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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.


Mutagenesis of residues in the substrate-binding pocket confers moderate resistance to fedratinib.(a) Surface depiction of JAK2 kinase in active conformation, showing the activation loop (green) and binding of fedratinib (yellow) in the substrate-binding pocket. (b) Substrate-binding pocket is comprised of an activation loop (green) and helices F (blue), FG (purple) and G (green). Amino-acid residues IFWY in the activation loop is a highly conserved kinase motif that serves as a hydrophobic platform for inhibitor and substrate-binding (green surface sandwiched between inhibitor and helix F). (c) Surface depiction of the IFWY motif (upper panel); an Ile 1018-to-tryptophan substitution results in steric clash with fedratinib (lower panel). (d) Surface depiction of Leu 1026 (upper panel); a Leu 1026-to-phenylalanine substitution results in steric clash with the benzene-sulfonamide group of fedratinib. (e) Dose-response cell proliferations showing moderate increase in IC50 values by JAK2 variants JAK2V617F-Y931C/I1018W and JAK2V617F-Y931C/L1026F; on the other hand, mutations at F1019L and W1020C resulted in inactive kinase (i.e., no change in IC50 values). (f) Mutations identified from drug resistant screen at low dose of fedratinib (2.5 μM) using BaF3 cells expressing JAK2-V617F/Y931C. The α-carbon of each resistant mutation is shown as a circle. (g) Surface depiction of Tyr 1045 showing the π-stacking with residues Val 1042 (helix-F), Leu1086 (helix H), Phe1061 (helix FG), and the IFWY activation-loop motif. A bulkier substitution at Y1045 would push the IFWY motif towards the inhibitor, thereby possibly conferring resistance. (h) A bulkier substitution at Pro 1058 would push the Phe 1019 of IFWY, which might affect fedratinib binding. (i) A bulkier substitution at Val1075 (e.g., to phenylalanine) would cause steric hindrance to fedratinib. (j) Dose-response growth curves showing higher IC50 values for S1025C, Y1045W, F1061W, and V1075F. Variants W1038C, S1039F and Y1045* encode weekend kinase (Fig. S14), but displayed subtle increases in IC50. (k) Immunoblot analysis of phospho STAT5 (upper panel) and total STAT5 (lower panel) in BaF3 cells expressing JAK2-V617/Y931C variants. Variants S1025C, Y1045W and V1075F are resistant to 5 μM fedratinib, and also conferred highest resistance in cell-proliferation assays.
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f8: Mutagenesis of residues in the substrate-binding pocket confers moderate resistance to fedratinib.(a) Surface depiction of JAK2 kinase in active conformation, showing the activation loop (green) and binding of fedratinib (yellow) in the substrate-binding pocket. (b) Substrate-binding pocket is comprised of an activation loop (green) and helices F (blue), FG (purple) and G (green). Amino-acid residues IFWY in the activation loop is a highly conserved kinase motif that serves as a hydrophobic platform for inhibitor and substrate-binding (green surface sandwiched between inhibitor and helix F). (c) Surface depiction of the IFWY motif (upper panel); an Ile 1018-to-tryptophan substitution results in steric clash with fedratinib (lower panel). (d) Surface depiction of Leu 1026 (upper panel); a Leu 1026-to-phenylalanine substitution results in steric clash with the benzene-sulfonamide group of fedratinib. (e) Dose-response cell proliferations showing moderate increase in IC50 values by JAK2 variants JAK2V617F-Y931C/I1018W and JAK2V617F-Y931C/L1026F; on the other hand, mutations at F1019L and W1020C resulted in inactive kinase (i.e., no change in IC50 values). (f) Mutations identified from drug resistant screen at low dose of fedratinib (2.5 μM) using BaF3 cells expressing JAK2-V617F/Y931C. The α-carbon of each resistant mutation is shown as a circle. (g) Surface depiction of Tyr 1045 showing the π-stacking with residues Val 1042 (helix-F), Leu1086 (helix H), Phe1061 (helix FG), and the IFWY activation-loop motif. A bulkier substitution at Y1045 would push the IFWY motif towards the inhibitor, thereby possibly conferring resistance. (h) A bulkier substitution at Pro 1058 would push the Phe 1019 of IFWY, which might affect fedratinib binding. (i) A bulkier substitution at Val1075 (e.g., to phenylalanine) would cause steric hindrance to fedratinib. (j) Dose-response growth curves showing higher IC50 values for S1025C, Y1045W, F1061W, and V1075F. Variants W1038C, S1039F and Y1045* encode weekend kinase (Fig. S14), but displayed subtle increases in IC50. (k) Immunoblot analysis of phospho STAT5 (upper panel) and total STAT5 (lower panel) in BaF3 cells expressing JAK2-V617/Y931C variants. Variants S1025C, Y1045W and V1075F are resistant to 5 μM fedratinib, and also conferred highest resistance in cell-proliferation assays.

