127 results on '"Durek, Thomas"'
Search Results
102. The tarantula toxin β/δ-TRTX-Pre1a highlights the importance of the S1-S2 voltage-sensor region for sodium channel subtype selectivity
- Author
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Wingerd, Joshua S., Mozar, Christine A., Ussing, Christine A., Murali, Swetha S., Chin, Yanni K.-Y., Cristofori-Armstrong, Ben, Durek, Thomas, Gilchrist, John, Vaughan, Christopher W., Bosmans, Frank, Adams, David J., Lewis, Richard J., Alewood, Paul F., Mobli, Mehdi, Christie, Macdonald J., and Rash, Lachlan D.
- Abstract
Voltage-gated sodium (NaV) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal NaV channels inhibiting peak current of hNaV1.1, rNaV1.2, hNaV1.6, and hNaV1.7 while concurrently inhibiting fast inactivation of hNaV1.1 and rNaV1.3. The DII and DIV S3-S4 loops of NaV channel voltage sensors are important for the interaction of Pre1a with NaV channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between NaV channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a’s potent and selective inhibitory effect on the fast inactivation process of NaV1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of NaV channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.
- Published
- 2017
- Full Text
- View/download PDF
103. In Vitro Semisynthesis and Applications of C-Terminally Modified Rab Proteins.
- Author
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Walker, John M., Niemeyer, Christof M., Durek, Thomas, Goody, Roger S., and Alexandrov, Kirill
- Abstract
Expressed protein ligation is a powerful tool for the generation of natively folded proteins composed of recombinantly generated and chemically synthesized polypeptides. Using this approach, we developed protocols for the production of prenylated and/or otherwise-labeled Rab GTPase. The protocols are generally applicable to most small GTPases that can be supplied with a variety of new chemical functionalities. We used semisynthetic fluorescently labeled Rab7 GTPase as a molecular probe to study protein-protein interactions with components of the prenylation machinery [ABSTRACT FROM AUTHOR]
- Published
- 2004
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104. Nicotiana alataDefensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant
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Bleackley, Mark R., Payne, Jennifer A. E., Hayes, Brigitte M. E., Durek, Thomas, Craik, David J., Shafee, Thomas M. A., Poon, Ivan K. H., Hulett, Mark D., van der Weerden, Nicole L., and Anderson, Marilyn A.
- Abstract
ABSTRACTThe plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-β motif which consists of an α helix and a triple-stranded β-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.
- Published
- 2016
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105. Isolation and Structural and Pharmacological Characterization of α-Elapitoxin-Dpp2d, an Amidated Three Finger Toxin from Black Mamba Venom.
- Author
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Anderson Wang, Ching-I., Reeks, Timothy, Vetter, Irina, Vergara, Irene, Kovtun, Oleksiy, Lewis, Richard J., Alewood, Paul F., and Durek, Thomas
- Published
- 2014
- Full Text
- View/download PDF
106. Chemical Synthesis, 3D Structure, and ASIC Binding Site of the Toxin Mambalgin-2.
- Author
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Schroeder, Christina I., Rash, Lachlan D., Vila‐Farrés, Xavier, Rosengren, K. Johan, Mobli, Mehdi, King, Glenn F., Alewood, Paul F., Craik, David J., and Durek, Thomas
- Subjects
THERAPEUTIC use of venom ,SNAKE venom ,SPIDER venom ,TOXIN analysis ,ANALGESIC synthesis ,ACID-sensing ion channels ,POLYPEPTIDES - Abstract
Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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107. Efficient chemical synthesis of human complement protein C3a.
- Author
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GhassemianThese authors contributed equally., Artin, Wang, Ching-I Anderson, Yau, Mei-Kwan, Reid, Robert C., Lewis, Richard J., Fairlie, David P., Alewood, Paul F., and Durek, Thomas
- Subjects
CRYSTAL structure ,POLYTYPIC transformations ,CRYSTALLOGRAPHY ,CONDENSED matter physics ,CHEMICAL synthesis - Abstract
We report the total chemical synthesis of human C3a by one-pot native chemical ligation of three unprotected peptide segments, followed by efficient in vitro folding that yielded the anaphylatoxin C3a in high yield and excellent purity. Synthetic C3a was fully active and its crystal structure at 2.1 Å resolution showed 3 helices and a C-terminal turn motif. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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108. Application of Protein Semisynthesis for the Construction of Functionalized Posttranslationally Modified Rab GTPases.
