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Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance.

Kesarwani M, Huber E, Kincaid Z, Evelyn CR, Biesiada J, Rance M, Thapa MB, Shah NP, Meller J, Zheng Y, Azam M - Sci Rep (2015)

Bottom Line: In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM).Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants.Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

View Article: PubMed Central - PubMed

Affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.

ABSTRACT
Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib. In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

No MeSH data available.


Fedratinib binds to ATP and substrate-binding sites in JAK2 kinase.(a) Surface depiction of JAK2 kinase in active conformation, showing activation loop (green), fedratinib binding (orange stick) to ATP-binding and substrate-binding sites (arrows). (b) Enlarged view of ATP-binding site, showing fedratinib anchorage by hydrogen bonding (dashed gray line) with Glu 930 (hinge region), Arg 980 and Asn 981 (catalytic site), and Asp 994 of the DFG motif. (c) Surface depiction of Tyr 931showing no direct interaction with fedratinib. However, resistant variant Y931C confers moderate resistance, possibly by modulating the architecture of the active site as a result of loss of hydrogen bonding with water molecules (Fig. 2a). (d) Surface depiction of Leu 983 from catalytic site. (e) Effect of phenylalanine substitution for Leu 983 on fedratinib binding and resistance. (f) Immunoblot analysis of BaF3 cells expressing JAK2-V617 variants Y931C and L983F, showing dose-dependent inhibition by fedratinib. Upper and lower panels show increasing phospho-JAK2 and decreasing phospho-STAT5, respectively, with increasing drug concentrations. Blots were stripped and reprobed for total JAK2 and STAT5 levels. (g) Steady-state kinetic analysis of purified JAK2 kinase inhibition by INCB01842 with increasing ATP concentrations (upper panel) and STAT5 peptide-substrate concentrations (lower panel). IC50 values for kinase inhibition are shown in parentheses against each concentration. (h) Purified JAK2 kinase inhibition by fedratinib, showing a ~20-fold change in IC50 value (upper panel) with increasing ATP concentration. Similarly, increasing substrate concentrations (lower panel) showed an even higher shift in IC50 value (~60 fold).
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f5: Fedratinib binds to ATP and substrate-binding sites in JAK2 kinase.(a) Surface depiction of JAK2 kinase in active conformation, showing activation loop (green), fedratinib binding (orange stick) to ATP-binding and substrate-binding sites (arrows). (b) Enlarged view of ATP-binding site, showing fedratinib anchorage by hydrogen bonding (dashed gray line) with Glu 930 (hinge region), Arg 980 and Asn 981 (catalytic site), and Asp 994 of the DFG motif. (c) Surface depiction of Tyr 931showing no direct interaction with fedratinib. However, resistant variant Y931C confers moderate resistance, possibly by modulating the architecture of the active site as a result of loss of hydrogen bonding with water molecules (Fig. 2a). (d) Surface depiction of Leu 983 from catalytic site. (e) Effect of phenylalanine substitution for Leu 983 on fedratinib binding and resistance. (f) Immunoblot analysis of BaF3 cells expressing JAK2-V617 variants Y931C and L983F, showing dose-dependent inhibition by fedratinib. Upper and lower panels show increasing phospho-JAK2 and decreasing phospho-STAT5, respectively, with increasing drug concentrations. Blots were stripped and reprobed for total JAK2 and STAT5 levels. (g) Steady-state kinetic analysis of purified JAK2 kinase inhibition by INCB01842 with increasing ATP concentrations (upper panel) and STAT5 peptide-substrate concentrations (lower panel). IC50 values for kinase inhibition are shown in parentheses against each concentration. (h) Purified JAK2 kinase inhibition by fedratinib, showing a ~20-fold change in IC50 value (upper panel) with increasing ATP concentration. Similarly, increasing substrate concentrations (lower panel) showed an even higher shift in IC50 value (~60 fold).

