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    On the Effect of Cyclization on Peptide Backbone Dynamics

    Conan K. Wang1, Joakim E. Swedberg1, Susan E. Northfield1, David J. Craik1,*

    1Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia

    *Corresponding Author:

    Professor David J. Craik

    Institute for Molecular Bioscience,

    The University of Queensland,

    Brisbane, QLD, 4072, Australia

    Tel: 61-7-3346 2019

    Fax: 61-7-3346 2101

    e-mail: [email protected]

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    Despite the widespread use of cyclization as a structure optimization tool in peptide chemistry, little is

    known about the effect of cyclization on peptide internal dynamics. Here we used a combination of multi-

    field NMR relaxation and molecular dynamics techniques to study monocyclic as well as polycyclic

    peptides that have promising biopharmaceutical properties – VH, SFTI-1 and cVc1.1 – and their less

    constrained analogues to study the effect of backbone cyclization (which forms a macrocycle) and disulfide

    bond cyclization (which forms internal cycles). We confirmed that backbone cyclization contributes to the

    rigidity of the monocyclic VH. Interestingly though, backbone cyclization of the bicyclic SFTI-1 had a

    limited effect on rigidity, with changes in internal dynamics localized around the ligation site. This suggests

    that the disulfide bond, which creates an internal cycle, has a insulating effect, protecting the internal cycle

    from external motional effects. An insulating effect was also observed for the polycyclic cVc1.1 – the

    rigidity of the core was not enhanced by macrocyclization. Additionally, we found that disulfide bonds had a

    greater contribution to overall rigidity than macrocyclization. Overall, our results suggest that, although

    backbone cyclization can improve rigidity, there is a complex interplay between dynamics and cyclization,

    particularly for polycyclic systems.

    Keywords: cyclic peptide, disulfide bond, model free, molecular dynamics, NMR relaxation

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    Cyclization is an important strategy that is widely used to improve the biopharmaceutical properties of

    peptide leads in drug discovery.1-3 Cyclization is generally believed to impart improved metabolic stability

    to a target peptide, enabling medicinal chemists to exploit the properties of high selectivity, high potency

    and low toxicity that are inherent to peptides and underpin the renewed interest in them as drugs. Peptides

    can be cyclized in many ways, including through head-to-tail (e.g. backbone cyclization) and side-chain-to-

    side-chain (e.g. disulfide bond formation) connections.1 One of the earliest examples of cyclization in drug

    design involved a potent cyclic hexapeptide analogue of somatostatin, also known as the Veber-Hirschmann

    peptide (VH; Figure 1a and 1c).4 Backbone cyclization not only constrained the peptide into a bioactive

    conformation but also improved its metabolic stability, resulting in a cyclic peptide that had both a long

    duration of action and oral activity. Subsequent structural studies on other monocyclic peptides have further

    shown how backbone cyclization can constrain peptides into bioactive conformations.5

    We recently extended the concept of cyclization to disulfide-rich peptides by re-engineering a conotoxin

    from the venom of Conus victoriae (cone snail) to make it backbone cyclic by adding a seven-residue linker

    to connect the N- and C-termini (Figure 1a and 1c).6 The engineered cyclic peptide showed potent and

    selective inhibition of voltage-activated calcium channels associated with pain responses. Whereas the

    backbone acyclic peptide was inactive via the oral delivery route in an animal model of neuropathic pain, the

    backbone cyclic peptide had oral activity and was more than two orders of magnitude more potent than

    gabapentin, the current leading therapy for neuropathic pain.6 Along with this example demonstrating the

    benefits of cyclization, there are now a growing number of cyclic peptides that have been reported to have

    potent bioactivities and/or high absorption within the gastro-intestinal tract.7-10

    The benefits of cyclization have been exploited not only by medicinal chemists but also by nature. To

    understand the advantages of cyclization, we have conducted structural studies on a wide range of cyclic

    peptides found in bacteria, fungi, plants and mammals.10 In one study, we examined the effect of backbone

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    cyclization on the structure and activity of sunflower trypsin inhibitor-1 (SFTI-1; Figure 1a and 1b), a cyclic

    peptide that comprises one cross-bracing disulfide bond and is the smallest and most potent known inhibitor

    of trypsin, and found that cyclization was integral to its proteolytic stability and inhibitory activity.11,12

