The Binding Mechanism of a Peptidic Cyclic Serine Protease Inhibitor

Jiang, Longguang; Svane, Anna S.P.; Sørensen, H.; Jensen, Jan K.; Hosseini, Masood; Chen, Zhuo; Weydert, Caroline; Nielsen, Jakob T.; Christensen, Anni; Yuan, Cai; Jensen, Knud J.; Nielsen, Niels Chr.; Malmendal, Anders; Huang, Mingdong; Andreasen, Peter A.

doi:10.1016/j.jmb.2011.07.028

Cited by 16 publications

(14 citation statements)

References 38 publications

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“…k d = 0.022 s −1 for 19 amino acid cyclic peptide (‘MGSADGA‐u2’ serine protease inhibitor candidate) binding to immobilized uPA (Jiang et al . ); k d = 0.008 s −1 for single‐chain antibody fragment (‘I2’ anti‐amyloid candidate) binding to immobilized Aβ42 fibrils (Munke et al . ); k d = 0.026 s −1 for 7 amino acid cyclic peptide (‘CP2’ anti‐infective candidate) binding to immobilized hSPSB2 (Sadek et al .…”

Section: Discussionmentioning

confidence: 99%

Label‐free distribution of anti‐amyloid D‐AIP in Drosophila melanogaster: prevention of Aβ42‐induced toxicity without side effects in transgenic flies

Zhong

Shobo

Hancock

et al. 2019

Journal of Neurochemistry

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Soluble oligomers of the 42‐amino acid amyloid beta (Aβ42) peptide are highly toxic and suspected as the causative agent of synaptic dysfunction and neuronal loss in Alzheimer's disease (AD). Previously, we have shown that a small, D‐amino acid Aβ42‐oligomer interacting peptide (D‐AIP) can neutralize human Aβ42‐mediated toxicity using in vitro and cell‐based assays. In the present longitudinal study using a transgenic Drosophila melanogaster model, advanced live confocal imaging and mass spectrometry imaging (MALDI‐MSI) showed that the eight amino acid D‐AIP can attenuate Aβ42‐induced toxicity in vivo. By separating male and female flies into distinct groups, the resultant distribution of ingested D‐AIP was different between the sexes. The Aβ42‐induced ‘rough eye’ phenotype could be rescued in the female transgenics, likely because of the co‐localization of D‐AIP with human Aβ42 in the female fly heads. Interestingly, the phenotype could not be rescued in the male transgenics, likely because of the co‐localization of D‐AIP with a confounding male‐specific sex peptide (Acp70A candidate in MSI spectra) in the gut of the male flies. As a novel, more cost‐effective strategy to prevent toxic amyloid formation during the early stages of AD (i.e. neutralization of toxic low‐order Aβ42 oligomers without creating larger aggregates in the process), our longitudinal study establishes that D‐AIP is a stable and highly effective neutralizer of toxic Aβ42 peptides in vivo. Cover Image for this issue: doi: .

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Section: Discussionmentioning

confidence: 99%

Label‐free distribution of anti‐amyloid D‐AIP in Drosophila melanogaster: prevention of Aβ42‐induced toxicity without side effects in transgenic flies

Zhong

Shobo

Hancock

et al. 2019

Journal of Neurochemistry

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“…The conformations of the mupain-1 peptides on the enzyme surface are perpendicular to the previously reported conformation of the peptide upain-1 (CSWRGLENHRMC), a competitive inhibitor of huPA ( [20] ). In the upain-1 – huPA complex, Glu 7 of the peptide blocks the oxyanion hole [15] , [16] , [32] . Thus, the inhibitory mechanism of mupain-1 is different from that of upain-1.…”

Section: Discussionmentioning

confidence: 99%

A Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide Flexibility

Zhao

Jiang

et al. 2014

PLoS ONE

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Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

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“…[14] The baseline-adjusted profile was fitted to a 1:1 binding model, and a thermodynamic analysis was performed to investigate the enthalpic and entropic contributions during binding. A reduced entropy burden was observed for the bicyclic peptides 24 and 29, compared to the monocyclic 1.…”

Section: Isothermal Titration Calorimetry Reveals Changes In Thermodymentioning

confidence: 99%

“…Isothermal titration calorimetry: [14] All ITC experiments were performed on a MicroCal VP-ITC instrument (GE Life Sciences) equilibrated at 25 8C. uPA was dissolved and dialysed into buffer (Na 2 HPO 4 (20 mm, pH 7.4), NaCl (140 mm)).…”

Section: Lc-msmentioning

confidence: 99%

“…This approach was validated by the knowledge that upain-1 exists as an ensemble of structures in solution, each of which differs substantially from that of the bound structure. [14] After identification of an equipotent bicyclic peptide we performed isothermal titration calorimetry (ITC) and showed that correctly positioned bicyclisation of the peptide indeed reduced the loss of entropy upon binding to uPA. However, the peptide also exhibited a reduction in enthalpy, due to slightly compromised binding.…”

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confidence: 99%

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Bicyclic Peptide Inhibitor of Urokinase‐Type Plasminogen Activator: Mode of Action

et al. 2013

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The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide-based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established monocyclic peptide, upain-2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across-the-ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain-2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain-2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X-ray structure of the bicyclic-peptide-uPA complex confirmed an interaction similar to that for the previous upain-1/upain-2-uPA complexes. These physical studies of the peptide-protease interactions will aid future designs of bicyclic peptide protease inhibitors.

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The Binding Mechanism of a Peptidic Cyclic Serine Protease Inhibitor

Cited by 16 publications

References 38 publications

Label‐free distribution of anti‐amyloid D‐AIP in Drosophila melanogaster: prevention of Aβ42‐induced toxicity without side effects in transgenic flies

Label‐free distribution of anti‐amyloid D‐AIP in Drosophila melanogaster: prevention of Aβ42‐induced toxicity without side effects in transgenic flies

A Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide Flexibility

Bicyclic Peptide Inhibitor of Urokinase‐Type Plasminogen Activator: Mode of Action

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