Top > Search of International Patents > METHOD FOR SCREENING SUBSTANCE HAVING HIGH AFFINITY FOR TARGET SUBSTANCE

METHOD FOR SCREENING SUBSTANCE HAVING HIGH AFFINITY FOR TARGET SUBSTANCE

Foreign code F180009514
File No. (S2017-0313-N0)
Posted date Nov 2, 2018
Country WIPO
International application number 2018JP003442
International publication number WO 2018143357
Date of international filing Feb 1, 2018
Date of international publication Aug 9, 2018
Priority data
  • P2017-016609 (Feb 1, 2017) JP
Title METHOD FOR SCREENING SUBSTANCE HAVING HIGH AFFINITY FOR TARGET SUBSTANCE
Abstract This method for screening a substance having a high affinity for a target substance includes: a step of preparing a measurement sample in which a sample containing a candidate substance is brought into contact with the target substance; a step in which the measurement sample is subjected to imaging mass spectrometry using matrix assisted laser desorption/ionization (MALDI) with the target substance as a matrix, to identify an ionized molecule; and a step of selecting the identified molecule as being a substance having high affinity for the target substance.
Outline of related art and contending technology BACKGROUND ART
In drug development, a huge amount of compound from a library of compounds having the expected therapeutic effect can be effectively selected in important.For this purpose, if the evaluation item is better is simple.For example, a variety of physiologic functions to affect the complexity of the target animal with the individual, by screening the candidate compound than in the case of a therapeutic effect, a simple cell physiology more preferable to be a target.Further, the therapeutic effect are directly involved in the interaction at the molecular level can be analyzed more, a compound having a therapeutic effect for the purpose of more efficiently can be selected.Such as a particular neuropsychiatric disease therapeutic affect a higher function of a living body is described, the action of the individual animal experiments to assess the large amount of time and investment, since the labor is required, a highly reliable evidence based on the obtained evaluation method and a molecular target.
Conventionally, as a method for evaluating the interaction between molecules, such as by immunoprecipitation and Western blot or affinity purification in combination with a detection system such as mass spectrometry, fluorescence resonance energy transfer (FRET) methods such as imaging techniques, surface plasmon resonance analysis and the analysis quantity change, such as nuclear magnetic resonance (NMR) of the high magnetic field such as spectra analysis is generally performed by the.In these methods, a sample of high purity, high quality antibodies, the fabrication of the probe, the isotope label is required and thus, in practice, the cost and labor, time, various constraints in terms of safety is present.For example, in some methods require the detection probe, specific for each target observation for designing a probe, only the interaction between the molecules of interest cannot be measured.In addition, methods which do not use probes to detect, for example, a binding molecule against a target substance such as liquid chromatography-mass spectrometry method that detects, as the pre-processing such as purification of the immunoprecipitation method and is generally, also in this purification step is limited due to the detectable molecule.For this reason, if the search of the binding molecule study, the non-target can be analyzed by a detection system is required.Further, if there is the step of extracting from the living organism, in vitro or refining operation in the living environment is changed or a case where a three-dimensional structure, only a limited location of the living body does not exist in the first place can be difficult to collect the trace molecules such as the problem.
In addition, in the drug development, in recent years, the cause of the disease specific in biological cells, involved in biological cells and in particular targeted to the physiologically active substance, a so-called molecular target agent is currently under development.This is, the cause of the disease to elucidate the molecular level or at the genomic level, disease related to the target material (a compound having a pharmacological effect) pharmaceutical molecular design is carried out from, a pharmaceutical is designed, the greater the effect the treatment of a disease, other diseases further less likely to be acting in biological cells, the effect of reducing the side effects are expected.On the other hand, (1) the cause of the disease at a molecular level or at the genomic level to ascertain the cause of a particular substance, (2) based on the chemical structure, molecular weight of the drug design, are required.However, (1) is also discussed in, (2) any of the days is required and, as a result the power consumption and long-term development.