- Author
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Goody, Roger S., Durek, Thomas, Waldmann, Herbert, Brunsveld, Lucas, and Alexandrov, Kirill
- Abstract
An abstract of the article "Application of Protein Semisynthesis for the Construction of Functionalized Posttranslationally Modified Rab GTPases," by Roger S. Goody and others is presented.
- Published
- 2005
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109. Neurotoxic and cytotoxic peptides underlie the painful stings of the tree nettle Urtica ferox.
- Author
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Jing Xie, Robinson, Samuel D., Gilding, Edward K., Jami, Sina, Deuis, Jennifer R., Rehm, Fabian B. H., Yap, Kuok, Ragnarsson, Lotten, Lai Yue Chan, Hamilton, Brett R., Harvey, Peta J., Craik, David J., Vetter, Irina, and Durek, Thomas
- Subjects
- *
STINGING nettle , *PEPTIDES , *SODIUM channels , *ACTIVATION energy , *CELL membranes , *TOXINS - Abstract
The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while β/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that β/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
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110. Isolation, synthesis and characterization of ω-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type CaV channels.
- Author
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Klint, Julie K., Berecki, Géza, Durek, Thomas, Mobli, Mehdi, Knapp, Oliver, King, Glenn F., Adams, David J., Alewood, Paul F., and Rash, Lachlan D.
- Subjects
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SPIDER venom , *TARANTULAS , *CALCIUM channels regulation , *VENOM , *CHEMICAL synthesis , *PROTEIN expression , *PHARMACOLOGY - Abstract
Abstract: Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba2+ currents (I Ba) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825nM and 2.24μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited I Ba mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1–E5) or C-terminus (ΔW35–V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels. [Copyright &y& Elsevier]
- Published
- 2014
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111. Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells
- Author
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Vetter, Irina, Mozar, Christine A., Durek, Thomas, Wingerd, Joshua S., Alewood, Paul F., Christie, Macdonald J., and Lewis, Richard J.
- Subjects
- *
NEUROBLASTOMA , *CANCER cells , *CELL lines , *SODIUM channels , *GENE expression , *SERUM albumin , *CALCIUM ions - Abstract
Abstract: The human neuroblastoma cell line SH-SY5Y is a potentially useful model for the identification and characterisation of Nav modulators, but little is known about the pharmacology of their endogenously expressed Navs. The aim of this study was to determine the expression of endogenous Nav α and β subunits in SH-SY5Y cells using PCR and immunohistochemical approaches, and pharmacologically characterise the Nav isoforms endogenously expressed in this cell line using electrophysiological and fluorescence approaches. SH-SY5Y human neuroblastoma cells were found to endogenously express several Nav isoforms including Nav1.2 and Nav1.7. Activation of endogenously expressed Navs with veratridine or the scorpion toxin OD1 caused membrane depolarisation and subsequent Ca2+ influx through voltage-gated L- and N-type calcium channels, allowing Nav activation to be detected with membrane potential and fluorescent Ca2 dyes. μ-Conotoxin TIIIA and ProTxII identified Nav1.2 and Nav1.7 as the major contributors of this response. The Nav1.7-selective scorpion toxin OD1 in combination with veratridine produced a Nav1.7-selective response, confirming that endogenously expressed human Nav1.7 in SH-SY5Y cells is functional and can be synergistically activated, providing a new assay format for ligand screening. [Copyright &y& Elsevier]
- Published
- 2012
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112. Convergent synthesis of proteins by kinetically controlled ligation
- Author
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Durek, Thomas [Chicago, IL]
