摘要
多发性骨髓瘤是第二大恶性血液肿瘤疾病，大约占血液系统肿瘤的10%。由于缺乏有效药物，多发性骨髓瘤的死亡率一直高居不下，占年癌症总死亡率的2%，因此急需开发新型的分子靶标抗骨髓瘤药物。PI3K/AKT通路与多发性骨髓瘤的存活、分化、迁移、血管生成以及耐药性都密切相关，因此PI3K/AKT已经成为开发抗骨髓瘤药物的一个理想靶标。目前已经有一些抑制剂被报道，有些已经进入了临床试验。随着组合化学、肿瘤生物学、生物信息学以及生物工程的高速发展，使得大规模筛选PI3K抑制剂的方法被越来越广泛地采用。与传统的高通量筛选相比，计算机虚拟筛选具有高效、快速、低成本的优点，使它成为高通量筛选的一个重要辅助方法。在本课题中，我们结合虚拟筛选和生物学实验的方法来发现新的PI3K抑制剂，并阐明其抗多发性骨髓瘤的分子机制。
研究方法：
(1)使用逐层虚拟筛选的方法来发现新的高效的PI3K虚拟抑制剂。
(2)使用MTT实验法检测虚拟化合物对细胞活性水平的初筛，发现在细胞水平具有活性的化合物C98。
(3)酶活性实验分析C98对PI3K的抑制作用。
(4) Western blotting方法检测经C98处理之后细胞蛋白水平的表达，包括对PI3K通路的抑制以及对骨髓瘤细胞的促凋亡作用。
(5)流式细胞技术检测C98对多发性骨髓瘤细胞的促凋亡作用。
(6)免疫荧光实验用来检测AKT的细胞表达水平及亚细胞定位。
(7)建立多发性骨髓瘤异种移植小鼠模型，检测C98对骨髓瘤生长的影响。
(8)使用分子对接和分子动力学模拟方法在分子水平阐明C98抑制PI3K的作用机理。
研究结果：
(1)通过逐层虚拟筛选的方法对Specs和Chembridge化合物文库的80万个化合物进行筛选，经成药性检测以及聚类分析之后，得到了148个潜在的PI3K抑制剂。
(2)采用MTT方法将上述148个化合物处理多发性骨髓瘤OPM2细胞，最后得到了6个具有抑制OPM2细胞增殖的化合物，然后使用IGF-1刺激法初步验证了这6个化合物中C98具有较好的PI3K抑制活性。
(3)酶活性实验发现C98可以抑制I型PI3Ks，其中对PI3K和γ抑制活性最高达到纳摩尔级，但是对AKT及mTOR没有明显抑制作用。
(4) Western blotting方法检测到C98特异性地抑制PI3K/AKT/mTOR通路，而对其他通路的蛋白（IGF-1、MAPK、ERK、p38、c-Src、STAT3等）没有抑制作用。
(5)免疫荧光实验发现C98可以阻止AKT细胞质膜转移，从而抑制它的磷酸化。
(6)伴随着对PI3K的抑制，C98可以诱导骨髓瘤细胞的凋亡。流式细胞术分析发现C98对骨髓瘤细胞的促凋亡作用呈浓度依赖性关系。Western blotting检测发现伴随着磷酸化AKT的抑制，C98可以引起细胞内Caspase-3和PARP的裂解激活。
(7)使用C98处理OPM2和JJN3肿瘤模型小鼠，发现灌胃给药C98在3周内可以显著抑制肿瘤的生长，并呈剂量效应关系。C98对肿瘤细胞的抑制与AKT的磷酸化关联。
(8)分子对接和分子动力学研究发现在C98与PI3K的相互作用中氢键以及范德华相互作用起了重要的作用。例如C98的氨基（-NH2）与各亚型的对应残基都形成了相应的氢键相互作用：PI3K的Ser854、PI3Kβ的Val848、PI3K的Ser831、PI3Kγ的Ala885。而PI3Ks与C98也形成了数目不等的π-H相互作用：PI3K的Tyr836、Ile848、Val850、Ile932；PI3Kβ的Ile930；PI3K的Tyr813、Ile930；PI3Kγ的Tyr867。这些相互作用对于稳定C98的结合同样起到了积极的作用。
结论
本课题结合逐层虚拟筛选和生物学实验的方法发现了I型PI3K广谱抑制剂C98。它可以与PI3K的ATP口袋相结合从而抑制PI3K的活性，并特异性地抑制PI3K/AKT信号通路，从而诱导多发性骨髓瘤细胞的凋亡，延缓肿瘤细胞的生长。由于C98表现出了良好的口服活性和较低的毒性，C98可以发展成为一个高效的抗多发性骨髓瘤药物。
Multiple myeloma is the second most hematological malignancy, accounting formore than10%of all blood cancers and2%of annual cancer-related deaths due tolack of curable drugs. Novel and molecularly targeted anti-MM drugs are in urgentneed. The phosphatidylinositol3-kinase (PI3K)/AKT signaling pathway plays acritical regulatory role in the pathophysiology of multiple myeloma, includingsurvival, proliferation, migration, angiogenesis, and chemoresistance. PI3K hastherefore emerged as a key therapeutic target. Many potent inhibitors targeting thispathway have been developed and some have been moved for clinical evaluations formultiple myeloma. With the advances of combinatorial chemistry, cancer biology,informatics technology and bioengineering, large-scale screening become more andmore common in PI3K-targeted drug discovery. Comparing to traditional highthroughput screening, virtual screening is reliable, cost-effective and time-saving, ithas become an important complementary approach to experimental large-scalescreening. In this project, we combined virtual screening and wet-lab experiments todiscover new PI3K inhibitors and found a potential new candidate for targeted therapyin multiple myeloma.
Methods:
(1) A hierarchical virtual screening was employed to find novel PI3K inhibitors.
(2) MTT assay was used to further identify potential PI3K inhibitors that inhibitMM cell proliferation.
(3) Cell-free assay was used to determine PI3K inhibitory effects of C98.
(4) Western blotting was performed to examine the expression of proteins after the treatment of C98that involved in cell apoptosis and PI3K/AKT signalingpathway.
(5) Flow cytometry was used to measure apoposis of myeloma cells induced byC98.
(6) Immunofluorescence analysis was used to determine the cell expression andcellular localization of p-AKT in OPM2cells.
(7) Human myeloma xenograft models in nude mice were used to evaluate thetherapeutic effects of C98in vivo.
(8) Molecular docking and molecular dynamic stimulation were employed toinvestigate the mechnism of PI3K inhibition by C98.
Results:
(1) The hierarchical virtual screening combined with drug-like test and clusteranalysis led to148virtual “top-hits” after screening against800,000of smallmolecule compounds from the Specs and Chembridge Chemicals Libraries.
(2) Six compounds showed potent anti-proliferation in the multiple myeloma cellline OPM2. These compounds were further subject to PI3K inhibition evaluated incells after IGF-1stimulation, from which C98was found to show potent inhibition ofAKT activation.
(3) Enzymatic reaction assays in the cell free system showed that C98inhibitshuman PI3K, β, and γ at nanomolar or low micromolar concentrations but does notsuppress AKT or mTOR activity.
(4) C98specifically inhibits the PI3K signaling pathway and has no inhibitoryeffects on other signal transduction pathways such as IGF-1, MAPK, ERK, p38, c-Srcand STAT3.
(5) Immunofluorescence assay showed that C98blocks AKT translocation to thecytoplamic membrane.
(6) C98induces apoptosis of MM cells in parallel with its PI3K inhibition. Cellapoptosis was found in C98-treated cells as measured by Annexin V-FITC and PIstaining. The results of Western blotting showed that C98activated Caspase-3andPARP in parallel with its p-AKT inhibition.
(7) C98significantly delayed myeloma tumor growth in OPM2and JJN3xenograft models. C98delayed MM tumor growth at least partly in associated with itsinhibition of PI3K/AKT signaling pathway.
(8) Molecular docking and MD stimulations confirm that the H-bonds and van deWaals contacts are important for the interference of C98with the ATP active pocket ofPI3Ks. These interactions established between specific groups of C98and conservedhydrophobic residues of the ATP-binding cleft may account for its potency againstPI3K and selectivity profile over protein kinases.
