Figure 3.
Pluripotin is a type II inhibitor of FLT3, ABL, and Jak2 and a potent inhibitor of gatekeeper FLT3 mutant, F691L. Ribbon depiction of a structure of FLT3 with quizartinib (A) and with pluripotin (B). Ribbon depiction of Jak2-coordinate with type II inhibitor BBT594 (C) and with pluripotin (D). A ribbon cartoon of ABL-coordinate with type II inhibitor nilotinib (E) and with pluripotin (F). A stick representation of active sites of FLT3 (G), JAK2 (H), and ABL (I) showing pluripotin interaction with residues from the hinge region, catalytic glutamate of helix-C, and the conserved aspartate from the HRD motif. Overlapping binding of pluripotin is shown below. (J) Pluripotin binding is incompatible with active kinase conformation as phenylalanine residue of the DFG motif will block the binding (shown on the red surface) whereas inactive conformation is accessible when Phe will move out (shown on the green surface). (K) A close-up view of the active site of the FLT3 kinase showing the interaction of pluripotin with the gatekeeper, Phe 691 (top). A model showing that the gatekeeper mutation, F691L, will not affect pluripotin binding (bottom). (L) Sigmoidal curve showing that pluripotin is equally active on FLT3ITD and its gatekeeper mutant, although mutants from the activation loop are resistant. Nonetheless, a significant therapeutic index (TI) exists between the activation loop mutants and normal Ba/F3 cells suggesting that dose escalation will suppress these variants. (M) Active site of Jak2 kinase showing the interaction of pluripotin with Leu 983 (top). A phenyl alanine substitution for Leu 983 will confer steric hindrance (bottom). (N) According to prediction, Jak2V617F/L983F mutant conferred resistance to pluripotin. Thus, confirming the model and on-target inhibitory activity of pluripotin. (O) Active site of ABL kinase domain showing the interaction of pluripotin with the gatekeeper residue, Thr 315 (top). An isoleucine substitution for Thr 315 will confer steric hindrance (bottom). (P) As expected from the modeling studies, BCR-ABLT315 mutant conferred resistance to pluripotin. Together, these modeling and in vitro validation data confirm that pluripotin directly binds with inactive confirmations of FLT3, JAK2, and ABL.

Pluripotin is a type II inhibitor of FLT3, ABL, and Jak2 and a potent inhibitor of gatekeeper FLT3 mutant, F691L. Ribbon depiction of a structure of FLT3 with quizartinib (A) and with pluripotin (B). Ribbon depiction of Jak2-coordinate with type II inhibitor BBT594 (C) and with pluripotin (D). A ribbon cartoon of ABL-coordinate with type II inhibitor nilotinib (E) and with pluripotin (F). A stick representation of active sites of FLT3 (G), JAK2 (H), and ABL (I) showing pluripotin interaction with residues from the hinge region, catalytic glutamate of helix-C, and the conserved aspartate from the HRD motif. Overlapping binding of pluripotin is shown below. (J) Pluripotin binding is incompatible with active kinase conformation as phenylalanine residue of the DFG motif will block the binding (shown on the red surface) whereas inactive conformation is accessible when Phe will move out (shown on the green surface). (K) A close-up view of the active site of the FLT3 kinase showing the interaction of pluripotin with the gatekeeper, Phe 691 (top). A model showing that the gatekeeper mutation, F691L, will not affect pluripotin binding (bottom). (L) Sigmoidal curve showing that pluripotin is equally active on FLT3ITD and its gatekeeper mutant, although mutants from the activation loop are resistant. Nonetheless, a significant therapeutic index (TI) exists between the activation loop mutants and normal Ba/F3 cells suggesting that dose escalation will suppress these variants. (M) Active site of Jak2 kinase showing the interaction of pluripotin with Leu 983 (top). A phenyl alanine substitution for Leu 983 will confer steric hindrance (bottom). (N) According to prediction, Jak2V617F/L983F mutant conferred resistance to pluripotin. Thus, confirming the model and on-target inhibitory activity of pluripotin. (O) Active site of ABL kinase domain showing the interaction of pluripotin with the gatekeeper residue, Thr 315 (top). An isoleucine substitution for Thr 315 will confer steric hindrance (bottom). (P) As expected from the modeling studies, BCR-ABLT315 mutant conferred resistance to pluripotin. Together, these modeling and in vitro validation data confirm that pluripotin directly binds with inactive confirmations of FLT3, JAK2, and ABL.

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