Mentions: To further probe the mechanism of fedratinib binding to the substrate-binding site, we created substrate-site mutations in the background of JAK2-V617F-Y931C by introducing bulkier, hydrophobic amino acids proximal to the inhibitor-binding site. We used the JAK2-V617F-Y931C, because blocking one binding site is probably insufficient to observe resistance conferred by a secondary site. As expected, mutations I1018F/W and L1026F reduced kinase activity, while F1019L, W1020C and Y1021H resulted in inactive kinase (Supplementary Fig. 17). I1018F/W and L1026F exhibited increased IC50 values in a dose-dependent cell proliferation assay (Fig. 8a–e) and a phospho-STAT5 inhibition assay (Fig. 8k). Next, we performed a resistant screen using 100 million BaF3 cells expressing JAK2-V617F-Y931C; cells were treated with fedratinib at 2.0 μM—with the assumption that a lower drug concentration may select for clones conferring mild resistance, which could inform the secondary binding site. This screen produced one resistant clone, sequencing of which revealed a total of 13 different mutations—all in the substrate-binding pocket (Fig. 8f). Structural modeling suggested that these mutations affect fedratinib binding either by direct steric hindrance (S1025C, S1039F, G1041R, V1042F, I1074F, and V1075F) or by affecting stability of the conserved IFWY motif of the activation loop in the substrate-binding site (W1038C, Y1045W/*, P1058F, F1061W, I1079L, and L1082R) (Fig. 8g–i). To validate these findings, we designed the mutations (S1025C, W1038C, S1039F, Y1045W/*, F1061W, V1075F) in the context of JAK2V617F-Y931C. As expected, all 13 variants displayed weaker kinase activity, compared to parental, JAK2-V617F/Y931C kinase (Supplementary Fig. 17). While 4 variants (S1025C, Y1045W, F1061W and V1075F) conferred strong biochemical and cellular resistance (Fig. 8J,K), others (W1038C, S1039F and Y1045*) conferred subtle changes in cellular IC50 but no change in STAT5 inhibition. This suggests either these variants are false positives or express a catalytically inactive kinase that acts as a sink for inhibitor, which is reflected in moderate change in drug response.