Conclusion: A successful Virtual Screen-based identification of PI3K inhibitorswas established which leads to discovery of C98, a pan-Class I PI3K inhibitor,confirmed by a series of biochemical, cellular and animal studies. By binding to andinterfering with the ATP active cleft in PI3K enzymes, C98specifically blocks thePI3K/AKT signaling pathway. The impressive oral activity and well tolerance of C98in in-vivo models highlights its candidacy in MM treatment.
引文
1. Anderson KC, Carrasco RD. Pathogenesis of myeloma. Annu Rev Pathol.2011;6:249-274.
2. Kuehl WM, Bergsagel PL. Multiple myeloma: evolving genetic events and host interactions.Nat Rev Cancer.2002;2(3):175-187.
3. Avet-Loiseau H, Attal M, Moreau P, et al. Genetic abnormalities and survival in multiplemyeloma: the experience of the Intergroupe Francophone du Myelome. Blood.2007;109(8):3489-3495.
4. International Myeloma Working G. Criteria for the classification of monoclonalgammopathies, multiple myeloma and related disorders: a report of the International MyelomaWorking Group. Br J Haematol.2003;121(5):749-757.
5. Siegel R, Naishadham D, Jemal A. Cancer statistics,2013. CA Cancer J Clin.2013;63(1):11-30.
6. Anderson KC. New insights into therapeutic targets in myeloma. Hematology Am SocHematol Educ Program.2011;2011:184-190.
7. Mahindra A, Laubach J, Raje N, Munshi N, Richardson PG, Anderson K. Latest advancesand current challenges in the treatment of multiple myeloma. Nat Rev Clin Oncol.2012;9(3):135-143.
8. Fonseca R, Barlogie B, Bataille R, et al. Genetics and cytogenetics of multiple myeloma: aworkshop report. Cancer Res.2004;64(4):1546-1558.
9. Cantrell DA. Phosphoinositide3-kinase signalling pathways. J Cell Sci.2001;114(Pt8):1439-1445.
10. Kawauchi K, Ogasawara T, Yasuyama M, Otsuka K, Yamada O. The PI3K/Akt pathway as atarget in the treatment of hematologic malignancies. Anticancer Agents Med Chem.2009;9(5):550-559.
11. San-Miguel J, Harousseau JL, Joshua D, Anderson KC. Individualizing treatment of patientswith myeloma in the era of novel agents. J Clin Oncol.2008;26(16):2761-2766.
12. Pene F, Claessens YE, Muller O, et al. Role of the phosphatidylinositol3-kinase/Akt andmTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma. Oncogene.2002;21(43):6587-6597.
13. Leevers SJ, Vanhaesebroeck B, Waterfield MD. Signalling through phosphoinositide3-kinases: the lipids take centre stage. Curr Opin Cell Biol.1999;11(2):219-225.
14. Vivanco I, Sawyers CL. The phosphatidylinositol3-Kinase AKT pathway in human cancer.Nat Rev Cancer.2002;2(7):489-501.
15. Pal I, Mandal M. PI3K and Akt as molecular targets for cancer therapy: current clinicaloutcomes. Acta Pharmacol Sin.2012;33(12):1441-1458.
16. Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway in human cancer:rationale and promise. Cancer Cell.2003;4(4):257-262.
17. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD. Cellular function ofphosphoinositide3-kinases: implications for development, homeostasis, and cancer. Annu RevCell Dev Biol.2001;17:615-675.
18. Vanhaesebroeck B, Guillermet-Guibert J, Graupera M, Bilanges B. The emergingmechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol.2010;11(5):329-341.
19. Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterfield MD. Phosphoinositide3-kinases: aconserved family of signal transducers. Trends Biochem Sci.1997;22(7):267-272.
20. Falasca M, Maffucci T. Role of class II phosphoinositide3-kinase in cell signalling. BiochemSoc Trans.2007;35(Pt2):211-214.
21. Vieira OV, Botelho RJ, Rameh L, et al. Distinct roles of class I and class IIIphosphatidylinositol3-kinases in phagosome formation and maturation. J Cell Biol.2001;155(1):19-25.
22. Simonsen A, Tooze SA. Coordination of membrane events during autophagy by multipleclass III PI3-kinase complexes. J Cell Biol.2009;186(6):773-782.
23. Mahindra A, Cirstea D, Raje N. Novel therapeutic targets for multiple myeloma. FutureOncol.2010;6(3):407-418.
24. Younes H, Leleu X, Hatjiharissi E, et al. Targeting the phosphatidylinositol3-kinase pathwayin multiple myeloma. Clin Cancer Res.2007;13(13):3771-3775.
25. Amzel LM, Huang CH, Mandelker D, Lengauer C, Gabelli SB, Vogelstein B. Structuralcomparisons of class I phosphoinositide3-kinases. Nat Rev Cancer.2008;8(9):665-669.
26. Maira SM, Voliva C, Garcia-Echeverria C. Class IA phosphatidylinositol3-kinase: from theirbiologic implication in human cancers to drug discovery. Expert Opin Ther Targets.2008;12(2):223-238.
27. Fruman DA. Regulatory subunits of class IA PI3K. Curr Top Microbiol Immunol.2010;346:225-244.
28. Whitehead MA, Bombardieri M, Pitzalis C, Vanhaesebroeck B. Isoform-selective inductionof human p110delta PI3K expression by TNFalpha: identification of a new and inducible PIK3CDpromoter. Biochem J.2012;443(3):857-867.
29. Samuels Y, Ericson K. Oncogenic PI3K and its role in cancer. Curr Opin Oncol.2006;18(1):77-82.
30. Ali K, Bilancio A, Thomas M, et al. Essential role for the p110delta phosphoinositide3-kinase in the allergic response. Nature.2004;431(7011):1007-1011.
31. Billottet C, Grandage VL, Gale RE, et al. A selective inhibitor of the p110delta isoform of PI3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic effects of VP16.Oncogene.2006;25(50):6648-6659.
32. Sujobert P, Bardet V, Cornillet-Lefebvre P, et al. Essential role for the p110delta isoform inphosphoinositide3-kinase activation and cell proliferation in acute myeloid leukemia. Blood.2005;106(3):1063-1066.
33. Liu P, Cheng H, Roberts TM, Zhao JJ. Targeting the phosphoinositide3-kinase pathway incancer. Nat Rev Drug Discov.2009;8(8):627-644.
34. Fruman DA, Rommel C. PI3K and cancer: lessons, challenges and opportunities. Nat RevDrug Discov.2014;13(2):140-156.
35. Suire S, Coadwell J, Ferguson GJ, Davidson K, Hawkins P, Stephens L. p84, a new G betagamma-activated regulatory subunit of the type IB phosphoinositide3-kinase p110gamma.Current Biology.2005;15(6):566-570.
36. Voigt P, Dorner MB, Schaefer M. Characterization of p87(PIKAP), a novel regulatorysubunit of phosphoinositide3-kinase gamma that is highly expressed in heart and interacts withPDE3B. Journal of Biological Chemistry.2006;281(15):9977-9986.
37. Patrucco E, Notte A, Barberis L, et al. PI3K gamma modulates the cardiac response tochronic pressure overload by distinct kinase-dependent and-independent effects. Cell.2004;118(3):375-387.
38. Sasaki T, Irie-Sasaki J, Jones RG, et al. Function of PI3K gamma in thymocyte development,T cell activation, and neutrophil migration. Science.2000;287(5455):1040-1046.
39. Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M.PI3K/Akt signalling pathway and cancer. Cancer Treat Rev.2004;30(2):193-204.
40. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell.2007;129(7):1261-1274.
41. Thomas CC, Deak M, Alessi DR, van Aalten DM. High-resolution structure of the pleckstrinhomology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)-trisphosphate.Curr Biol.2002;12(14):1256-1262.
42. Tsuchiya A, Kanno T, Nishizaki T. PI3kinase directly phosphorylates Akt1/2at Ser473/474in the insulin signal transduction pathway. J Endocrinol.2013;220(1):49-59.
43. Downward J. Mechanisms and consequences of activation of protein kinase B/Akt. CurrentOpinion in Cell Biology.1998;10(2):262-267.
