High-throughput turbidimetric screening for heparin-neutralizing agents and low-molecular-weight heparin mimetics

Atsushi Sekiya, Shinya Oishi, Nobutaka Fujii, Takaki Koide

    Research output: Contribution to journalArticle

    2 Citations (Scopus)

    Abstract

    Safer heparin-neutralizing agents are currently required to replace protamine, the use of which causes adverse effects such as anaphylaxia. Low-molecular-weight (LMW) heparin mimetics that potentiate antithrombin III (AT) action are also valuable as anti-thrombotics. This paper describes a high-throughput assay for both heparin-neutralizing agents and LMW heparin mimetics without the use of blood preparations. The assay is based on turbidimetric measurement of a solution of collagen, heparin, and a test compound. Native collagen molecules spontaneously form insoluble fibrils when transferred to a physiological buffer, and this process is inhibited by heparin. In the presence of a heparin-neutralizing agent or an LMW heparin mimetic, the inhibitory effect of heparin is canceled and turbidity increase is retrieved. We demonstrated that this assay is effective in detecting potential agents with high reliability (Z′ factor=0.9). The screening of a chemical library (34400 compounds) was further performed in a 384-well format, and led to the identification of a novel heparin-neutralizing agent. Since this assay protocol is feasible for an automated highthroughput screening (HTS) system, it could enhance the lead seeking process for drugs related to heparin/ heparan sulfate (HS) functions.

    Original languageEnglish
    Pages (from-to)371-376
    Number of pages6
    JournalChemical and Pharmaceutical Bulletin
    Volume60
    Issue number3
    DOIs
    Publication statusPublished - 2012 Mar

    Fingerprint

    Low Molecular Weight Heparin
    Heparin
    Screening
    Throughput
    Assays
    Collagen
    Small Molecule Libraries
    Physiological Phenomena
    Protamines
    Heparitin Sulfate
    Antithrombin III
    Turbidity
    Buffers
    Blood
    Molecules
    Pharmaceutical Preparations

    Keywords

    • Collagen
    • Heparin
    • Heparin mimetic
    • High-throughput screening

    ASJC Scopus subject areas

    • Drug Discovery
    • Chemistry(all)

    Cite this

    High-throughput turbidimetric screening for heparin-neutralizing agents and low-molecular-weight heparin mimetics. / Sekiya, Atsushi; Oishi, Shinya; Fujii, Nobutaka; Koide, Takaki.

    In: Chemical and Pharmaceutical Bulletin, Vol. 60, No. 3, 03.2012, p. 371-376.

    Research output: Contribution to journalArticle

    @article{470d47ca0e6746a5b7cb0f83085e3729,
    title = "High-throughput turbidimetric screening for heparin-neutralizing agents and low-molecular-weight heparin mimetics",
    abstract = "Safer heparin-neutralizing agents are currently required to replace protamine, the use of which causes adverse effects such as anaphylaxia. Low-molecular-weight (LMW) heparin mimetics that potentiate antithrombin III (AT) action are also valuable as anti-thrombotics. This paper describes a high-throughput assay for both heparin-neutralizing agents and LMW heparin mimetics without the use of blood preparations. The assay is based on turbidimetric measurement of a solution of collagen, heparin, and a test compound. Native collagen molecules spontaneously form insoluble fibrils when transferred to a physiological buffer, and this process is inhibited by heparin. In the presence of a heparin-neutralizing agent or an LMW heparin mimetic, the inhibitory effect of heparin is canceled and turbidity increase is retrieved. We demonstrated that this assay is effective in detecting potential agents with high reliability (Z′ factor=0.9). The screening of a chemical library (34400 compounds) was further performed in a 384-well format, and led to the identification of a novel heparin-neutralizing agent. Since this assay protocol is feasible for an automated highthroughput screening (HTS) system, it could enhance the lead seeking process for drugs related to heparin/ heparan sulfate (HS) functions.",
    keywords = "Collagen, Heparin, Heparin mimetic, High-throughput screening",
    author = "Atsushi Sekiya and Shinya Oishi and Nobutaka Fujii and Takaki Koide",
    year = "2012",
    month = "3",
    doi = "10.1248/cpb.60.371",
    language = "English",
    volume = "60",
    pages = "371--376",
    journal = "Chemical and Pharmaceutical Bulletin",
    issn = "0009-2363",
    publisher = "Pharmaceutical Society of Japan",
    number = "3",

    }

    TY - JOUR

    T1 - High-throughput turbidimetric screening for heparin-neutralizing agents and low-molecular-weight heparin mimetics

    AU - Sekiya, Atsushi

    AU - Oishi, Shinya

    AU - Fujii, Nobutaka

    AU - Koide, Takaki

    PY - 2012/3

    Y1 - 2012/3

    N2 - Safer heparin-neutralizing agents are currently required to replace protamine, the use of which causes adverse effects such as anaphylaxia. Low-molecular-weight (LMW) heparin mimetics that potentiate antithrombin III (AT) action are also valuable as anti-thrombotics. This paper describes a high-throughput assay for both heparin-neutralizing agents and LMW heparin mimetics without the use of blood preparations. The assay is based on turbidimetric measurement of a solution of collagen, heparin, and a test compound. Native collagen molecules spontaneously form insoluble fibrils when transferred to a physiological buffer, and this process is inhibited by heparin. In the presence of a heparin-neutralizing agent or an LMW heparin mimetic, the inhibitory effect of heparin is canceled and turbidity increase is retrieved. We demonstrated that this assay is effective in detecting potential agents with high reliability (Z′ factor=0.9). The screening of a chemical library (34400 compounds) was further performed in a 384-well format, and led to the identification of a novel heparin-neutralizing agent. Since this assay protocol is feasible for an automated highthroughput screening (HTS) system, it could enhance the lead seeking process for drugs related to heparin/ heparan sulfate (HS) functions.

    AB - Safer heparin-neutralizing agents are currently required to replace protamine, the use of which causes adverse effects such as anaphylaxia. Low-molecular-weight (LMW) heparin mimetics that potentiate antithrombin III (AT) action are also valuable as anti-thrombotics. This paper describes a high-throughput assay for both heparin-neutralizing agents and LMW heparin mimetics without the use of blood preparations. The assay is based on turbidimetric measurement of a solution of collagen, heparin, and a test compound. Native collagen molecules spontaneously form insoluble fibrils when transferred to a physiological buffer, and this process is inhibited by heparin. In the presence of a heparin-neutralizing agent or an LMW heparin mimetic, the inhibitory effect of heparin is canceled and turbidity increase is retrieved. We demonstrated that this assay is effective in detecting potential agents with high reliability (Z′ factor=0.9). The screening of a chemical library (34400 compounds) was further performed in a 384-well format, and led to the identification of a novel heparin-neutralizing agent. Since this assay protocol is feasible for an automated highthroughput screening (HTS) system, it could enhance the lead seeking process for drugs related to heparin/ heparan sulfate (HS) functions.

    KW - Collagen

    KW - Heparin

    KW - Heparin mimetic

    KW - High-throughput screening

    UR - http://www.scopus.com/inward/record.url?scp=84857849902&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=84857849902&partnerID=8YFLogxK

    U2 - 10.1248/cpb.60.371

    DO - 10.1248/cpb.60.371

    M3 - Article

    VL - 60

    SP - 371

    EP - 376

    JO - Chemical and Pharmaceutical Bulletin

    JF - Chemical and Pharmaceutical Bulletin

    SN - 0009-2363

    IS - 3

    ER -