In Silico Screening Of Novel PI3K Inhibitors And Their Applications In The Treatment Of Multiple Myeloma

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.