44. Stephens L, Anderson K, Stokoe D, et al. Protein kinase B kinases that mediatephosphatidylinositol3,4,5-trisphosphate-dependent activation of protein kinase B. Science.1998;279(5351):710-714.
45. Alessi DR, James SR, Downes CP, et al. Characterization of a3-phosphoinositide-dependentprotein kinase which phosphorylates and activates protein kinase B alpha. Current Biology.1997;7(4):261-269.
46. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature.2001;411(6835):355-365.
47. Dong LQ, Liu F. PDK2: the missing piece in the receptor tyrosine kinase signaling pathwaypuzzle. Am J Physiol Endocrinol Metab.2005;289(2):E187-196.
48. Currie RA, Walker KS, Gray A, et al. Role of phosphatidylinositol3,4,5-trisphosphate inregulating the activity and localization of3-phosphoinositide-dependent protein kinase-1.Biochemical Journal.1999;337:575-583.
49. Cully M, You H, Levine AJ, Mak TW. Beyond PTEN mutations: the PI3K pathway as anintegrator of multiple inputs during tumorigenesis. Nat Rev Cancer.2006;6(3):184-192.
50. Komander D, Fairservice A, Deak M, et al. Structural insights into the regulation of PDK1byphosphoinositides and inositol phosphates. EMBO J.2004;23(20):3918-3928.
51. Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell.2007;12(1):9-22.
52. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation ofAkt/PKB by the rictor-mTOR complex. Science.2005;307(5712):1098-1101.
53. Sale EM, Hodgkinson CP, Jones NP, Sale GJ. A new strategy for studying protein kinase Band its three isoforms. Role of protein kinase B in phosphorylating glycogen synthase kinase-3,tuberin, WNK1, and ATP citrate lyase. Biochemistry.2006;45(1):213-223.
54. Kumar A, Purohit R. Cancer associated E17K mutation causes rapid conformational drift inAKT1pleckstrin homology (PH) domain. PLoS One.2013;8(5):e64364.
55. Cheng JQ, Lindsley CW, Cheng GZ, Yang H, Nicosia SV. The Akt/PKB pathway: moleculartarget for cancer drug discovery. Oncogene.2005;24(50):7482-7492.
56. Rohrschneider LR, Fuller JF, Wolf I, Liu Y, Lucas DM. Structure, function, and biology ofSHIP proteins. Genes&Development.2000;14(5):505-520.
57. Kalesnikoff J, Sly LM, Hughes MR, et al. The role of SHIP in cytokine-induced signaling.Reviews of Physiology, Biochemistry and Pharmacology, Vol149.2004;149:87-103.
58. Stambolic V, Suzuki A, de la Pompa JL, et al. Negative regulation of PKB/Akt-dependentcell survival by the tumor suppressor PTEN. Cell.1998;95(1):29-39.
59. Liu QR, Sasaki T, Kozieradzki I, et al. SHIP is a negative regulator of growth factorreceptor-mediated PKB/Akt activation and myeloid cell survival. Genes&Development.1999;13(7):786-791.
60. Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated inhuman brain, breast, and prostate cancer. Science.1997;275(5308):1943-1947.
61. Cantley LC, Neel BG. New insights into tumor suppression: PTEN suppresses tumorformation by restraining the phosphoinositide3-kinase AKT pathway. Proceedings of the NationalAcademy of Sciences of the United States of America.1999;96(8):4240-4245.
62. Liu S, Knapp S, Ahmed AA. The Structural Basis of PI3K Cancer Mutations: FromMechanism to Therapy. Cancer Res.2014;74(3):641-646.
63. Hassan B, Akcakanat A, Holder AM, Meric-Bernstam F. Targeting the PI3-kinase/Akt/mTORsignaling pathway. Surg Oncol Clin N Am.2013;22(4):641-664.
64. Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: its functions and alterations in humancancer. Apoptosis.2004;9(6):667-676.
65. Braccini L, Ciraolo E, Martini M, et al. PI3K keeps the balance between metabolism andcancer. Adv Biol Regul.2012;52(3):389-405.
66. Podar K, Chauhan D, Anderson KC. Bone marrow microenvironment and the identificationof new targets for myeloma therapy. Leukemia.2009;23(1):10-24.
67. Bianchi G, Ghobrial IM. Molecular mechanisms of effectiveness of novel therapies inmultiple myeloma. Leuk Lymphoma.2013;54(2):229-241.
68. Chauhan D, Uchiyama H, Akbarali Y, et al. Multiple myeloma cell adhesion-inducedinterleukin-6expression in bone marrow stromal cells involves activation of NF-kappa B. Blood.1996;87(3):1104-1112.
69. Chauhan D, Uchiyama H, Urashima M, Yamamoto K, Anderson KC. Regulation ofinterleukin6in multiple myeloma and bone marrow stromal cells. Stem Cells.1995;13Suppl2:35-39.
70. Ara T, Declerck YA. Interleukin-6in bone metastasis and cancer progression. Eur J Cancer.2010;46(7):1223-1231.
71. Qiang YW, Yao L, Tosato G, Rudikoff S. Insulin-like growth factor I induces migration andinvasion of human multiple myeloma cells. Blood.2004;103(1):301-308.
72. Ge NL, Rudikoff S. Insulin-like growth factor I is a dual effector of multiple myeloma cellgrowth. Blood.2000;96(8):2856-2861.
73. Saile B, DiRocco P, Dudas J, et al. IGF-I induces DNA synthesis and apoptosis in rat liverhepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts (rMF).Lab Invest.2004;84(8):1037-1049.
74. Mairet-Coello G, Tury A, DiCicco-Bloom E. Insulin-like growth factor-1promotes G(1)/Scell cycle progression through bidirectional regulation of cyclins and cyclin-dependent kinaseinhibitors via the phosphatidylinositol3-kinase/Akt pathway in developing rat cerebral cortex. JNeurosci.2009;29(3):775-788.
75. Tu YP, Gardner A, Lichtenstein A. The phosphatidylinositol3-kinase/AKT kinase pathway inmultiple myeloma plasma cells: Roles in cytokine-dependent survival and proliferative responses.Cancer Research.2000;60(23):6763-6770.
76. Akiyama M, Hideshima T, Hayashi T, et al. Cytokines modulate telomerase activity in ahuman multiple myeloma cell line. Cancer Research.2002;62(13):3876-3882.
77. Chesi M, Nardini E, Lim RS, Smith KD, Kuehl WM, Bergsagel PL. The t(4;14) translocationin myeloma dysregulates both FGFR3and a novel gene, MMSET, resulting in IgH/MMSEThybrid transcripts. Blood.1998;92(9):3025-3034.
78. Ismail SI, Mahmoud IS, Msallam MM, Sughayer MA. Hotspot mutations of PIK3CA andAKT1genes are absent in multiple myeloma. Leuk Res.2010;34(6):824-826.
79. Ikeda H, Hideshima T, Fulciniti M, et al. PI3K/p110{delta} is a novel therapeutic target inmultiple myeloma. Blood.2010;116(9):1460-1468.
80. An HJ, Cho NH, Yang HS, et al. Targeted RNA interference of phosphatidylinositol3-kinasep110-beta induces apoptosis and proliferation arrest in endometrial carcinoma cells. J Pathol.2007;212(2):161-169.
81. Zhang J, Choi Y, Mavromatis B, Lichtenstein A, Li W. Preferential killing of PTEN-nullmyelomas by PI3K inhibitors through Akt pathway. Oncogene.2003;22(40):6289-6295.
82. Ge NL, Rudikoff S. Expression of PTEN in PTEN-deficient multiple myeloma cellsabolishes tumor growth in vivo. Oncogene.2000;19(36):4091-4095.
83. Alkan S, Izban KF. Immunohistochemical localization of phosphorylated AKT in multiplemyeloma. Blood.2002;99(6):2278-2279.
84. Hsu J, Shi Y, Krajewski S, et al. The AKT kinase is activated in multiple myeloma tumorcells. Blood.2001;98(9):2853-2855.
85. Mitsiades CS, Mitsiades N, Poulaki V, et al. Activation of NF-kappaB and upregulation ofintracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells:therapeutic implications. Oncogene.2002;21(37):5673-5683.
86. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating andinhibiting a Forkhead transcription factor. Cell.1999;96(6):857-868.
87. Hideshima T, Catley L, Raje N, et al. Inhibition of Akt induces significant downregulation ofsurvivin and cytotoxicity in human multiple myeloma cells. Br J Haematol.2007;138(6):783-791.
88. Hideshima T, Catley L, Yasui H, et al. Perifosine, an oral bioactive novel alkylphospholipid,inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells. Blood.2006;107(10):4053-4062.
89. Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB. Exploiting the PI3K/AKT pathway forcancer drug discovery. Nat Rev Drug Discov.2005;4(12):988-1004.
90. Bergsagel PL, Kuehl WM. Critical roles for immunoglobulin translocations and cyclin Ddysregulation in multiple myeloma. Immunol Rev.2003;194:96-104.
91. Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N. CyclinD expression is controlled post-transcriptionally via a phosphatidylinositol3-kinase/Akt-dependent pathway. J Biol Chem.1998;273(45):29864-29872.
92. Bergsagel PL, Kuehl WM, Zhan FH, Sawyer J, Barlogie B, Shaughnessy J. Cyclin Ddysregulation: an early and unifying pathogenic event in multiple myeloma. Blood.2005;106(1):296-303.
93. Flinn W, Byrd J, Furman R, et al. Preliminary Evidence of Clinical Activity in a Phase1Study of Cal-101, a Potent Selective Inhibitor of the P110delta Isoform of Phosphatidylinositol3-Kinase, in Patient with B-Cell Malignancies. Haematologica-the Hematology Journal.2009;94:303-303.
94. Herman SE, Gordon AL, Wagner AJ, et al. Phosphatidylinositol3-kinase-delta inhibitorCAL-101shows promising preclinical activity in chronic lymphocytic leukemia by antagonizingintrinsic and extrinsic cellular survival signals. Blood.2010;116(12):2078-2088.
95. Castillo JJ, Furman M, Winer ES. CAL-101: a phosphatidylinositol-3-kinase p110-deltainhibitor for the treatment of lymphoid malignancies. Expert Opin Investig Drugs.2012;21(1):15-22.
96. Zheng YH, Yang J, Qian JF, et al. Novel phosphatidylinositol3-kinase inhibitorNVP-BKM120induces apoptosis in myeloma cells and shows synergistic anti-myeloma activitywith dexamethasone. Journal of Molecular Medicine-Jmm.2012;90(6):695-706.
97. Rodon J, Bendell J, Razak ARA, et al. A Phase I Dose Escalation and Expansion Trial ofBkm120, an Oral Pan-Pi3k Inhibitor, in Patients with Advanced Solid Tumors: Analysis ofPharmacodynamic Biomarker Data. Annals of Oncology.2012;23:158-159.
98. McMillin DW, Ooi M, Delmore J, et al. Antimyeloma activity of the orally bioavailable dualphosphatidylinositol3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235. CancerRes.2009;69(14):5835-5842.
99. Maira SM, Furet P, Stauffer F. Discovery of novel anticancer therapeutics targeting thePI3K/Akt/mTOR pathway. Future Med Chem.2009;1(1):137-155.
100. Fokas E, Yoshimura M, Prevo R, et al. NVP-BEZ235and NVP-BGT226, dualphosphatidylinositol3-kinase/mammalian target of rapamycin inhibitors, enhance tumor andendothelial cell radiosensitivity. Radiation Oncology.2012;7.
101. Chang KY, Tsai SY, Wu CM, Yen CJ, Chuang BF, Chang JY. Novel Phosphoinositide3-Kinase/mTOR Dual Inhibitor, NVP-BGT226, Displays Potent Growth-Inhibitory Activityagainst Human Head and Neck Cancer Cells In Vitro and In Vivo. Clinical Cancer Research.2011;17(22):7116-7126.
102. Glienke W, Maute L, Wicht J, Bergmann L. The dual PI3K/mTOR inhibitor NVP-BGT226induces cell cycle arrest and regulates Survivin gene expression in human pancreatic cancer celllines. Tumor Biology.2012;33(3):757-765.
103. Baumann P, Schneider L, Mandl-Weber S, Oduncu F, Schmidmaier R. Simultaneoustargeting of PI3K and mTOR with NVP-BGT226is highly effective in multiple myeloma.Anti-Cancer Drugs.2012;23(1):131-138.
104. Mao X, Cao B, Wood TE, et al. A small-molecule inhibitor of D-cyclin transactivationdisplays preclinical efficacy in myeloma and leukemia via phosphoinositide3-kinase pathway.Blood.2011;117(6):1986-1997.
105. Harvey RD, Lonial S. PI3kinase/AKT pathway as a therapeutic target in multiple myeloma.Future Oncol.2007;3(6):639-647.
106. De P, Dey N, Terakedis B, et al. An integrin-targeted, pan-isoform, phosphoinositide-3kinaseinhibitor, SF1126, has activity against multiple myeloma in vivo. Cancer Chemother Pharmacol.2013;71(4):867-881.
107. Fan QW, Knight ZA, Goldenberg DD, et al. A dual PI3kinase/mTOR inhibitor revealsemergent efficacy in glioma. Cancer Cell.2006;9(5):341-349.
108. Glassford J, Kassen D, Quinn J, et al. Inhibition of cell cycle progression by dualphosphatidylinositol-3-kinase and mTOR blockade in cyclin D2positive multiple myelomabearing IgH translocations. Blood Cancer Journal.2012;2.
109. Raynaud FI, Eccles SA, Patel S, et al. Biological properties of potent inhibitors of class Iphosphatidylinositide3-kinases: from PI-103through PI-540, PI-620to the oral agent GDC-0941.Mol Cancer Ther.2009;8(7):1725-1738.
110. Munugalavadla V, Mariathasan S, Slaga D, et al. The PI3K inhibitor GDC-0941combineswith existing clinical regimens for superior activity in multiple myeloma. Oncogene.2013.
111. Maira SM, Pecchi S, Huang A, et al. Identification and Characterization of NVP-BKM120,an Orally Available Pan-Class I PI3-Kinase Inhibitor. Molecular Cancer Therapeutics.2012;11(2):317-328.
112. Garlich JR, De P, Dey N, et al. A vascular targeted pan phosphoinositide3-kinase inhibitorprodrug, SF1126, with antitumor and antiangiogenic activity. Cancer Res.2008;68(1):206-215.
113. Wagner AJ, Von Hoff DH, LoRusso PM, et al. A first-in-human phase I study to evaluate thepan-PI3K inhibitor GDC-0941administered QD or BID in patients with advanced solid tumors.Journal of Clinical Oncology.2009;27(15).
114. Maira SM, Stauffer F, Brueggen J, et al. Identification and characterization of NVP-BEZ235,a new orally available dual phosphatidylinositol3-kinase/mammalian target of rapamycininhibitor with potent in vivo antitumor activity. Molecular Cancer Therapeutics.2008;7(7):1851-1863.
115. Raynaud FI, Eccles S, Clarke PA, et al. Pharmacologic characterization of a potent inhibitorof class I phosphatidylinositide3-kinases. Cancer Research.2007;67(12):5840-5850.
116. Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol3-kinase,2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem.1994;269(7):5241-5248.
117. Wymann MP, Bulgarelli-Leva G, Zvelebil MJ, et al. Wortmannin inactivatesphosphoinositide3-kinase by covalent modification of Lys-802, a residue involved in thephosphate transfer reaction. Mol Cell Biol.1996;16(4):1722-1733.
118. Schultz RM, Merriman RL, Andis SL, et al. In vitro and in vivo antitumor activity of thephosphatidylinositol-3-kinase inhibitor, wortmannin. Anticancer Res.1995;15(4):1135-1139.
119. Hu L, Zaloudek C, Mills GB, Gray J, Jaffe RB. In vivo and in vitro ovarian carcinomagrowth inhibition by a phosphatidylinositol3-kinase inhibitor (LY294002). Clin Cancer Res.2000;6(3):880-886.
120. Knight ZA, Chiang GG, Alaimo PJ, et al. Isoform-specific phosphoinositide3-kinaseinhibitors from an arylmorpholine scaffold. Bioorg Med Chem.2004;12(17):4749-4759.