    Another example of a plant-derived cyclic disulfide-rich peptide is kalata B1, originally discovered as the

    active uterotonic agent in an African medicinal tea and later shown to be resistant to adverse enzymatic,

    thermal and chemical conditions.13 The remarkable in vitro enzymatic, chemical and thermal stability of

    these peptides, which have been attributed to their cyclic backbones and disulfide-rich nature, makes them

    excellent scaffolds in drug development.10 Indeed, they have led to the design of several leads with potent

    activities in animal models of disease, including pain, multiple sclerosis and cancer.10

    Although the importance of cyclization as a design tool is well-established, the interplay between cyclization

    and peptide backbone dynamics is poorly understood. For proteins, dynamics is intimately linked to the

    folding free energy14 and other aspects of protein behavior in solution, including thermostability15 and

    solubility.16 Dynamics can also regulate protein activity and, importantly, in ways that sometimes cannot be

    predicted based on the protein's ground-state structure.17 For example, internal dynamics can have a

    significant effect on binding to a target protein even if no conformational changes are observed upon

    binding.17 Specifically, changes in internal dynamics can have a marked effect on the strength of the

    enthalpy-entropy compensation that contributes to the free energy of binding. Therefore, a study of

    dynamics can uncover cryptic structural information that is fundamental to understanding function.

    Nuclear magnetic resonance (NMR) relaxation is a powerful technique that is well-suited for the

    characterization of the internal and overall (diffusion controlled) motions of molecules (Figure 1b).18-20

    Combined with advances in molecular dynamics simulation, NMR relaxation can provide a comprehensive

    and informative picture of the dynamics of a biomolecular system.21 Here we compared the backbone

    dynamics of a series of representative cyclic peptides with that of their acyclic derivatives (i.e. backbone and

    disulfide variants). We focused on three sets of peptides of varying size and amino acid content: the Veber-

    Hirschmann peptide (VH) and its acyclic analogue ([lin]VH); SFTI-1, its backbone acyclic analogue

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    ([lin]SFTI-1), and a disulfide bond-deleted mutant ([C3A,C11A]SFTI-1); and cVc1.1 and its linear analogue

    Vc1.1, as well as a series of disulfide-bond deletion/substitution analogues ([C2A,C8A]cVc1.1,

    [C2A,C8A]Vc1.1 and [C2H,C8F]cVc1.1). We obtained multi-field NMR relaxation measurements for these

    peptides and derived parameters describing their backbone dynamics. We evaluated these results with

    respect to molecular dynamics simulations in explicit solvent.

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    Figure 1: Structures and sequences of the peptides in this study. a) Structures of cyclic peptides with

    selected residues labeled. The site where the linear peptides are joined to create the cyclic analogue is

    shown for VH and SFTI-1. The linker sequence is highlighted for cVc1.1 with a dotted line. A

    schematic of each cyclic peptide is shown to the right of its structure. b) Schematic of some of the

    types of motion that can be measured using NMR. For internal motion, the order parameter, S2, for the

    Cα-Hα backbone vector can be used as an indicator of molecular flexibility. c) Sequences and disulfide

    bond connectivity. Backbone cyclic peptides are indicated with the term 'cyclo'. Residues are shown as

    single letter amino acid codes. Cysteine residues and substituted residues are highlighted in bold. The

    cystine connectivity is shown. The asterisk indicates an amidated C-terminus.

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    Methods and Materials


    N-terminal protected amino acids, resins and coupling agents were purchased from ChemImpex

    International, and all other reagents were purchased from Auspep, Merck and Sigma, and used without

    further purification. D2O (D, 99.96%) and acetonitrile-d3 (D, 99.8%) were purchased from Cambridge

    Isotope Laboratories, Inc.

    Peptide synthesis and purification

    VH, [lin]VH, [lin]SFTI-1, [C3A,C11A]SFTI-1, Vc1.1, [C2A,C8A]cVc1.1, and [C2A,C8A]Vc1.1 were

    synthesized on 2-chlorotrityl chloride (2CTC) resin on