擺動胺基酸支鍊與氫鍵Constraint 對改善SRC激酶虛擬篩選的影響

Abstract

SRC Kinase為細胞中訊息傳遞的重要成員，其活性大小除了對細胞的增殖速率具調控作用外，同時也直接影響了“癌細胞的轉移”，若能以虛擬篩選的方式搜尋出可對SRC Kinase進行抑制的藥物分子，便可在研發資源有限的條件下，協助找出有效控制癌細胞生長及轉移的標靶藥物。本研究主要以改善SRC Kinase虛擬篩選效果為目的，利用DUD資料庫配合擺動胺基酸支鍊與氫鍵Constraint的方法，找出增進SRC Kinase篩選效果的合適條件，並實際應用於虛擬篩選的資料庫。 首先，對同序列的三個SRC Kinase結晶結構 – PDB碼: 2SRC、1Y57及1FMK 進行活性中心10 Å範圍內所有胺基酸支鍊的擺動，雖然三者間並沒有共同可使Enrichment Factor (EF) 值明顯提升之胺基酸，但可以知道位於Hinge、Gatekeeper以及Activation Loop上那些胺基酸對計算所得的EF值有重要影響。接著對SRC Kinase進行兩種不同結合方式的氫鍵Constraint，分別為Type A與B，兩者的差別主要在於Type A同時對Hinge上兩個胺基酸主鍊具氫鍵連結，Type B則是對單一胺基酸，結果發現Type B氫鍵Constraint可將EF值平均上升至原本的兩倍。於是便將以上兩部分可將EF值改善之條件進行組合，得知Type B氫鍵Constraint配合擺動Asp404胺基酸可使三結晶結構EF值平均提升至原本的將近三倍左右。最後把以上所得結論運用於DUD以外的資料庫，本研究是以ZINC資料庫來進行篩選驗證，而驗證結果與使用DUD資料庫結果類似。

SRC kinase plays a significant role in signal transduction. Its activity level is associated with cell proliferation and the migration of cancer cells. Virtual Screening is one of widely used computational tools in reducing research cost to develop drugs. Our aim is to develop computational protocols for improving the Virtual Screening of SRC kinase by side chain flexibility and hydrogen bonds constraint(s) in docking computation. First, we used DUD database to benchmark these effects, and later applied the protocols to one larger database, ZINC database. In the first place, we used three SRC Kinase crystal structures (PDB codes: 2SRC, 1Y57, and 1FMK) for docking computations and allowed all side chains within 10 Å centered at a selected point of the active site to move. Although there is no common residue’s side chain movement that can improve EF value among the examined three structures, individual effect of each examined side chain located at hinge, gatekeeper segments, and activation loop on calculated EF values are reported. After that, we applied two different kinds of hydrogen bond constraints in docking against SRC kinase. They are called Type A and B. Type A is that the ligands having hydrogen bonds with two residues of SRC kinase, and Type B is just connecting with one residue on the hinge segment. The results show that Type B hydrogen bond constraint approximately doubles the average EF value for those three crystal structures. Subsequently, we combined both side chain flexibility and hydrogen bond constraint and found that Type B hydrogen bond constraint with Asp404 side chain flexibility enhance the average EF value approximately in three-fold. Finally, we applied the above optimized conditions to the ZINC database. The calculated results are similar to the results above, and are analyzed and discussed.