121. Ward SG, Finan P. Isoform-specific phosphoinositide3-kinase inhibitors as therapeuticagents. Curr Opin Pharmacol.2003;3(4):426-434.
122. Wymann MP, Zvelebil M, Laffargue M. Phosphoinositide3-kinase signalling--which way totarget? Trends Pharmacol Sci.2003;24(7):366-376.
123. Kong DX, Yamori T. ZSTK474, a novel phosphatidylinositol3-kinase inhibitor identifiedusing the JFCR39drug discovery system. Acta Pharmacologica Sinica.2010;31(9):1189-1197.
124. Crabbe T. Exploring the potential of PI3K inhibitors for inflammation and cancer.Biochemical Society Transactions.2007;35:253-256.
125. Safavi-Sohi R, Ghasemi JB. Quasi4D-QSAR and3D-QSAR study of the pan class Iphosphoinositide-3-kinase (PI3K) inhibitors. Medicinal Chemistry Research.2013;22(4):1587-1596.
126. Frederick R, Mawson C, Kendall JD, et al. Phosphoinositide-3-kinase (PI3K) inhibitors:Identification of new scaffolds using virtual screening. Bioorganic&Medicinal Chemistry Letters.2009;19(20):5842-5847.
127. Hou T, Xu X. Recent development and application of virtual screening in drug discovery: anoverview. Curr Pharm Des.2004;10(9):1011-1033.
128. Marshall GR. Computer-aided drug design. Annu Rev Pharmacol Toxicol.1987;27:193-213.
129. Zhang S. Computer-aided drug discovery and development. Methods Mol Biol.2011;716:23-38.
130. Ou-Yang SS, Lu JY, Kong XQ, Liang ZJ, Luo C, Jiang H. Computational drug discovery.Acta Pharmacol Sin.2012;33(9):1131-1140.
131. Sliwoski G, Kothiwale S, Meiler J, Lowe EW, Jr. Computational methods in drug discovery.Pharmacol Rev.2014;66(1):334-395.
132. Singla D, Dhanda SK, Chauhan JS, et al. Open source software and web services fordesigning therapeutic molecules. Curr Top Med Chem.2013;13(10):1172-1191.
133. Kar S, Roy K. How far can virtual screening take us in drug discovery? Expert Opin DrugDiscov.2013;8(3):245-261.
134. Loew GH, Villar HO, Alkorta I. Strategies for indirect computer-aided drug design. PharmRes.1993;10(4):475-486.
135. Kitchen DB, Decornez H, Furr JR, Bajorath J. Docking and scoring in virtual screening fordrug discovery: methods and applications. Nat Rev Drug Discov.2004;3(11):935-949.
136. Alonso H, Bliznyuk AA, Gready JE. Combining docking and molecular dynamic simulationsin drug design. Med Res Rev.2006;26(5):531-568.
137. Schneider G, Bohm HJ. Virtual screening and fast automated docking methods. Drug DiscovToday.2002;7(1):64-70.
138. Gschwend DA, Good AC, Kuntz ID. Molecular docking towards drug discovery. J MolRecognit.1996;9(2):175-186.
139. Huang CH, Mandelker D, Schmidt-Kittler O, et al. The structure of a humanp110alpha/p85alpha complex elucidates the effects of oncogenic PI3Kalpha mutations. Science.2007;318(5857):1744-1748.
140. Zhang X, Vadas O, Perisic O, et al. Structure of lipid kinase p110beta/p85beta elucidates anunusual SH2-domain-mediated inhibitory mechanism. Mol Cell.2011;41(5):567-578.
141. Berndt A, Miller S, Williams O, et al. The p110delta structure: mechanisms for selectivityand potency of new PI(3)K inhibitors. Nat Chem Biol.2010;6(3):244.
142. Camps M, Ruckle T, Ji H, et al. Blockade of PI3Kgamma suppresses joint inflammation anddamage in mouse models of rheumatoid arthritis. Nat Med.2005;11(9):936-943.
143. Frederick R, Mawson C, Kendall JD, et al. Phosphoinositide-3-kinase (PI3K) inhibitors:identification of new scaffolds using virtual screening. Bioorg Med Chem Lett.2009;19(20):5842-5847.
144. Giordanetto F, Kull B, Dellsen A. Discovery of novel class1phosphatidylinositide3-kinases(PI3K) fragment inhibitors through structure-based virtual screening. Bioorg Med Chem Lett.2011;21(2):829-835.
145. Park H, Lee S, Hong S. Discovery of MEK/PI3K dual inhibitor via structure-based virtualscreening. Bioorg Med Chem Lett.2012;22(15):4946-4950.
146. Berman HM, Westbrook J, Feng Z, et al. The Protein Data Bank. Nucleic Acids Res.2000;28(1):235-242.
147. Ohwada J, Ebiike H, Kawada H, et al. Discovery and biological activity of a novel class IPI3K inhibitor, CH5132799. Bioorg Med Chem Lett.2011;21(6):1767-1772.
148. Kaminski GA, Friesner RA, Tirado-Rives J, Jorgensen WL. Evaluation and reparametrizationof the OPLS-AA force field for proteins via comparison with accurate quantum chemicalcalculations on peptides. J Phys Chem B.2001;105(28):6474-6487.
149. Chen X, Liu M, Gilson MK. BindingDB: A web-accessible molecular recognition database.Combinatorial Chemistry&High Throughput Screening.2001;4(8):719-725.
150. Discovery Studio2.5Guide, Accelrys Inc., San Diego, http://www.accelrys.com.2009.
151. MOE. Chemical Computing Group Inc.2010.10.
152. SYBYL8.1. Tripos, Inc, St. Louis, MO. USA.2010.
153. Bohm HJ. The computer program LUDI: a new method for the de novo design of enzymeinhibitors. J Comput Aided Mol Des.1992;6(1):61-78.
154. Bohm HJ. LUDI: rule-based automatic design of new substituents for enzyme inhibitor leads.J Comput Aided Mol Des.1992;6(6):593-606.
155. Jorgensen WL, Maxwell DS, TiradoRives J. Development and testing of the OPLS all-atomforce field on conformational energetics and properties of organic liquids. Journal of the AmericanChemical Society.1996;118(45):11225-11236.
156. Friesner RA, Murphy RB, Repasky MP, et al. Extra precision glide: Docking and scoringincorporating a model of hydrophobic enclosure for protein-ligand complexes. Journal ofMedicinal Chemistry.2006;49(21):6177-6196.
157. Cross JB, Thompson DC, Rai BK, et al. Comparison of several molecular docking programs:pose prediction and virtual screening accuracy. J Chem Inf Model.2009;49(6):1455-1474.
158. Hansson T, Oostenbrink C, van Gunsteren W. Molecular dynamics simulations. Curr OpinStruct Biol.2002;12(2):190-196.
159. Karplus M, McCammon JA. Molecular dynamics simulations of biomolecules. Nat StructBiol.2002;9(9):646-652.
160. Norberg J, Nilsson L. Advances in biomolecular simulations: methodology and recentapplications. Q Rev Biophys.2003;36(3):257-306.
161. Adcock SA, McCammon JA. Molecular dynamics: survey of methods for simulating theactivity of proteins. Chem Rev.2006;106(5):1589-1615.
162. McCammon JA, Gelin BR, Karplus M. Dynamics of folded proteins. Nature.1977;267(5612):585-590.
163. Durrant JD, McCammon JA. Molecular dynamics simulations and drug discovery. BMC Biol.2011;9:71.
164. Cornell WD, Cieplak P, Bayly CI, et al. A second generation force field for the simulation ofproteins, nucleic acids, and organic molecules (vol117, pg5179,1995). Journal of the AmericanChemical Society.1996;118(9):2309-2309.
165. Ma BY, Kumar S, Tsai CJ, Nussinov R. Folding funnels and binding mechanisms. ProteinEngineering.1999;12(9):713-720.
166. Kumar S, Ma BY, Tsai CJ, Wolfson H, Nussinov R. Folding funnels and conformationaltransitions via hinge-bending motions. Cell Biochemistry and Biophysics.1999;31(2):141-164.
167. Ma BY, Shatsky M, Wolfson HJ, Nussinov R. Multiple diverse ligands binding at a singleprotein site: A matter of pre-existing populations. Protein Science.2002;11(2):184-197.
