Identification of non-charged 7.44 analogs interacting with the NHR2 domain of RUNX1-ETO and exhibiting an improved, selective antiproliferative effect in RUNX-ETO positive cells

Abstract

AbstractThe RUNX1/ETO fusion protein is a chimeric transcription factor in acute myeloid leukemia (AML) created by chromosomal translocation t(8;21)(q22;q22). t(8;21) abnormality is associated with 12% ofde novoAML cases and up to 40% in the AML subtype M2. Previously, we identified the small-molecule inhibitor7.44, which specifically interferes with NHR2 domain tetramerization of RUNX1/ETO, restores gene expression down-regulated by RUNX1/ETO, inhibits proliferation, and reduces RUNX1/ETO-related tumor growth in a mouse model. However, despite generally favorable physicochemical, pharmacokinetic, and toxicological properties,7.44is negatively charged at physiological pH and was predicted to have low to medium membrane permeability. Here, we identifiedM23,M27,andM10as non-charged analogs of7.44using ligand-based virtual screening,in vivohit identification, biophysical andin vivohit validation, and integrative modeling and ADMET predictions. All three compounds interact with the NHR2 domain and showKD,appvalues of 39-114 µM in Microscale Thermophoresis experiments as well asIC50values of 33-77 μM as to cell viability in RUNX1/ETO-positive KASUMI cells, i.e., are ∼5 to 10-fold more potent than7.44.M23is ∼10-fold more potent than7.44in inhibiting cell proliferation of RUNX1/ETO-positive cells.M23andM27are negligibly protonated or in a ∼1:1 ratio at physiological pH, whileM10has no (de-)protonatable group. The non-protonated species are predicted to be highly membrane-permeable, along with other favorable pharmacokinetic and toxicological properties. These compounds might serve as lead structures for the optimization of binding affinity, bioavailability, and anti-leukemic effects of compounds inhibiting RUNX1/ETO oncogenic function in t(8;21) AML.