On the other hand, in recent years, on the biomolecules of the tissue sample to be visualized as a two-dimensional IMS 2 (also referred to as Mass microscopy.) Have been developed for the device.MALDI method (MALDI-IMS) is used in the IMS, such as a tissue section sample to be analyzed, the biomolecule absorbs the laser light and to facilitate the ionization of a low molecular weight compound (matrix) is applied, scanning the laser light is irradiated to, the three-dimensional coordinates 2 of the ions detected at each point on the basis of the image to reconstruct an image.In this method, three-dimensional laser scanning of tissue sections for analysis by 2 as it is, the position information on the tissue sample of the biological molecules can be ionized being maintained.Biological molecule is ionized, and analyzed by time-of-flight mass spectrometer, in accordance with the mass to charge ratio are identified.Thus, in the MALDI-IMS, the distribution of the biomolecules on the tissue sample, between the measurement points by the relative value of the signal intensity of the image can be reduced.As the matrix, ionization of the biomolecules is empirically suitable compounds are used.For example, methoxy 3,5-4-hydroxycinnamic acid, 9-(9AA) such as aminoacridine 50 molecular weight of less than 300 or more is used as the ionic dissociation of the substance, biomolecule on the tissue sample is ionized and a state in which the matrix molecules are coupled and released from the tissue sample, and thereby, in the MALDI-IMS analyzer, mass to charge ratio (m/z) separately for, is identified.
Patent Document 1 is for example, using the MALDI-IMS, the treatment of breast cancer therapeutic agent in the assessment of the effect of a candidate compound has been disclosed a method.In this method, in the breast, a specific molecular species of the phosphatidylcholine lipid metabolism (PC) mammary tissue from there are a large number, the candidate compound has been administered to the subject the presence of the molecular species of phosphatidylcholine in the mammary gland tissue MALDI-IMS is measured by the ratio, there are a number of breast cancer patient and the presence of molecular species in the case where the ratio is low, the candidate compounds are evaluated as effective as treatment of breast cancer.That is, the method, the candidate compound to the subject directly, through metabolism exhibit anti-cancer effects before they are compared, can be used for screening of a candidate substance is naturally on the type of restriction, there has been a limit to the efficiency of screening.In addition, the prior art, using the MALDI-IMS, the intermolecular interaction is not to mention, the in-vivo substance is involved in a method to determine the affinity is not found.
Scope of claims (In Japanese)請求の範囲
[請求項1]
 標的物質と候補物質を含む試料を接触させた測定用試料を調製する工程と、
 前記測定用試料に対して、前記標的物質をマトリクスとしたMALDI法を用いたイメージング質量分析法を行い、イオン化された分子を同定する工程と、
 前記同定した分子を、前記標的物質との親和性の高い物質であると選出する工程と、を有する、標的物質と親和性の高い物質をスクリーニングする方法。
[請求項2]
 前記測定用試料を調製する工程は、
 前記候補物質を含む被検試料と、前記標的物質と、を接触させる工程と、
 前記接触させた試料と標的物質を、接触状態のまま乾燥させ、測定用試料とする乾燥工程と、を含み、
 前記同定工程は、前記測定用試料に対して、MALDI法を用いたイメージング質量分析法を行い、イオン化された分子のスペクトルを得るスペクトルスキャン工程と、
 前記スペクトルより、ピーク物質を同定する工程と、を含む、
 請求項1に記載の方法。
[請求項3]
 前記標的物質が、分子量10,000以下の化合物である、請求項1又は2に記載の方法。
[請求項4]
 前記候補物質が、リン脂質である、請求項1~3のいずれか一項に記載の方法。
[請求項5]
 前記候補物質を含む試料が組織切片であり、前記測定用試料が、前記候補物質を含む試料の表面に前記標的物質を付着させたものである、請求項1~4のいずれか一項に記載の方法。
[請求項6]
 前記MALDI法を用いたイメージング質量分析法に用いるレーザーの波長が200~400nmであり、
前記標的物質が、芳香環及び/または複素環を含む分子量300~10,000の化合物である請求項1~5のいずれか一項に記載の方法。
[請求項7]
 前記標的物質とは異なる物質を対照物質とし、さらに、
 前記対照物質と前記候補物質を含む試料を接触させた測定用対照試料を調製する工程と、
 前記測定用対照試料に対して、前記対照物質をマトリクスとしたMALDI法を用いたイメージング質量分析法を行い、イオン化された分子を同定する工程と、
 前記同定した分子を、前記対照物質との親和性の高い物質であると選出する工程と、を有し、
 前記測定用試料からイオン化された分子のうち、前記対照物質との親和性の高い物質であると選出された分子以外を、前記標的物質との親和性の高い物質であると選定する、請求項1~6のいずれか一項に記載の方法。
[請求項8]
 前記対照物質が、3,5-メトキシ-4-ヒドロキシケイ皮酸、α-シアノ-4-ヒドロキシケイ皮酸、trans-4-ヒドロキシ-3-メトキシケイ皮酸、2,5-ジヒドロキシ安息香酸、3-ヒドロキシピコリン酸、9-アミノアクリジン、2,5-ジヒドロキシアセトフェノン、又は1,8-ジヒドロキシ-9,10-ジヒドロアントラセン-9-オンである、請求項7に記載の方法。
[請求項9]
 標的物質を含む被検試料と、候補物質と、を接触させる工程と、
 前記被検試料と前記候補物質とを、接触状態のまま乾燥させ、測定用試料とする乾燥工程と、
 前記測定用試料に対して、MALDI法を用いたイメージング質量分析法を行い、イオン化された分子のうち、前記標的物質に対応するm/zをスキャンするスキャン工程と、
 前記m/zにおけるピーク強度を測定するピーク強度測定工程と、
 前記ピーク強度が検出された候補物質を、前記標的物質との親和性の高い物質であると選出する工程と、を含む、
標的物質と親和性の高い物質をスクリーニングする方法。
[請求項10]
 前記候補物質が、分子量10,000以下の化合物である、請求項9に記載の方法。
[請求項11]
 前記標的物質が、リン脂質である、請求項9又は10記載の方法。
[請求項12]
 前記標的物質を含む試料が組織切片であり、前記測定用試料が、前記標的物質を含む試料の表面に前記候補物質を付着させたものである、請求項9~11のいずれか一項に記載の方法。
[請求項13]
 前記MALDI法を用いたイメージング質量分析法に用いるレーザーの波長が200~400nmであり、
前記候補物質が、芳香環及び/または複素環を含む分子量300~10,000の化合物である請求項9~12のいずれか一項に記載の方法。
[請求項14]
 請求項9~13のいずれか一項に記載の標的物質と親和性の高い物質のスクリーニング工程と、
前記スクリーニング工程により選出された候補物質を有効成分として含有させることを含む、医薬の設計方法。
[請求項15]
 請求項9~13のいずれか一項に記載の標的物質と親和性の高い物質のスクリーニング工程と、
前記スクリーニング工程により選出された候補物質を有効成分として含有させることを含む、医薬の製造方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION HAMAMATSU UNIVERSITY SCHOOL OF MEDICINE
  • Inventor
  • SETOU Mitsutoshi
  • KONDO Takeshi
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
Please contact us by E-mai if you have any interests on this patent.

PAGE TOP

close
close
close
close
close
close