168. Koshland DE. Application of a Theory of Enzyme Specificity to Protein Synthesis. Proc NatlAcad Sci U S A.1958;44(2):98-104.
169. Schames JR, Henchman RH, Siegel JS, Sotriffer CA, Ni H, McCammon JA. Discovery of anovel binding trench in HIV integrase. J Med Chem.2004;47(8):1879-1881.
170. Amaro RE, Baron R, McCammon JA. An improved relaxed complex scheme for receptorflexibility in computer-aided drug design. J Comput Aided Mol Des.2008;22(9):693-705.
171. Schwab F, van Gunsteren WF, Zagrovic B. Computational study of the mechanism and therelative free energies of binding of anticholesteremic inhibitors to squalene-hopene cyclase.Biochemistry.2008;47(9):2945-2951.
172. Han M, Zhang JZ. Class I phospho-inositide-3-kinases (PI3Ks) isoform-specific inhibitionstudy by the combination of docking and molecular dynamics simulation. J Chem Inf Model.2010;50(1):136-145.
173. Sabbah DA, Vennerstrom JL, Zhong H. Docking studies on isoform-specific inhibition ofphosphoinositide-3-kinases. J Chem Inf Model.2010;50(10):1887-1898.
174. Zhu J, Pan P, Li Y, et al. Theoretical studies on beta and delta isoform-specific bindingmechanisms of phosphoinositide3-kinase inhibitors. Mol Biosyst.2014;10(3):454-466.
175. Nacht M, Qiao L, Sheets MP, et al. Discovery of a potent and isoform-selective targetedcovalent inhibitor of the lipid kinase PI3Kalpha. J Med Chem.2013;56(3):712-721.
176. Certal V, Halley F, Virone-Oddos A, et al. Discovery and optimization of new benzimidazole-and benzoxazole-pyrimidone selective PI3Kbeta inhibitors for the treatment of phosphatase andTENsin homologue (PTEN)-deficient cancers. J Med Chem.2012;55(10):4788-4805.
177. Case DA, Cheatham TE,3rd, Darden T, et al. The Amber biomolecular simulation programs.J Comput Chem.2005;26(16):1668-1688.
178. Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and testing of ageneral amber force field. J Comput Chem.2004;25(9):1157-1174.
179. Bayly CI, Cieplak P, Cornell WD, Kollman PA. A Well-Behaved Electrostatic PotentialBased Method Using Charge Restraints for Deriving Atomic Charges-the Resp Model. Journal ofPhysical Chemistry.1993;97(40):10269-10280.
180. Wang JM, Wang W, Kollman PA, Case DA. Automatic atom type and bond type perceptionin molecular mechanical calculations. Journal of Molecular Graphics&Modelling.2006;25(2):247-260.
181. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of SimplePotential Functions for Simulating Liquid Water. Journal of Chemical Physics.1983;79(2):926-935.
182. Darden T, York D, Pedersen L. Particle Mesh Ewald-an N.Log(N) Method for Ewald Sumsin Large Systems. Journal of Chemical Physics.1993;98(12):10089-10092.
183. Ryckaert J-P, Ciccotti G, Berendsen HJ. Numerical integration of the cartesian equations ofmotion of a system with constraints: molecular dynamics of n-alkanes. Journal ofComputational Physics.1977;23(3):327-341.
184. Gohlke H, Kiel C, Case DA. Insights into protein-protein binding by binding free energycalculation and free energy decomposition for the Ras-Raf and Ras-RaIGDS complexes. Journalof Molecular Biology.2003;330(4):891-913.
185. Hou TJ, Zhang W, Case DA, Wang W. Characterization of domain-peptide interactioninterface: A case study on the amphiphysin-1SH3domain. Journal of Molecular Biology.2008;376(4):1201-1214.
186. Onufriev A, Bashford D, Case DA. Modification of the generalized Born model suitable formacromolecules. Journal of Physical Chemistry B.2000;104(15):3712-3720.
187. Walker EH, Pacold ME, Perisic O, et al. Structural determinants of phosphoinositide3-kinaseinhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Molecular Cell.2000;6(4):909-919.
188. Amzel LM, Huang CH, Mandelker D, Lengauer C, Gabelli SB, Vogelstein B. Structuralcomparisons of class I phosphoinositide3-kinases. Nature Reviews Cancer.2008;8(9):665-669.
189.Fleming FF, Yao LH, Ravikumar PC, Funk L, Shook BC. Nitrile-Containing Pharmaceuticals:Efficacious Roles of the Nitrile Pharmacophore. Journal of Medicinal Chemistry.2010;53(22):7902-7917.
1. Smith A, Howell D, Patmore R, Jack A, Roman E. Incidence of haematologicalmalignancy by sub-type: a report from the Haematological Malignancy Research Network. Br JCancer2011;105:1684-1692.
2. Peyrade F, Gastaud L, Re D, Pacquelet-Cheli S, Thyss A. Treatment decisions forelderly patients with haematological malignancies: a dilemma. Lancet Oncol2012;13:e344-352.
3. Hamilton A, Gallipoli P, Nicholson E, Holyoake TL. Targeted therapy in haematologicalmalignancies. J Pathol2010;220:404-418.
4. Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N.Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol3-kinase/Akt-dependent pathway. J Biol Chem1998;273:29864-29872.
5. Liang J, Slingerland JM. Multiple roles of the PI3K/PKB (Akt) pathway in cell cycleprogression. Cell Cycle2003;2:339-345.
6. So L, Fruman DA. PI3K signalling in B-and T-lymphocytes: new developments andtherapeutic advances. Biochem J2012;442:465-481.
7. Li J, Zhu J, Cao B, Mao X. The mTOR Signaling Pathway is an Emerging TherapeuticTarget in Multiple Myeloma. Curr Pharm Des.2013.
8. Mao X, Cao B, Wood TE, et al. A small-molecule inhibitor of D-cyclin transactivationdisplays preclinical efficacy in myeloma and leukemia via phosphoinositide3-kinase pathway.Blood2011;117:1986-1997.
9. Fruman DA, Rommel C. PI3Kdelta inhibitors in cancer: rationale and serendipity mergein the clinic. Cancer Discov2012;1:562-572.
10. Mahadevan D, Chiorean EG, Harris WB, et al. Phase I pharmacokinetic andpharmacodynamic study of the pan-PI3K/mTORC vascular targeted pro-drug SF1126in patientswith advanced solid tumours and B-cell malignancies. Eur J Cancer2012;48:3319-3327.
11. Liu P, Cheng H, Roberts TM, Zhao JJ. Targeting the phosphoinositide3-kinase pathwayin cancer. Nat Rev Drug Discov.2009;8:627-644.(**This article comprehensively discussed PI3Ksignaling pathway and its significance in PI3K inhibitors for cancer therapy)
12. Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB. Exploiting the PI3K/AKT pathwayfor cancer drug discovery. Nat Rev Drug Discov2005;4:988-1004.(**This articlecomprehensively discussed PI3K signaling pathway and its significance cancer therapy)
13. Rossi DJ, Weissman IL. Pten, tumorigenesis, and stem cell self-renewal. Cell2006;125:229-231.
14. Min YH, Eom JI, Cheong JW, et al. Constitutive phosphorylation of Akt/PKB protein inacute myeloid leukemia: its significance as a prognostic variable. Leukemia2003;17:995-997.
15. Sujobert P, Bardet V, Cornillet-Lefebvre P, et al. Essential role for the p110delta isoformin phosphoinositide3-kinase activation and cell proliferation in acute myeloid leukemia. Blood2005;106:1063-1066.
16. Billottet C, Banerjee L, Vanhaesebroeck B, Khwaja A. Inhibition of class Iphosphoinositide3-kinase activity impairs proliferation and triggers apoptosis in acutepromyelocytic leukemia without affecting atra-induced differentiation. Cancer Res2009;69:1027-1036.
17. Ikeda H, Hideshima T, Fulciniti M, et al. PI3K/p110{delta} is a novel therapeutic targetin multiple myeloma. Blood2010;116:1460-1468.
