A1 retains Bcl-2 inhibition ability and inhibits cell growth with apoptosis induction in VEN-refractory AML cell lines. (A) The cocrystal structure of VEN bound to Bcl-2 (Protein Data Bank [PDB] code: 6O0K). (B) The design of A1 by conjugating VEN with DHA. (C) Affinity of A1 for Bcl-2, Bcl-xL, and Mcl-1 based on FPA. (D) The predicted position of VEN (pink structure) and A1 (blue structure) bound to Bcl-2 (PDB code: 6O0K) and Bcl-xL (PDB code: 4QNQ). (E) Growth inhibition. U937, Mono Mac 6, THP-1, Kasumi-1, and ME-1 cells were treated with 0.05 to 0.8 μM of DHA, VEN, DHA + VEN, and A1 for 72 hours. Cell growth inhibition was measured by counting the cell number and comparing it with the untreated group. (F) Apoptosis induction. The cells were treated with 0.8 μM DHA, VEN, DHA + VEN, and A1 for 24 hours. The number of apoptotic cells was determined by fluorescence-activated cell sorting (FACS) after staining with annexin V/propidium iodide (PI). (G) Cell cycle distribution. The cells were treated as labeled in panel F, followed by FACS after PI staining. The values show the mean ± standard deviation (SD) of 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 when compared with the control group. Con, control; IC₅₀, 50% inhibitory concentration; N.A., no activity (IC₅₀>200 μM).