- Published
- 2010
113. A Chemoenzymatic Approach To Produce a Cyclic Analogue of the Analgesic Drug MVIIA (Ziconotide).
- Author
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Zhou Y, Harvey PJ, Koehbach J, Chan LY, Jones A, Andersson Å, Vetter I, Durek T, and Craik DJ
- Subjects
- Humans, Analgesics pharmacology, Analgesics therapeutic use, Calcium Channels chemistry, Calcium Channel Blockers pharmacology, omega-Conotoxins pharmacology, omega-Conotoxins therapeutic use, Conotoxins pharmacology
- Abstract
Ziconotide (ω-conotoxin MVIIA) is an approved analgesic for the treatment of chronic pain. However, the need for intrathecal administration and adverse effects have limited its widespread application. Backbone cyclization is one way to improve the pharmaceutical properties of conopeptides, but so far chemical synthesis alone has been unable to produce correctly folded and backbone cyclic analogues of MVIIA. In this study, an asparaginyl endopeptidase (AEP)-mediated cyclization was used to generate backbone cyclic analogues of MVIIA for the first time. Cyclization using six- to nine-residue linkers did not perturb the overall structure of MVIIA, and the cyclic analogues of MVIIA showed inhibition of voltage-gated calcium channels (Ca
V 2.2) and substantially improved stability in human serum and stimulated intestinal fluid. Our study reveals that AEP transpeptidases are capable of cyclizing structurally complex peptides that chemical synthesis cannot achieve and paves the way for further improving the therapeutic value of conotoxins., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)- Published
- 2023
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114. Low potency inhibition of Na V 1.7 by externally applied QX-314 via a depolarizing shift in the voltage-dependence of activation.
- Author
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Klasfauseweh T, Israel MR, Ragnarsson L, Cox JJ, Durek T, Carter DA, Leffler A, Vetter I, and Deuis JR
- Subjects
- HEK293 Cells, Humans, Sodium metabolism, Sodium Channel Blockers pharmacology, Anesthetics, Local pharmacology, Lidocaine analogs & derivatives, Lidocaine pharmacology
- Abstract
QX-314 is a quaternary permanently charged lidocaine derivative that inhibits voltage-gated sodium channels (Na
V ). As it is membrane impermeable, it is generally considered that QX-314 applied externally is inactive, unless it can gain access to the internal local anesthetic binding site via another entry pathway. Here, we characterized the electrophysiological effects of QX-314 on NaV 1.7 heterologously expressed in HEK293 cells, and found that at high concentrations, external QX-314 inhibited NaV 1.7 current (IC50 2.0 ± 0.3 mM) and shifted the voltage-dependence to more depolarized potentials (ΔV50 +10.6 mV). Unlike lidocaine, the activity of external QX-314 was not state- or use-dependent. The effect of externally applied QX-314 on NaV 1.7 channel biophysics differed to that of internally applied QX-314, suggesting QX-314 has an additional externally accessible site of action. In line with this hypothesis, disruption of the local anesthetic binding site in a [F1748A]NaV 1.7 mutant reduced the potency of lidocaine by 40-fold, but had no effect on the potency or activity of externally applied QX-314. Therefore, we conclude, using an expression system where QX-314 was unable to cross the membrane, that externally applied QX-314 is able to inhibit NaV 1.7 peak current at low millimolar concentrations., (Copyright © 2022 Elsevier B.V. All rights reserved.)- Published
- 2022
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115. Cystine Knot Peptides with Tuneable Activity and Mechanism.
- Author
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Li CY, Rehm FBH, Yap K, Zdenek CN, Harding MD, Fry BG, Durek T, Craik DJ, and de Veer SJ
- Subjects
- Cystine, Humans, Peptides metabolism, Peptides pharmacology, Proteins, Streptavidin, Cystine-Knot Miniproteins metabolism
- Abstract
Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)
- Published
- 2022
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116. Enzymatic C-to-C Protein Ligation.
- Author
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Rehm FBH, Tyler TJ, de Veer SJ, Craik DJ, and Durek T
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- Amino Acids chemistry, Biocatalysis, Cysteine Endopeptidases chemistry, Protein Engineering, Amino Acids biosynthesis, Cysteine Endopeptidases metabolism
- Abstract
Transpeptidase-catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N-to-C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l-/d- or α-/β-amino acid junctions, we find C-terminal Leu-ethylenediamine (Leu-Eda) motifs to be bona fide mimetics of native N-terminal Gly-Leu sequences. Appending a C-terminal Leu-Eda to synthetic peptides or, via an intein-splicing approach, to recombinant proteins enables direct transpeptidase-catalyzed C-to-C ligations. This work significantly expands the synthetic scope of enzyme-catalyzed protein transpeptidation reactions., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)
- Published
- 2022
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- View/download PDF
117. Improved Asparaginyl-Ligase-Catalyzed Transpeptidation via Selective Nucleophile Quenching.
- Author
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Rehm FBH, Tyler TJ, Yap K, Durek T, and Craik DJ
- Subjects
- Amino Acid Motifs, Biocatalysis, Copper chemistry, Copper metabolism, Humans, Nickel chemistry, Nickel metabolism, Peptides chemistry, Protein Binding, Protein Engineering, Serum Albumin chemistry, Serum Albumin metabolism, Cysteine Endopeptidases metabolism, Peptides metabolism
- Abstract
The use of enzymes for the site-specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP-catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving-group peptide. Our metal-complexation-based strategy enables efficient peptide/protein labeling at the N- or C-terminus with near-equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein-protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable., (© 2020 Wiley-VCH GmbH.)