18. Uddin S, Hussain AR, Siraj AK, et al. Role of phosphatidylinositol3'-kinase/AKTpathway in diffuse large B-cell lymphoma survival. Blood2006;108:4178-4186.
19. Psyrri A, Papageorgiou S, Liakata E, et al. Phosphatidylinositol3'-kinase catalyticsubunit alpha gene amplification contributes to the pathogenesis of mantle cell lymphoma. ClinCancer Res2009;15:5724-5732.
20. Martelli AM, Evangelisti C, Chappell W, et al. Targeting the translational apparatus toimprove leukemia therapy: roles of the PI3K/PTEN/Akt/mTOR pathway. Leukemia2011;25:1064-1079.
21. Gutierrez A, Sanda T, Grebliunaite R, et al. High frequency of PTEN, PI3K, and AKTabnormalities in T-cell acute lymphoblastic leukemia. Blood2009;114:647-650.
22. Hyun T, Yam A, Pece S, et al. Loss of PTEN expression leading to high Akt activation inhuman multiple myelomas. Blood2000;96:3560-3568.
23. Silva A, Yunes JA, Cardoso BA, et al. PTEN posttranslational inactivation andhyperactivation of the PI3K/Akt pathway sustain primary T cell leukemia viability. J Clin Invest2008;118:3762-3774.
24. Zhang L, Yang J, Qian J, et al. Role of the microenvironment in mantle cell lymphoma:IL-6is an important survival factor for the tumor cells. Blood2012;120:3783-3792.
25. Chiron D, Maiga S, Surget S, et al. Autocrine insulin-like growth factor1and stem cellfactor but not interleukin6support self-renewal of human myeloma cells. Blood Cancer J2013;3:e120.
26. Wang YY, Zhou GB, Yin T, et al. AML1-ETO and C-KIT mutation/overexpression int(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec. Proc NatlAcad Sci U S A2005;102:1104-1109.
27. Gilliland DG, Griffin JD. Role of FLT3in leukemia. Curr Opin Hematol2002;9:274-281.
28. Martelli AM, Nyakern M, Tabellini G, et al. Phosphoinositide3-kinase/Akt signalingpathway and its therapeutical implications for human acute myeloid leukemia. Leukemia2006;20:911-928.(*This article discussed PI3K signaling pathway in AML)
29. Workman P, Clarke PA, Raynaud FI, van Montfort RL. Drugging the PI3kinome: fromchemical tools to drugs in the clinic. Cancer Res2010;70:2146-2157.
30. Knight ZA. Small molecule inhibitors of the PI3-kinase family. Curr Top MicrobiolImmunol2010;347:263-278.
31. Kawauchi K, Ogasawara T, Yasuyama M, Otsuka K, Yamada O. The PI3K/Akt pathwayas a target in the treatment of hematologic malignancies. Anticancer Agents Med Chem2009;9:550-559.
32. Ameriks MK, Venable JD. Small molecule inhibitors of phosphoinositide3-kinase(PI3K) delta and gamma. Curr Top Med Chem2009;9:738-753.
33. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of somecommonly used protein kinase inhibitors. Biochem J2000;351:95-105.
34. Vlahos CJ, Matter WF, Hui KY, Brown RF. A Specific Inhibitor of Phosphatidylnositol3-Kinase,2-(4-Morpholinyl)-8-Phenyl-4h-1-Benzopyran-4-One (Ly294002). J Biol Chem1994;269:5241-5248.(*This is one of the earliest reports about PI3K inhibitor LY294002)
35. Walker EH, Pacold ME, Perisic O, et al. Structural determinants of phosphoinositide3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell2000;6:909-919.(**This article dissects the interaction of PI3K inhibitor LY294002andwortmannin with PI3K, which helps in-depth understanding of PI3K inhibition and guides thedesign of novel PI3K inhibitors)
36. Garlich JR, De P, Dey N, et al. A vascular targeted pan phosphoinositide3-kinaseinhibitor prodrug, SF1126, with antitumor and antiangiogenic activity. Cancer Res2008;68:206-215.
37. Bird JE, Smith PL, Bostwick JS, Shipkova P, Schumacher WA. Bleeding responseinduced by anti-thrombotic doses of a phosphoinositide3-kinase (PI3K)-beta inhibitor in mice.Throm Res2011;127:560-564.
38. Tanaka H, Yoshida M, Tanimura H, et al. The selective class I PI3K inhibitorCH5132799targets human cancers harboring oncogenic PIK3CA mutations. Clin Cancer Res2011;17:3272-3281.
39. Raynaud FI, Eccles S, Clarke PA, et al. Pharmacologic characterization of a potentinhibitor of class I phosphatidylinositide3-kinases. Cancer Res2007;67:5840-5850.
40. Wagner AJ, Von Hoff DH, LoRusso PM, et al. A first-in-human phase I study to evaluatethe pan-PI3K inhibitor GDC-0941administered QD or BID in patients with advanced solidtumors. J Clin Oncol2009;27.
41. Yamori T, Yaguchi S, Toshiyuki M, Fukui Y, Hirono S, Yamazaki K. ZSTK474, a novelanticancer drug candidate targeted at phosphatidylinositol-3kinase: Identification of the moleculartarget using COMPARE analysis. Clin Cancer Res2005;11:9115s-9115s.
42. Maira SM, Pecchi S, Huang A, et al. Identification and Characterization ofNVP-BKM120, an Orally Available Pan-Class I PI3-Kinase Inhibitor. Mol Cancer Ther2012;11:317-328.
43. Arcaro A, Wymann MP. Wortmannin Is a Potent Phosphatidylinositol3-Kinase Inhibitor-the Role of Phosphatidylinositol3,4,5-Trisphosphate in Neutrophil Responses. Biochem J1993;296:297-301.
44. Wymann MP, BulgarelliLeva G, Zvelebil MJ, et al. Wortmannin inactivatesphosphoinositide3-kinase by covalent modification of Lys-802, a residue involved in thephosphate transfer reaction. Mol Cell Biol1996;16:1722-1733.
45. Knight ZA. Small Molecule Inhibitors of the PI3-Kinase Family. Curr Top MicrobiolImmunol2010;347:263-78.
46. Hong DS, Bowles DW, Falchook GS, et al. A multicenter phase I trial of PX-866, anoral irreversible phosphatidylinositol3-kinase inhibitor, in patients with advanced solid tumors.Clin Cancer Res2012;18:4173-4182.
47. Bowles DW, Ma WW, Senzer N, et al. A multicenter phase1study of PX-866incombination with docetaxel in patients with advanced solid tumours. Br J Cancer2013;109:1085-1092.
48. Ihle NT, Williams R, Chow S, et al. Molecular pharmacology and antitumor activity ofPX-866, a novel inhibitor of phosphoinositide-3-kinase signaling. Mol Cancer Ther2004;3:763-772.
49. Le Cras TD, Korfhagen TR, Davidson C, et al. Inhibition of PI3K by PX-866PreventsTransforming Growth Factor-alpha-Induced Pulmonary Fibrosis. Am J Path2010;176:679-686.
50. Yu K, Lucas J, Zhu T, et al. PWT-458, a novel pegylated-17-hydroxywortmannin,inhibits phosphatidylinositol3-kinase signaling and suppresses growth of solid tumors. CancerBiol Ther2005;4:538-545.(*This article reported a modified wortmannin for cancer therapy)
51. Garcia-Echeverria C, Sellers WR. Drug discovery approaches targeting the PI3K/Aktpathway in cancer. Oncogene2008;27:5511-5526.
52. Workman P, Clarke PA, Raynaud FI, van Montfort RLM. Drugging the PI3Kinome:From Chemical Tools to Drugs in the Clinic. Cancer Res2010;70:2146-2157.
53. Shoemaker RH. The NCI60human tumour cell line anticancer drug screen. Nat RevCancer2006;6:813-823.(*This article introduces the NCI60drug screening system)
54. Kong D, Yamori T. JFCR39, a panel of39human cancer cell lines, and its application inthe discovery and development of anticancer drugs. Bioorg Med Chem2012;20:1947-1951.(*This article discusses the JFCR39drug screening system)
55. Yaguchi S, Fukui Y, Koshimizu I, et al. Antitumor activity of ZSTK474, a newphosphatidylinositol3-kinase inhibitor. J Natl Cancer Inst2006;98:545-556.
