A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility

Baoyu Zhao; Peng Xu; Longguang Jiang; Berit Paaske Jensen; Tobias Kromann-Hansen; Jan Kristian Jensen; Hans Peter Sorensen; Zhuo Liu; Jakob T. Nielsen; Anni Christensen; Masood Hosseini; Kasper Kildegaard Sørensen; Niels Christian Nielsen; Knud Jørgen Jensen; Mingdong Huang; Peter Andreasen

doi:10.1371/journal.pone.0115872

A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility

Baoyu Zhao, Peng Xu, Longguang Jiang, Berit Paaske Jensen, Tobias Kromann-Hansen, Jan Kristian Jensen, Hans Peter Sorensen, Zhuo Liu, Jakob T. Nielsen, Anni Christensen, Masood Hosseini, Kasper Kildegaard Sørensen, Niels Christian Nielsen, Knud Jørgen Jensen, Mingdong Huang, Peter Andreasen

Department of Chemistry

14 Citations (Scopus)

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Abstract

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.

Original language	English
Article number	e115872
Journal	PLOS ONE
Volume	9
Issue number	12
Number of pages	27
ISSN	1932-6203
DOIs	https://doi.org/10.1371/journal.pone.0115872
Publication status	Published - 2014

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A Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide FlexibilityFinal published version, 1.79 MB

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Zhao, B., Xu, P., Jiang, L., Jensen, B. P., Kromann-Hansen, T., Jensen, J. K., Sorensen, H. P., Liu, Z., Nielsen, J. T., Christensen, A., Hosseini, M., Sørensen, K. K., Nielsen, N. C., Jensen, K. J., Huang, M., & Andreasen, P. (2014). A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility. PLOS ONE, 9(12), Article e115872. https://doi.org/10.1371/journal.pone.0115872

Zhao, B, Xu, P, Jiang, L, Jensen, BP, Kromann-Hansen, T, Jensen, JK, Sorensen, HP, Liu, Z, Nielsen, JT, Christensen, A, Hosseini, M, Sørensen, KK, Nielsen, NC, Jensen, KJ, Huang, M & Andreasen, P 2014, 'A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility', PLOS ONE, vol. 9, no. 12, e115872. https://doi.org/10.1371/journal.pone.0115872

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title = "A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility",

abstract = "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.",

author = "Baoyu Zhao and Peng Xu and Longguang Jiang and Jensen, {Berit Paaske} and Tobias Kromann-Hansen and Jensen, {Jan Kristian} and Sorensen, {Hans Peter} and Zhuo Liu and Nielsen, {Jakob T.} and Anni Christensen and Masood Hosseini and S{\o}rensen, {Kasper Kildegaard} and Nielsen, {Niels Christian} and Jensen, {Knud J{\o}rgen} and Mingdong Huang and Peter Andreasen",

year = "2014",

doi = "10.1371/journal.pone.0115872",

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issn = "1932-6203",

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T1 - A cyclic peptidic serine protease inhibitor

T2 - increasing affinity by increasing peptide flexibility

AU - Zhao, Baoyu

AU - Xu, Peng

AU - Jiang, Longguang

AU - Jensen, Berit Paaske

AU - Kromann-Hansen, Tobias

AU - Jensen, Jan Kristian

AU - Sorensen, Hans Peter

AU - Liu, Zhuo

AU - Nielsen, Jakob T.

AU - Christensen, Anni

AU - Hosseini, Masood

AU - Sørensen, Kasper Kildegaard

AU - Nielsen, Niels Christian

AU - Jensen, Knud Jørgen

AU - Huang, Mingdong

AU - Andreasen, Peter

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

U2 - 10.1371/journal.pone.0115872

DO - 10.1371/journal.pone.0115872

M3 - Journal article

C2 - 25545505

AN - SCOPUS:84920147460

SN - 1932-6203

VL - 9

JO - PLOS ONE

JF - PLOS ONE

IS - 12

M1 - e115872

ER -

A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility

Abstract

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