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In the present study we used the earlier characterized line 42 of T-cell lymphoma from Akt2-transgenic mice (42-936, 42-577, and 42-588) and line 72 (wtl36). Screening for the new ligand molecules specific to DLX5 has been carried out with The QUANTUM software suite, based on the analysis of the protein crystal structure. The search was made more complicated by the absence of preliminary data on the binding of the known compounds with DLX5 protein; therefore, blind studies were performed. The best molecules and all their structural analogues from the original ENAMINE library were sorted on the basis of their predicted binding energy. According to the results of molecular docking, 100 ligands were selected; 14 of those with the best predicted binding energy of DLX5 protein were ordered and synthesized at ENAMINE company; then, they were tested on cell cultures. The cells of the earlier characterized line 42 of T-cell lymphoma from Akt2-transgenic mice were used as a model to verify the specific activity of the selected ligands. As can be seen in Fig. 1, the ligands demonstrate different efficacies of impact on the proliferation of lymphoma cells; compounds Q8, Q12, Q9, and Q13 manifested the best inhibitory activity.
The possible nonspecific cytotoxic action of the selected compounds was tested on normal human ovarian epithelial cells without DLX5 expression. When comparing with the control, it can be seen that most ligand molecules, with the exception of the compounds Q8 and Q13, manifest no significant cytotoxicity. Since Q8 and Q13 manifested a cytotoxic effect, they were eliminated from further consideration. Compounds Q12 and Q9 were selected for further studies as the most promising ones.
In order to eliminate the possibility of a nonspecific impact of compounds Q12 and Q9 on cells of the lymphoid series, their action was tested on T-cell lymphoma cells of line 72 with absent expression of Dlx5 from Akt2-transgenic mice . Than we check the effect of compound Q12 on the proliferation of an additional two subtypes of lymphoma cells expressing Dlx5 (42-577 and 42-588) , as well as the proliferation of the human lymphoma cells Jurkat and Molt16 not expressing DLX5.
It is known that the DLX5 transcription factor can directly control the expression of protooncogene c-myc. The impact of Q12 on the expression of c-myc in the lymphoma cells 42-936 expressing Dlx5 was studied by real-time RT- PCR. Figure 2 shows the levels of mRNA of c-myc with respect to the endogenous control, mRNA of TATA-binding protein (Tbp) or mRNA of Dlx5, as well as mRNA of Dlx5 with respect to mRNA of Tbp in the presence of 10 μM Q12 and without any addition of it. It can be seen that the expression of c-myc decreases considerably under the action of Q12, while the expression of Dlx5 remains intact. These results agree with the conception of the inhibitory effect of ligand Q12 on the transcription activity of the Dlx5 factor. Although these data need to be tested on a larger number of cell lines, it is tempting to make a preliminary conclusion on the specificity of binding between the transcription factor DLX5 and ligand Q12 based on the results of this study.
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