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  • br The docking scores for binding of to three sites

    2019-10-21


    The docking scores for binding of 1 to three sites of HSA are shown in Table S2 (Supplementary material). Docking scores are very similar for all three binding sites. Complex 1 binds to the site IIA with slight preference. The majority of docking solutions places 1 at the surface of HSA. The large portion of bound molecule is exposed to the solvent, and this may explain relatively weak binding constant found for 1. Binding mode of 1 to the binding site IIA is shown in Fig. 15. The acetate ligand
    of 1 interacts with Lys195 and Arg222 through hydrogen bonding (at distances 2.84 and 3.11 Å, respectively). It also interacts with Ala291 and Glu292 through non-specific, hydrophobic contacts. Complex 1 is located far from Trp214 (distance is 4.7 Å) and does not interact with
    this residue. The fluorescence quenching observed is probably due to the conformational changes in the surroundings of Trp214, induced by the binding of 1. The moderate binding constant of 1 to HSA is in line with the binding mode found through the molecular docking, as there is a small number of interactions that could stabilize HSA-1 aduct. A large portion of the bound molecule is solvent-exposed, and water molecules from the medium can compete for the ligand molecule and destabilize protein-ligand complex.
    4. Conclusion
    Current results on biological activity reveal that our novel bi-nuclear, hydrazone-based cadmium complex 1 is a very strong pro-apoptotic inducer in both, MCF-7 and AsPC-1 cells, even more powerful than CDDP, particularly for AsPC-1 CSCs. While changes in Dihexa (PNB-0408) distribution signify that 1 interferes in the process of DNA replication, comparative analysis indicates that its mechanism of action differs from the one of CDDP. That hypothesis is confirmed in DNA interaction study where results show non-covalent low binding strength of 1 into DNA minor groove together with the lack of DNA cleavage in pUC19 ex-periment, which indicates that its target is rather related to protein(s)  Journal of Inorganic Biochemistry 190 (2019) 45–66
    involved in the control of replication than DNA itself. Furthermore, apoptotic death caused by 1 was maintained mostly in caspase-in-dependent manner and included activation of intrinsic apoptotic cas-cade, which was quite the opposite of CDDP. The underlying cause for suppressed activation of caspase-8, accompanied with significant in-hibition of cross-talk between intrinsic and extrinsic caspase pathways in both cell lines treated with 1, remains to be further investigated. Although 1 has proved to be a powerful inducer of mitochondrial O2%−, it still remains unclear whether this mechanism could have an im-portant role in apoptosis induction. Finally, results on 3D spheroids displayed substantial activity of 1 against CSCs. All these results make 1 as a serious candidate for further investigations on other CSC models and as a putative treatment of e.g. highly resistant pancreatic cancer.
    Abbreviations
    CDDP cisplatin
    haOEt × HCl ethyl hydrazinoacetate hydrochloride
    2‑ap 2‑acetylpyridine MCF-7 human mammary adenocarcinoma cell line
    CSC cancer stem cell
    AsPC-1 human pancreatic adenocarcinoma cell line
    2D two-dimensional
    3D three-dimensional
    ATR Attenuated Total Reflection
    RCF relative centrifugal force
    FBS fetal bovine serum
    RPMI Roswell Park Memorial Institute DMEM Dulbecco's modified Eagle's medium
    PI propidium iodide
    Annexin V Ca2+-dependent phospholipid-binding protein with high affinity for phosphatidylserine Z-VAD-fmk carbobenzoxy‑valyl‑alanyl‑aspartyl‑[O‑methyl]‑fluor-
    HSA ketone
    human serum albumin
    ROS reactive oxygen species