PIM2蛋白在脫輔基和抑制劑結合狀態下的結構研究

Abstract

PIM蛋白的致癌效果主要是透過增生機制使細胞不斷的生長，如透過增強MYC蛋白的轉錄活性、增強cap-dependent translation以及細胞週期的進行、透過活化MCL1的基因以及磷酸化Bad蛋白來達到抑制細胞凋亡。PIM蛋白是一種經由轉譯後即有活性的絲氨酸/蘇氨酸激酵素，且PIM1經常廣泛的表現於造血細胞，而PIM2則表現於血液惡性腫瘤，包括了急性骨髓性白血病、慢性淋巴細胞白血病、彌漫性大B細胞淋巴瘤。重要的是，在已含有抗藥性的癌症當中，PIM2的抑制可以使得癌細胞恢復細胞凋亡，這是目前癌症治療當中極具潛力的療法之一。而近日來與PIM蛋白相關的抑制劑設計以及合成，均是以PIM1為主要目標，主要是因為PIM1不管是脫輔基態或是抑制劑結合態的結構均廣為人知。然而PIM2目前僅只有兩篇抑制劑結合態的結構解出，而根據此兩篇解出之結構與PIM1相比，發現確實有些許的不同可以用來作為PIM2專一抑制劑的參考。因此我們的研究目標便是解出PIM2的脫輔基態以及抑制劑結合態的結構，以便更進一步提供日後藥物設計的重要參考。首先我們使大腸桿菌成功的表現出PIM2，並利用鎳離子親和性樹脂以及膠體過濾法純化出PIM2蛋白。接著便使用前結晶測試法來測出PIM2適合的結晶濃度，並利用機械手臂測試了市售的結晶試劑，然而並未發現任何晶體，因此改用其它策略。一：利用TEV酵素切去PIM2蛋白N端的his-tag，以減少可動端。二：根據隨機微晶種法，利用PIM1晶體做為PIM2養晶時之晶種。三：利用多種截短型之PIM2來幫助晶體篩選(如N端截短型以及N,C端截短型)。至此為止，我們仍致力於上述養晶實驗。我們同時也利用了恆溫滴定微卡計以及差式掃描量熱儀來探測SGI-1776、5-carboxamidoindole以及Imatinib mesylate是否會和PIM2進行結合，藉此即可利用可結合之藥物進行共結晶。測試結果出來僅有SGI-1776以及5- carboxamidoindole可和PIM2進行結合。因此我們便將此兩種抑制劑與上述各種型式之PIM2蛋白進行共結晶，以期結出多種型式下的PIM2結構而利於日後人們對於PIM2的藥物設計。

The tumorigenic effects of PIM kinases are perpetuated through proliferative mechanisms including potentiation of MYC transcriptional activity, enhancement of cap-dependent translation, and cell cycle progression, as well as antiapoptotic effects with up-regulation of MCL1 and Bad phosphorylation. The PIM proteins are constitutively active serine/threonine kinases that are ubiquitously expressed with predominance for PIM1 in hematopoietic cells and for PIM2 in hematological malignancies including acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and diffuse large B-cell. Importantly, PIM2 inactivation can restore apoptosis to otherwise drug-resistant cancers and is therefore an attractive therapy to supplement current drug regimen. Several recent publications report the design and synthesis of new PIM kinase inhibitors targeting mainly PIM1. The availability of PIM1 kinase crystal structures at apo and drig-bound statues is playing a key role in the identification of new inhibitors. However, there were only two crystal structures of PIM2 kinase in complex with inhibitor until now and it revealed some differences to PIM1 which may be explored further to generate isoform selective inhibitors. Therefore, we decide to solve the structure of PIM-2 at apo and drug-bound states and it will provide significant structural and biological insights that should greatly benefit a structure-based drug design. We have successfully expressed PIM2 in E. coli and purified it with Ni column and gel-filtration. Then we used Pre-Crystallization Test (PCT) to test the suitable concentration of PIM2 for crystallization and crystal screening kit to find the suitable crystal condition. However, it did not grow any crystals. So we adopted other strategies to obtain crystal: 1. His-tag of PIM2 N-terminal was removed by TEV protease so it can reduce flexible part; 2. we used PIM1 crystal as seed to co-crystallize with PIM2 (random Microseed Matrix Screening, rMMS); 3. the various truncated constructs of PIM2 including N-terminal truncated forms and N,C-terminal truncated forms were used to crystal screening . Up to now, we still exert our best to get crystal. We also used Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC) to detect which kinds of inhibitors (SGI-1776、5-carboxamidoindole and Imatinib mesylate) will interact with PIM2 kinase, so we can use theses inhibitors to co-crystallize with PIM2 kinase. Then Only SGI-1776 and 5-carboxamidoindole can interact with PIM2 kinase. Then we adopted various PIM2 (including full length、N-terminal truncated form and N,C-terminal truncated form) to co-crystallize with SGI-1776 and 5- carboxamidoindole. We hope to get more kinds of PIM2 structure then assist people to generate further isoform selective inhibitors.