56. Kong DX, Yamori T. ZSTK474, a novel phosphatidylinositol3-kinase inhibitoridentified using the JFCR39drug discovery system. Acta Pharm Sin2010;31:1189-1197.
57. Albanese C, Wu K, D'Amico M, et al. IKKalpha regulates mitogenic signaling throughtranscriptional induction of cyclin D1via Tcf. Mol Biol Cell2003;14:585-599.
58. Gera JF, Mellinghoff IK, Shi Y, et al. AKT activity determines sensitivity to mammaliantarget of rapamycin (mTOR) inhibitors by regulating cyclin D1and c-myc expression. J BiolChem2004;279:2737-2746.
59. Yin SQ, Shi M, Kong TT, et al. Preparation of S14161and its analogues and thediscovery of6-bromo-8-ethoxy-3-nitro-2H-chromene as a more potent antitumor agent in vitro.Bioorg Med Chem Lett2013;23:3314-3319.
60. Crabbe T. Exploring the potential of PI3K inhibitors for inflammation and cancer.Biochem Soc Trans2007;35:253-256.
61. Giuliano KA, Haskins JR, Taylor DL. Advances in high content screening for drugdiscovery. Assay Drug Dev Tech2003;1:565-577.
62. Wolff M, Haasen D, Merk S, et al. Automated high content screening forphosphoinositide3kinase inhibition using an AKT1redistribution assay. Comb Chem High ThrScr2006;9:339-350.(**This article introduces the AKT1-GFP targeted HCS screening of PI3Kinhibitors)
63. Rabal O, Link W, Serelde BG, Bischoff JR, Oyarzabal J. An integrated one-step systemto extract, analyze and annotate all relevant information from image-based cell screening ofchemical libraries. Mol Biosyst2010;6:711-720.
64. Hou TJ, Xu XJ. Recent development and application of virtual screening in drugdiscovery: An overview. Curr Pharm Design2004;10:1011-1033.
65. Frederick R, Mawson C, Kendall JD, et al. Phosphoinositide-3-kinase (PI3K) inhibitors:Identification of new scaffolds using virtual screening. Bioorg Med Chem Lett2009;19:5842-5847.
66. Shuttleworth SJ, Silva FA, Cecil AR, et al. Progress in the preclinical discovery andclinical development of class I and dual class I/IV phosphoinositide3-kinase (PI3K) inhibitors.Curr Med Chem2011;18:2686-2714.
67. Doweyko AM.3D-QSAR illusions. J Comput Aided Mol Des.2004;18:587-596.
68. Safavi-Sohi R, Ghasemi JB. Quasi4D-QSAR and3D-QSAR study of the pan class Iphosphoinositide-3-kinase (PI3K) inhibitors. Med Chem Res2013;22:1587-1596.(**This articlediscusses PI3K inhibitors discovery by virtual screening)
69. Polak R, Buitenhuis M. The PI3K/PKB signaling module as key regulator ofhematopoiesis: implications for therapeutic strategies in leukemia. Blood.2012;119:911-923.
70. Blunt MD, Ward SG. Targeting PI3K isoforms and SHIP in the immune system: newtherapeutics for inflammation and leukemia. Curr Opin Pharmacol2012;12:444-451.
71. Wiestner A. Emerging role of kinase-targeted strategies in chronic lymphocyticleukemia. Blood.2012;120:4684-4691.
72. Jin L, Tabe Y, Kojima K, et al. PI3K inhibitor GDC-0941enhances apoptotic effects ofBH-3mimetic ABT-737in AML cells in the hypoxic bone marrow microenvironment. J Mol Med(Berl).2013.
73. Munugalavadla V, Mariathasan S, Slaga D, et al. The PI3K inhibitor GDC-0941combines with existing clinical regimens for superior activity in multiple myeloma. Oncogene2013; doi:10.1038/onc.2012.594.
74. Zang C, Eucker J, Liu H, et al. Inhibition of pan-class I phosphatidyl-inositol-3-kinaseby NVP-BKM120effectively blocks proliferation and induces cell death in diffuse large B-celllymphoma. Leuk Lymphoma2013;2013Jul25.[Epub ahead of print].
75. Rosich L, Saborit-Villarroya I, Lopez-Guerra M, et al. Thephosphatidylinositol-3-kinase inhibitor NVP-BKM120overcomes resistance signals derived frommicroenvironment by regulating the Akt/FoxO3a/Bim axis in chronic lymphocytic leukemia cells.Haematologica2013;98(11):1739-47.
76. Zheng Y, Yang J, Qian J, et al. Novel phosphatidylinositol3-kinase inhibitorNVP-BKM120induces apoptosis in myeloma cells and shows synergistic anti-myeloma activitywith dexamethasone. J Mol Med (Berl).2012;90:695-706.
77. De P, Dey N, Terakedis B, et al. An integrin-targeted, pan-isoform, phosphoinositide-3kinase inhibitor, SF1126, has activity against multiple myeloma in vivo. Cancer ChemotherPharmacol2013;71:867-881.
78. Reynolds CP, Kang MH, Carol H, et al. Initial testing (stage1) of thephosphatidylinositol3' kinase inhibitor, SAR245408(XL147) by the pediatric preclinical testingprogram. Pediatr Blood Cancer2013;60:791-798.
79. Glauer J, Pletz N, Schon M, et al. A novel selective small-molecule PI3K inhibitor iseffective against human multiple myeloma in vitro and in vivo. Blood Cancer J2013;3:e141.
80. Herman SE, Johnson AJ. Molecular pathways: targeting phosphoinositide3-kinasep110-delta in chronic lymphocytic leukemia. Clin Cancer Res2012;18:4013-4018.
81. Meadows SA, Vega F, Kashishian A, et al. PI3Kdelta inhibitor, GS-1101(CAL-101),attenuates pathway signaling, induces apoptosis, and overcomes signals from themicroenvironment in cellular models of Hodgkin lymphoma. Blood2012;119:1897-1900.
82. Lannutti BJ, Meadows SA, Herman SE, et al. CAL-101, a p110delta selectivephosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3Ksignaling and cellular viability. Blood2011;117:591-594.
83. Billottet C, Grandage VL, Gale RE, et al. A selective inhibitor of the p110delta isoformof PI3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic effects ofVP16. Oncogene2006;25:6648-6659.
84. Roschewski M, Farooqui M, Aue G, Wilhelm F, Wiestner A. Phase I study of ON01910.Na (Rigosertib), a multikinase PI3K inhibitor in relapsed/refractory B-cell malignancies.Leukemia2013;27:1920-1923.
85. Chapman CM, Sun X, Roschewski M, et al. ON01910.Na is selectively cytotoxic forchronic lymphocytic leukemia cells through a dual mechanism of action involving PI3K/AKTinhibition and induction of oxidative stress. Clin Cancer Res2012;18:1979-1991.
86. Thomas D, Powell JA, Vergez F, et al. Targeting acute myeloid leukemia by dualinhibition of PI3K signaling and Cdk9-mediated Mcl-1transcription. Blood2013;122:738-748.
87. Glassford J, Kassen D, Quinn J, et al. Inhibition of cell cycle progression by dualphosphatidylinositol-3-kinase and mTOR blockade in cyclin D2positive multiple myelomabearing IgH translocations. Blood Cancer J2012;2:e50.
88. Shehata M, Schnabl S, Demirtas D, et al. Reconstitution of PTEN activity by CK2inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect ofhuman stromal cells in chronic lymphocytic leukemia. Blood2010;116:2513-2521.
89. Chapuis N, Tamburini J, Green AS, et al. Dual inhibition of PI3K and mTORC1/2signaling by NVP-BEZ235as a new therapeutic strategy for acute myeloid leukemia. Clin CancerRes2010;16:5424-5435.
90. McMillin DW, Ooi M, Delmore J, et al. Antimyeloma activity of the orally bioavailabledual phosphatidylinositol3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235.Cancer Res2009;69:5835-5842.
91. Qian C, Lai CJ, Bao R, et al. Cancer network disruption by a single molecule inhibitortargeting both histone deacetylase activity and phosphatidylinositol3-kinase signaling. ClinCancer Res2012;18:4104-4113.
