Intrinsically disordered proteins exist as highly dynamic conformational ensembles of diverse forms. However, the majority of virtual screening only focuses on proteins with defined structures. This means that computer-aided drug discovery is restricted. As a breakthrough, understanding the structural characteristics of intrinsically disordered proteins and its application can open the gate for unrestricted drug discovery. First, we segmented the target disorder-to-order transition region into a series of overlapping 20-amino-acid-long peptides. Folding prediction generated diverse conformations of these peptides. Next, we applied molecular docking, new evaluation score function, and statistical analysis. This approach successfully distinguished known compounds and their corresponding binding regions. Especially, Myc proto-oncogene protein (MYC) inhibitor 10058F4 was well distinguished from others of the chemical compound library. We also studied differences between the two Methyl-CpG-binding domain protein 2 (MBD2) inhibitors (ABA (2-amino-N-[[(3S)-2,3-dihydro-1,4-benzodioxin-3-yl]methyl]-acetamide) and APC ((R)-(3-(2-Amino-acetylamino)-pyrrolidine-1-carboxylic acid tert-butyl ester))). Both compounds bind MBD2 through electrostatic interaction behind its p66α-binding site. ABA is also able to bind p66α through electrostatic interaction behind its MBD2-binding site while APC-p66α binding was nonspecific. Therefore, structural heterogeneity mimicking of the disorder-to-order transition region at the peptide level and utilization of the new docking score function represent a useful approach that can efficiently discriminate compounds for expanded virtual screening toward intrinsically disordered proteins.

Murine erythroleukemia (MEL) cells are often employed as a model to dissect mechanisms of erythropoiesis and erythroleukemia in vitro. Here, an allograft model using MEL cells resulting in splenomegaly was established to develop a diagnostic model for isolation/quantification of metastatic cells, anti-cancer drug screening, and evaluation of the tumorigenic or metastatic potentials of molecules in vivo. In this animal model, circulating MEL cells from the blood stream were successfully isolated and quantified with an additional in vitro cultivation step. In terms of the molecular-pathological analysis, we were able to successfully evaluate the functional discrimination between methyl-CpG-binding domain 2 (Mbd2) and p66α in erythroid differentiation, and tumorigenic potential in spleen and blood stream of allograft model mice. In addition, we found that the number of circulating MEL cells in anti-cancer drug-treated mice was dose-dependently decreased. Our data demonstrate that the newly established allograft model is useful to dissect erythroleukemia pathologies and non-invasively provides valuable means for isolation of metastatic cells, screening of anti-cancer drugs, and evaluation of the tumorigenic potentials.