- Published
- 2021
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118. Application and Structural Analysis of Triazole-Bridged Disulfide Mimetics in Cyclic Peptides.
- Author
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White AM, de Veer SJ, Wu G, Harvey PJ, Yap K, King GJ, Swedberg JE, Wang CK, Law RHP, Durek T, and Craik DJ
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Cyclization, Nuclear Magnetic Resonance, Biomolecular, Ruthenium chemistry, Disulfides chemistry, Molecular Mimicry, Peptides, Cyclic chemistry, Triazoles chemistry
- Abstract
Ruthenium-catalysed azide-alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2020
- Full Text
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119. Characterization of Synthetic Tf2 as a Na V 1.3 Selective Pharmacological Probe.
- Author
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Israel MR, Dash TS, Bothe SN, Robinson SD, Deuis JR, Craik DJ, Lampert A, Vetter I, and Durek T
- Abstract
Na
V 1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of Tityus fasciolatus , has been reported to selectively activate NaV 1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for NaV 1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of NaV 1.3 activation in the hyperpolarizing direction by -11.3 mV, with no activity at NaV 1.1, NaV 1.2, and NaV 1.4-NaV 1.8. Additional activity was found at NaV 1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by -6.3 mV. In an attempt to convert Tf2 into an NaV 1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at NaV 1.3, although it still caused a small -5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the NaV 1.1/1.3 inhibitor ICA-121431 nor in NaV 1.9-/- mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at NaV 1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.- Published
- 2020
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120. NMR Structure of μ-Conotoxin GIIIC: Leucine 18 Induces Local Repacking of the N-Terminus Resulting in Reduced Na V Channel Potency.
- Author
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Harvey PJ, Kurniawan ND, Finol-Urdaneta RK, McArthur JR, Van Lysebetten D, Dash TS, Hill JM, Adams DJ, Durek T, and Craik DJ
- Subjects
- Amino Acid Sequence, Conotoxins chemical synthesis, Conotoxins pharmacology, Disulfides chemistry, Leucine chemistry, Models, Molecular, Peptides chemical synthesis, Peptides chemistry, Protein Conformation, Protein Interaction Domains and Motifs, Sodium Channel Blockers chemical synthesis, Sodium Channel Blockers chemistry, Sodium Channel Blockers pharmacology, Sodium Channels chemistry, Sodium Channels metabolism, Structure-Activity Relationship, Conotoxins chemistry, Magnetic Resonance Spectroscopy
- Abstract
μ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of μ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of μ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical μ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (Na
V ), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of NaV 1.4 channels, but the same NaV selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.- Published
- 2018
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121. Combining Sense and Nonsense Codon Reassignment for Site-Selective Protein Modification with Unnatural Amino Acids.
- Author
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Cui Z, Mureev S, Polinkovsky ME, Tnimov Z, Guo Z, Durek T, Jones A, and Alexandrov K
- Subjects
- Amino Acyl-tRNA Synthetases metabolism, Azides pharmacology, Calcium metabolism, Cell-Free System drug effects, Cell-Free System metabolism, Codon, Terminator genetics, Escherichia coli drug effects, Escherichia coli genetics, Escherichia coli metabolism, Eukaryota drug effects, Eukaryota genetics, Eukaryota metabolism, Phenylalanine analogs & derivatives, Phenylalanine pharmacology, Protein Biosynthesis drug effects, Protein Processing, Post-Translational drug effects, RNA, Transfer genetics, Amino Acids genetics, Codon, Nonsense genetics, Protein Biosynthesis genetics, Protein Processing, Post-Translational genetics
- Abstract
Incorporation of unnatural amino acids (uAAs) via codon reassignment is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. However, the site-selective incorporation of multiple uAAs into polypeptides is hampered by the limited number of reassignable nonsense codons. This challenge is addressed in the current work by developing Escherichia coli in vitro translation system depleted of specific endogenous tRNAs. The translational activity in this system is dependent on the addition of synthetic tRNAs for the chosen sense codon. This allows site-selective uAA incorporation via addition of tRNAs pre- or cotranslationally charged with uAA. We demonstrate the utility of this system by incorporating the BODIPY fluorophore into the unique AGG codon of the calmodulin(CaM) open reading frame using in vitro precharged BODIPY-tRNA
Cys CCU . The deacylated tRNACys CCU is a poor substrate for Cysteinyl-tRNA synthetase, which ensures low background incorporation of Cys into the chosen codon. Simultaneously, p-azidophenylalanine mediated amber-codon suppression and its post-translational conjugation to tetramethylrhodamine dibenzocyclooctyne (TAMRA-DIBO) were performed on the same polypeptide. This simple and robust approach takes advantage of the compatibility of BODIPY fluorophore with the translational machinery and thus requires only one post-translational derivatization step to introduce two fluorescent labels. Using this approach, we obtained CaM nearly homogeneously labeled with two FRET-forming fluorophores. Single molecule FRET analysis revealed dramatic changes in the conformation of the CaM probe upon its exposure to Ca2+ or a chelating agent. The presented approach is applicable to other sense codons and can be directly transferred to eukaryotic cell-free systems.- Published
- 2017
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122. Approaches to the stabilization of bioactive epitopes by grafting and peptide cyclization.
