Understanding the Species Selectivity of Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors

To test for on target toxicity of a new chemical entity, it is important to have comparable binding affinitiesof the compound in the target proteins from humans and the test species. To evaluate our myeloid cell leukemia-1 (Mcl-1) inhibitors, we tested them against rodent Mcl-1 and found a significant loss of binding affinitywhen compared to that seen with human Mcl-1. To understand the affinity loss, we used sequence alignmentsand structures of human Mcl-1/inhibitor complexes to identify the important differences in the amino acid sequences. One difference is human L246 (F226 in rat, F227 in mouse) in the ligand binding pocket. Mutating rat F226 to a Leu restores affinity, but the mouse F227L mutant still has a ligand affinitythat is lower than that of human Mcl-1. Another mutation of mouse F267, located ∼12 A from the ligand pocket, to the human/rat cysteine, F267C, improved the affinityand combined with F227L resulted in a mutant mouse protein with a binding affinitysimilar to that of human Mcl-1...