Mentions: To better understand the molecular mechanism of fedratinib inhibition, we performed fedratinib molecular-docking experiments, by targeted docking to the ATP-site, substrate site and blind docking to the whole kinase domain (PDB:2B7A) by SWissDock35. These docking analyses revealed that fedratinib can simultaneously bind to two different sites in the kinase domain, the ATP site and the peptide substrate-binding site (Fig. 5a). Interestingly, dual binding of a kinase inhibitor with a similar chemical scaffold has been described previously (Supplementary Fig. 13)40. In order to validate the dual binding we further performed additional docking on another platform (AutoDock) and molecular dynamic simulations (MDS). Simulations using Autodock predict that fedratinib binds preferentially to the substrate-binding site. At the same time, in agreement with known experimental data, ruxolitinib is predicted to bind to the ATP binding site. Preferential binding of fedratinib to the substrate-binding pocket is indicated by both the predicted binding energies and the relative paucity of contacts unique to ATP binding site, i.e., those involving residues 930–940 (Supplementary Fig. 14 and supplementary table 2). This is in contrast to results obtained for ruxolitinib, which shows strong preference for the ATP binding site even when using the large simulation box that enables sampling both pockets (Supplementary Fig. 15 and supplementary table 3). In addition, predicted binding energies for the two compounds and the two alternative binding sites are also consistent with preferential binding of fedratinib to the substrate-binding site. The results shown in supplementary Figs 14 and 15 were obtained using 2B7A as the structure of the protein. Similar results were obtained also using 3TJD (data not shown). Molecular dynamics simulations provide further support that fedratinib binds at the substrate-binding site. The ligand remains bound to the protein in the course of 50 ns simulations, starting from an initial docking pose predicted by AutoDock (Supplementary Fig. 16A). Moreover, while both protein and ligand fluctuate, a pattern of relatively stable hydrogen bond was observed with Glu 1016 within the substrate-binding pocket (Supplementary figure 16B).


Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance.

Kesarwani M, Huber E, Kincaid Z, Evelyn CR, Biesiada J, Rance M, Thapa MB, Shah NP, Meller J, Zheng Y, Azam M - Sci Rep (2015)

