H. Kimura, Zhen ChengAbstract:Cystine knot (Knottin) peptides, engineered to bind with high affinity to integrin receptors, have shown promise as molecular imaging agents in living subjects. The aim of the current study was to evaluate tumor uptake and in vivo biodistribution of (18)F-labeled Knottins in a U87MG glioblastoma model. Engineered Knottin mutants 2.5D and 2.5F were synthesized using solid phase peptide synthesis and were folded in vitro, followed by radiolabeling with 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP). The resulting probes, (18)F-FP-2.5D and (18)F-FP-2.5F, were evaluated in nude mice bearing U87MG tumor xenografts using microPET and biodistribution studies. MicroPET imaging studies with (18)F-FP-2.5D and (18)F-FP-2.5F demonstrated high tumor uptake in U87MG xenograft mouse models. The probes exhibited rapid clearance from the blood and kidneys, thus leading to excellent tumor-to-normal tissue contrast. Specificity studies confirmed that (18)F-FP-2.5D and (18)F-FP-2.5F had reduced tumor uptake when co-injected with a large excess of the peptidomimetic c(RGDyK) as a blocking agent. (18)F-FP-2.5D and (18)F-FP-2.5F showed reduced gallbladder uptake compared with previously published (18)F-FB-2.5D. (18)F-FP-2.5D and (18)F-FP-2.5F enabled integrin-specific PET imaging of U87MG tumors with good imaging contrasts. (18)F-FP-2.5D demonstrated more desirable pharmacokinetics compared to (18)F-FP-2.5F, and thus has greater potential for clinical translation.
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Functional Mutation of Multiple Solvent-Exposed Loops in the Ecballium elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein
PloS one, 2011Co-Authors: Richard H. Kimura, Zheng Miao, Lei Jiang, Zhen Cheng, Douglas Jones, James H CochranAbstract:BACKGROUND The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the Knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops. METHODOLOGY/PRINCIPAL FINDINGS Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting Knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This Knottin peptide bound to α(v)β(3) and α(v)β(5) integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α(5)β(1) and α(iib)β(3). In addition, the engineered Knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α(v)β(3) and α(v)β(5) integrins. A (64)Cu radiolabeled version of this Knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3-5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys. CONCLUSIONS/SIGNIFICANCE We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting Knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this Knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II Knottin peptide containing only one mutated loop.