- Author
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Conibear AC, Chaousis S, Durek T, Rosengren KJ, Craik DJ, and Schroeder CI
- Subjects
- Amino Acid Sequence, Cell Line, Tumor, Cyclization, Humans, Peptides blood, Peptides chemical synthesis, Proton Magnetic Resonance Spectroscopy, Epitopes chemistry, Peptides chemistry
- Abstract
Peptides are attracting increasing interest from the pharmaceutical industry because of their specificity and ability to address novel targets, including protein-protein interactions. However, typically they require stabilization for therapeutic applications owing to their susceptibility to degradation by proteases. Advances in the ability to chemically synthesize peptides and the development of new side-chain and backbone ligation strategies provide new tools to stabilize bioactive peptide epitopes. Two such epitopes are LyP1, a nine residue peptide that localizes to tumor cells and has potential as an anticancer therapeutic, and RGDS, a tetrapeptide shown to bind to survivin and induce apoptosis. Here we applied a variety of strategies for the stabilization of LyP1 and RGDS, including side-chain cyclization using "click" chemistry and "grafting" the epitopes into two naturally occurring cyclic peptide scaffolds, i.e., θ-defensins and cyclotides. NMR data showed that the three-disulfide θ-defensin and cyclotide scaffolds accommodated the LyP1 and RGDS epitopes but that scaffolds with fewer disulfide bonds were structurally compromised by inclusion of the LyP1 epitope. LyP1, LyP1-, and RGDS-grafted peptides that were largely unstructured also had reduced resistance to degradation in human serum, showing that grafting into a stable cyclic scaffold is an effective strategy for increasing the stability of a bioactive peptide epitope. Overall, the study demonstrates several methods for stabilizing peptide epitopes using side-chain or backbone cyclization and illustrates their potential in peptide drug design., (© 2015 Wiley Periodicals, Inc.)
- Published
- 2016
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123. Chemical synthesis, 3D structure, and ASIC binding site of the toxin mambalgin-2.
- Author
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Schroeder CI, Rash LD, Vila-Farrés X, Rosengren KJ, Mobli M, King GF, Alewood PF, Craik DJ, and Durek T
- Subjects
- Acid Sensing Ion Channels genetics, Animals, Binding Sites drug effects, Dose-Response Relationship, Drug, Elapid Venoms chemistry, Models, Molecular, Molecular Structure, Peptides chemistry, Rats, Structure-Activity Relationship, Acid Sensing Ion Channels metabolism, Elapid Venoms pharmacology, Peptides pharmacology
- Abstract
Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
- Full Text
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124. Synthesis of photoactive analogues of a cystine knot trypsin inhibitor protein.
- Author
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Durek T, Zhang J, He C, and Kent SB
- Subjects
- Chromatography, Liquid, Spectrometry, Mass, Electrospray Ionization, Trypsin Inhibitors chemistry, Cystine Knot Motifs, Trypsin Inhibitors chemical synthesis
- Abstract
We describe the preparation of a recently described photoactive amino acid analogue (photoMethionine) by two novel synthetic routes, one of which is flexible and enantiospecific, and the site-specific chemical incorporation of photoMethionine into a defined and functionally active protein using a combination of solid-phase peptide synthesis and modern chemical ligation methodology. Site-specific labeling of proteins with this amino acid analogue through chemical synthesis provides valuable probes for photoaffinity cross-linking studies.
