サイズ排除クロマトグラフィ(SEC: Size Exclusion Chromatography)を利用したコンビナトリアル・ライブラリーのハイスループット・スクリーニング (HTS: High-Throughput Screening)

弊社ホームページのトピックスでも以前紹介しましたが、過去20年間において多くの製薬会社などが、PolyLCのポリハイドロキシエチルA (PolyHYDROXYETHYL A) サイズ排除クロマトグラフィ(SEC) カラムを用いて、コンビナトリアル・ライブラリーから、一回の実験ごとに最大2,000個までの化合物をスクリーニングし、標的高分子と高い親和性で結合する低分子化合物を特定してきました。このスクリーニング方法は、誘導体化も特別な装置も必要有りません。標的高分子は例えば、タンパク質、リボソームなどの細胞小器官、リボスイッチ、折りたたまれたDNAのプロモーター部位などになります[図1.(出典文献1.)]。

図1. Screening for compounds that bind with high affinity to varieties of RNA but not to proteins: RNA promiscuity is not correlated with protein promiscuity (R2 = 0.001). Number of protein targets bound vs. number of RNA targets bound plotted for each compound.

標的高分子と強力に結合した低分子化合物をSECカラムに注入しますと、ボイド容積 (Void Volume) のVoピークとして溶出し、結合していない低分子化合物は全容積 (Total Volume) のVtピークとして溶出します。Voピークとして溶出した低分子化合物は特定が行われ、高い親和性の部分集合体を共通にもつ特徴のもと、新しいライブラリーが合成されます。これらの反復工程で、非常に高い親和性 (Kd < 100nM) と選択性を持つ医薬品候補が造られます。


図2. ALIS Process and ALIS System Schematic. ALIS (Automated Ligand Identification System) enables screening of compound mixtures with soluble macromolecular targets by 1. Equilibrating the target with a mixture of compounds; 2. Fast size exclusion chromatography (SEC) selects the target-ligand complex from the unbound mixture (blue trace is target-ligand complex eluting from SEC, red trace is residual sample remaining after target-ligand complex is collected and transferred to reverse-phase chromatography (RPC) column); 3. Reverse-phase HPLC dissociates and separates the bound ligands from target using low pH and high temperature (40ºC) and 4. The Mass spectrometer identifies hits using full scan mass spectrometry.


図3. Affinity competition experiments enable protein-ligand binding affinity measurements in compound mixtures. As simulated in (A), a library of compounds of varying affinity (blue) is embedded with calibrant ligands of known Kd (red) and titrated with a compound of known Kd (purple) to yield MS-measured ACE50 curves. (B) A calibration curve generated from the calibrants’ ACE50 and Kd values yields the other mixture components' Kds.

図4. Compound binding affinity characterization to WT FMN Riboswitch Target. (A) Ribocil-B ligand (ALIS Kd=26 nM [11]; 0–50 µM) is used to titrate against constant FMN (ALIS Kd=1.7 nM [11]; 4 µM) (top) and compared to FMN ligand (0–200 µM) titrated against constant ribocil-B (4 µM), bottom. (B) Structures of FMN riboswitch ligands. (C) Example of relative ranking competition in ALIS. FMN is used to titrate a mixture of ribocil, ribocil-C and roseoflavin on the FMN WT riboswitch. Compounds are competed away from the riboswitch target in order of decreasing binding affinities (data from [11]).

出典文献1. Targeting RNA with Small Molecules: Identification of Selective, RNA- Binding Small Molecules Occupying Drug-Like Chemical Space; Supplementary Figure 5, Noreen F. Rizvi, John P. Santa Maria Jr., Ali Nahvi, Joel Klappenbach, Daniel J. Klein, Patrick J. Curran, Matthew P. Richards, Chad Chamberlin, Peter Saradjian, Julja Burchard, Rodrigo Aguilar, Jeannie T. Lee, Peter J. Dandliker, Graham F. Smith, Peter Kutchukian, and Elliott B. Nickbarg, SLAS Discovery 1-13, Online 11-8-2019
出典文献2. RNA-ALIS: Methodology for screening soluble RNAs as small molecule targets using ALIS affinity-selection mass spectrometry, Noreen F. Rizvi, and Elliott B. Nickbarg, Methods 167 (2019) 28-38
出典文献3. Method for Quantitative Protein-Ligand Affinity Measurements in Compound Mixtures, D. Allen Annis, Gerald W. Shipps Jr., Yongqi Deng, Janeta Popovici-Muller, M. Arshad Siddiqui, Patrick J. Curran, Matthew Gowen, and William T. Windsor, Analytical Chemistry, Vol.79, No.12, (June 15, 2007) 4538-4542