Complexes formed by DNA and polyethylenimine (PEI) are of great research interest because of their application in gene therapy. In this work, we carried out all-atom molecular dynamics simulations to study eight types of DNA/PEI complexes, each of which was formed by one DNA duplex d(CGCGAATTCGCG)(2) and one PEI. We used eight different PEIs with four different degrees of branching and two protonation ratios of amine groups (23% and 46%) in the simulations to investigate how the branching degree and protonation state can affect the binding. We found that 46% protonated PEIs form more stable complexes with DNA, and the binding is achieved mainly through direct interaction between the protonated amine groups on PEI and the electronegative oxygens on the DNA backbone, with some degree of interaction with electronegative groove nitrogens/oxygens. For the 23% protonated PEIs, indirect interaction mediated by one or more water molecules plays an important role in binding. Compared with the protonation state, the degree of branching has a smaller effect on binding, which essentially diminishes at the protonation ratio of 46%. These simulations shed light on the detailed mechanism(s) of PEI binding to DNA, and may facilitate the design of PEI-based gene delivery carriers.

Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases. Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora kinases (Aurora-A IC(50) = 0.015 +/- 0.003 muM, Aurora-B IC(50) = 0.025 muM, Aurora-C IC(50) = 0.019 muM). Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620 colon carcinoma xenografts following oral administration with no observed toxicities as defined by body weight loss.

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Acquired resistance to selective FLT3 inhibitors, is an emerging clinical problem in the treatment of FLT3-ITD+ acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has limited investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD+ human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD+ allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD+ patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13- RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild type allele and duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.