- Published
- 2007
- Full Text
- View/download PDF
125. Synthesis of functionalized rab GTPases by a combination of solution- or solid-phase lipopeptide synthesis with expressed protein ligation.
- Author
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Brunsveld L, Watzke A, Durek T, Alexandrov K, Goody RS, and Waldmann H
- Subjects
- Chromatography, Gel, Electrophoresis, Polyacrylamide Gel, Lipoproteins chemistry, Magnetic Resonance Spectroscopy, Models, Molecular, Protein Prenylation, Spectrometry, Mass, Electrospray Ionization, rab GTP-Binding Proteins chemistry, rab7 GTP-Binding Proteins, Lipoproteins chemical synthesis, rab GTP-Binding Proteins chemical synthesis
- Abstract
Prenylated proteins with non-native functionalities are generally very difficult to obtain by recombinant or enzymatic means. The semisynthesis of preparative amounts of prenylated Rab guanosine triphosphatases (GTPases) from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' native structure or modifications thereof. Here, we describe and compare solution-phase and solid-phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase. The solid-phase with utilisation of a hydrazide linker emerges as the more favourable approach. It allows a fast and practical synthesis of pure peptides and gives a high degree of flexibility in their modification. To facilitate the analysis of semisynthetic proteins, the synthesised peptides were equipped with a fluorescent group. Using the described approach, we introduced fluorophores at several different positions of the Rab7 C terminus. The position of the incorporated fluorescent groups in the peptides did not influence the protein-ligation reaction, as the generated peptides could be ligated onto thioester-tagged Rab7. However, it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins; analysis of the interaction of the semisynthetic Rab7 proteins with REP (Rab escort protein) and GDI (guanosine diphosphate dissociation inhibitor) molecules revealed that modification of the peptide side chains or of the C-terminal isoprenoid did not significantly interfere with complex formation. However, functionalisation of the C terminus was found to have an adverse effect on complex formation and stability, possibly reflecting low structural flexibility of the Rab GDI/REP molecules in the vicinity of the lipid-binding site.
- Published
- 2005
- Full Text
- View/download PDF
126. Chemical biology of protein lipidation: semi-synthesis and structure elucidation of prenylated RabGTPases.
- Author
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Watzke A, Brunsveld L, Durek T, Alexandrov K, Rak A, Goody RS, and Waldmann H
- Subjects
- Models, Molecular, Protein Conformation, Protein Engineering, Protein Prenylation, rab GTP-Binding Proteins chemical synthesis, rab GTP-Binding Proteins chemistry, rab GTP-Binding Proteins metabolism
- Abstract
Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells. For their function Rab/Ypt proteins require double modification with two covalently bound geranylgeranyl lipid moieties at the C-terminus. Generally, prenylated proteins are very difficult to obtain by recombinant or enzymatic methods. We generated prenylated RabGTPases using a combination of chemical synthesis and protein engineering. This semi-synthesis depends largely on the availability of functionalized prenylated peptides corresponding to the proteins' native structure or modifications. We developed solution phase and solid phase strategies for the generation of peptides corresponding to the prenylated C-terminus of Rab7 GTPase in preparative amounts enabling us to crystallize the mono-prenylated Ypt1:RabGDI complex. The structure of the complex provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins and a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.
- Published
- 2005
- Full Text
- View/download PDF
127. In vitro semisynthesis and applications of C-terminally modified rab proteins.
- Author
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Durek T, Goody RS, and Alexandrov K
- Subjects
- Electrophoresis, Polyacrylamide Gel, In Vitro Techniques, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Staining and Labeling, rab GTP-Binding Proteins chemistry, rab7 GTP-Binding Proteins, Protein Processing, Post-Translational, rab GTP-Binding Proteins chemical synthesis
- Abstract
Expressed protein ligation is a powerful tool for the generation of natively folded proteins composed of recombinantly generated and chemically synthesized polypeptides. Using this approach, we developed protocols for the production of prenylated and/or otherwise-labeled Rab GTPase. The protocols are generally applicable to most small GTPases that can be supplied with a variety of new chemical functionalities. We used semisynthetic fluorescently labeled Rab7 GTPase as a molecular probe to study protein-protein interactions with components of the prenylation machinery
- Published
- 2004
- Full Text
- View/download PDF
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