Fedratinib binds to ATP and substrate-binding sites in JAK2 kinase.(a) Surface depiction of JAK2 kinase in active conformation, showing activation loop (green), fedratinib binding (orange stick) to ATP-binding and substrate-binding sites (arrows). (b) Enlarged view of ATP-binding site, showing fedratinib anchorage by hydrogen bonding (dashed gray line) with Glu 930 (hinge region), Arg 980 and Asn 981 (catalytic site), and Asp 994 of the DFG motif. (c) Surface depiction of Tyr 931showing no direct interaction with fedratinib. However, resistant variant Y931C confers moderate resistance, possibly by modulating the architecture of the active site as a result of loss of hydrogen bonding with water molecules (Fig. 2a). (d) Surface depiction of Leu 983 from catalytic site. (e) Effect of phenylalanine substitution for Leu 983 on fedratinib binding and resistance. (f) Immunoblot analysis of BaF3 cells expressing JAK2-V617 variants Y931C and L983F, showing dose-dependent inhibition by fedratinib. Upper and lower panels show increasing phospho-JAK2 and decreasing phospho-STAT5, respectively, with increasing drug concentrations. Blots were stripped and reprobed for total JAK2 and STAT5 levels. (g) Steady-state kinetic analysis of purified JAK2 kinase inhibition by INCB01842 with increasing ATP concentrations (upper panel) and STAT5 peptide-substrate concentrations (lower panel). IC50 values for kinase inhibition are shown in parentheses against each concentration. (h) Purified JAK2 kinase inhibition by fedratinib, showing a ~20-fold change in IC50 value (upper panel) with increasing ATP concentration. Similarly, increasing substrate concentrations (lower panel) showed an even higher shift in IC50 value (~60 fold).
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f5: Fedratinib binds to ATP and substrate-binding sites in JAK2 kinase.(a) Surface depiction of JAK2 kinase in active conformation, showing activation loop (green), fedratinib binding (orange stick) to ATP-binding and substrate-binding sites (arrows). (b) Enlarged view of ATP-binding site, showing fedratinib anchorage by hydrogen bonding (dashed gray line) with Glu 930 (hinge region), Arg 980 and Asn 981 (catalytic site), and Asp 994 of the DFG motif. (c) Surface depiction of Tyr 931showing no direct interaction with fedratinib. However, resistant variant Y931C confers moderate resistance, possibly by modulating the architecture of the active site as a result of loss of hydrogen bonding with water molecules (Fig. 2a). (d) Surface depiction of Leu 983 from catalytic site. (e) Effect of phenylalanine substitution for Leu 983 on fedratinib binding and resistance. (f) Immunoblot analysis of BaF3 cells expressing JAK2-V617 variants Y931C and L983F, showing dose-dependent inhibition by fedratinib. Upper and lower panels show increasing phospho-JAK2 and decreasing phospho-STAT5, respectively, with increasing drug concentrations. Blots were stripped and reprobed for total JAK2 and STAT5 levels. (g) Steady-state kinetic analysis of purified JAK2 kinase inhibition by INCB01842 with increasing ATP concentrations (upper panel) and STAT5 peptide-substrate concentrations (lower panel). IC50 values for kinase inhibition are shown in parentheses against each concentration. (h) Purified JAK2 kinase inhibition by fedratinib, showing a ~20-fold change in IC50 value (upper panel) with increasing ATP concentration. Similarly, increasing substrate concentrations (lower panel) showed an even higher shift in IC50 value (~60 fold).
Mentions: To better understand the molecular mechanism of fedratinib inhibition, we performed fedratinib molecular-docking experiments, by targeted docking to the ATP-site, substrate site and blind docking to the whole kinase domain (PDB:2B7A) by SWissDock35. These docking analyses revealed that fedratinib can simultaneously bind to two different sites in the kinase domain, the ATP site and the peptide substrate-binding site (Fig. 5a). Interestingly, dual binding of a kinase inhibitor with a similar chemical scaffold has been described previously (Supplementary Fig. 13)40. In order to validate the dual binding we further performed additional docking on another platform (AutoDock) and molecular dynamic simulations (MDS). Simulations using Autodock predict that fedratinib binds preferentially to the substrate-binding site. At the same time, in agreement with known experimental data, ruxolitinib is predicted to bind to the ATP binding site. Preferential binding of fedratinib to the substrate-binding pocket is indicated by both the predicted binding energies and the relative paucity of contacts unique to ATP binding site, i.e., those involving residues 930–940 (Supplementary Fig. 14 and supplementary table 2). This is in contrast to results obtained for ruxolitinib, which shows strong preference for the ATP binding site even when using the large simulation box that enables sampling both pockets (Supplementary Fig. 15 and supplementary table 3). In addition, predicted binding energies for the two compounds and the two alternative binding sites are also consistent with preferential binding of fedratinib to the substrate-binding site. The results shown in supplementary Figs 14 and 15 were obtained using 2B7A as the structure of the protein. Similar results were obtained also using 3TJD (data not shown). Molecular dynamics simulations provide further support that fedratinib binds at the substrate-binding site. The ligand remains bound to the protein in the course of 50 ns simulations, starting from an initial docking pose predicted by AutoDock (Supplementary Fig. 16A). Moreover, while both protein and ligand fluctuate, a pattern of relatively stable hydrogen bond was observed with Glu 1016 within the substrate-binding pocket (Supplementary figure 16B).

Bottom Line: In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM).Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants.Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

View Article: PubMed Central - PubMed

Affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.

ABSTRACT
Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib. In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

No MeSH data available.