鑑尋BTK抑制劑的新作用標的與其作用機制之探討

Abstract

對於標靶藥物而言，了解它完整的作用標的族群是非常重要的，這可以幫助我們研究藥物作用的機制、增強藥效、開發新的適應症、以及減少藥物的副作用。BI1 是一種第二代Bruton’s tyrosine kinase (BTK)抑制劑，目前用於慢性淋巴性白血病(CLL)的治療，雖然BI1於臨床使用中有很好的藥效，但有別於大多數抗癌藥物，BI1對於表達有BTK這個作用標的的CLL細胞於生理濃度時幾乎沒有毒殺性，因此它的藥物作用機制仍有許多部分等待探討。本研究的目的就是希望能夠找到BI1新的藥物作用標的，並了解其在BI1抗癌的作用機制中是否扮演角色。我首先測試了BI1對於B細胞淋巴癌與前列腺癌細胞的藥效，並發現它對於抑制癌細胞生長與移動能力的藥效並沒有和癌細胞中的BTK表達量呈正相關，由此可以看出BI1存在有除了BTK以外新的藥物作用標的的可能性。我們與台大生化科學研究所的張震東教授合作，使用了一種BI1專一性的抗體將癌細胞當中所有被BI1結合的蛋白質以免疫沉澱的方式進行純化，並以LC-MS/MS進行蛋白質身分鑑定，獲得了在細胞中BI1作用蛋白的清單，並從中篩選出了L-plastin作為新的藥物標的進行後續的研究。實驗結果顯示，BI1與L-plastin有直接的共價鍵連接，並且可以抑制L-plastin綑綁肌動蛋白(actin bundling)的功能。這些結果共同證實了L-plastin是BI1抑制前列腺癌細胞侵襲能力中的新作用標的。這個發現或許能讓我們對於BI1藥物作用機制有更進一步的瞭解，也提出了前列腺癌作為BI1新適應症的可能性。

Identification of a drug target profile is crucial for clearly understanding of the drug mechanism of action, improvement of the drug efficacy, new indications of the drug, or reduction of side effects. BI1 is one of the second-generation Bruton’s tyrosine kinase (BTK) inhibitors and currently used in clinic for the treatment of CLL. Although BI1 exhibits a good efficacy on CLL, it still remains elusive because of its no or low cytotoxicity on BTK-positive CLL cancer cells at the physiological concentrations. The purpose of this study is to identify a novel drug target profile of BI1 and to understand its mechanism of action in human cancer. I found that the inhibitory efficacy of BI1 on the cancer cell proliferation and mobility is not dependent on the BTK levels in the B cell and prostate cancer cells, and encouraged us to identify novel target(s) of BI1. We collaborated with Dr. Geen-Dong Chang and used an antibody-based approach to pull down the BI1-labeled proteins in human B cell and prostate cancer cells, and applied LC-MS/MS analysis to reveal the identity of the target candidates. With this approach, I isolated L-plastin to be a novel target of BI1. BI1 can form a covalent linkage with L-plastin and suppress its actin-bundling function. The data indicate that L-plastin is a novel target for BI1 to suppress prostate cancer cell invasion. This findings provide more insights into the mechanism which lays behind the drug actions of this newly-developed BTK inhibitor, and give a new opportunity for indication of BI1 against prostate cancer.