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)

Mutagenesis of residues in the substrate-binding pocket confers moderate resistance to fedratinib.(a) Surface depiction of JAK2 kinase in active conformation, showing the activation loop (green) and binding of fedratinib (yellow) in the substrate-binding pocket. (b) Substrate-binding pocket is comprised of an activation loop (green) and helices F (blue), FG (purple) and G (green). Amino-acid residues IFWY in the activation loop is a highly conserved kinase motif that serves as a hydrophobic platform for inhibitor and substrate-binding (green surface sandwiched between inhibitor and helix F). (c) Surface depiction of the IFWY motif (upper panel); an Ile 1018-to-tryptophan substitution results in steric clash with fedratinib (lower panel). (d) Surface depiction of Leu 1026 (upper panel); a Leu 1026-to-phenylalanine substitution results in steric clash with the benzene-sulfonamide group of fedratinib. (e) Dose-response cell proliferations showing moderate increase in IC50 values by JAK2 variants JAK2V617F-Y931C/I1018W and JAK2V617F-Y931C/L1026F; on the other hand, mutations at F1019L and W1020C resulted in inactive kinase (i.e., no change in IC50 values). (f) Mutations identified from drug resistant screen at low dose of fedratinib (2.5 μM) using BaF3 cells expressing JAK2-V617F/Y931C. The α-carbon of each resistant mutation is shown as a circle. (g) Surface depiction of Tyr 1045 showing the π-stacking with residues Val 1042 (helix-F), Leu1086 (helix H), Phe1061 (helix FG), and the IFWY activation-loop motif. A bulkier substitution at Y1045 would push the IFWY motif towards the inhibitor, thereby possibly conferring resistance. (h) A bulkier substitution at Pro 1058 would push the Phe 1019 of IFWY, which might affect fedratinib binding. (i) A bulkier substitution at Val1075 (e.g., to phenylalanine) would cause steric hindrance to fedratinib. (j) Dose-response growth curves showing higher IC50 values for S1025C, Y1045W, F1061W, and V1075F. Variants W1038C, S1039F and Y1045* encode weekend kinase (Fig. S14), but displayed subtle increases in IC50. (k) Immunoblot analysis of phospho STAT5 (upper panel) and total STAT5 (lower panel) in BaF3 cells expressing JAK2-V617/Y931C variants. Variants S1025C, Y1045W and V1075F are resistant to 5 μM fedratinib, and also conferred highest resistance in cell-proliferation assays.
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f8: Mutagenesis of residues in the substrate-binding pocket confers moderate resistance to fedratinib.(a) Surface depiction of JAK2 kinase in active conformation, showing the activation loop (green) and binding of fedratinib (yellow) in the substrate-binding pocket. (b) Substrate-binding pocket is comprised of an activation loop (green) and helices F (blue), FG (purple) and G (green). Amino-acid residues IFWY in the activation loop is a highly conserved kinase motif that serves as a hydrophobic platform for inhibitor and substrate-binding (green surface sandwiched between inhibitor and helix F). (c) Surface depiction of the IFWY motif (upper panel); an Ile 1018-to-tryptophan substitution results in steric clash with fedratinib (lower panel). (d) Surface depiction of Leu 1026 (upper panel); a Leu 1026-to-phenylalanine substitution results in steric clash with the benzene-sulfonamide group of fedratinib. (e) Dose-response cell proliferations showing moderate increase in IC50 values by JAK2 variants JAK2V617F-Y931C/I1018W and JAK2V617F-Y931C/L1026F; on the other hand, mutations at F1019L and W1020C resulted in inactive kinase (i.e., no change in IC50 values). (f) Mutations identified from drug resistant screen at low dose of fedratinib (2.5 μM) using BaF3 cells expressing JAK2-V617F/Y931C. The α-carbon of each resistant mutation is shown as a circle. (g) Surface depiction of Tyr 1045 showing the π-stacking with residues Val 1042 (helix-F), Leu1086 (helix H), Phe1061 (helix FG), and the IFWY activation-loop motif. A bulkier substitution at Y1045 would push the IFWY motif towards the inhibitor, thereby possibly conferring resistance. (h) A bulkier substitution at Pro 1058 would push the Phe 1019 of IFWY, which might affect fedratinib binding. (i) A bulkier substitution at Val1075 (e.g., to phenylalanine) would cause steric hindrance to fedratinib. (j) Dose-response growth curves showing higher IC50 values for S1025C, Y1045W, F1061W, and V1075F. Variants W1038C, S1039F and Y1045* encode weekend kinase (Fig. S14), but displayed subtle increases in IC50. (k) Immunoblot analysis of phospho STAT5 (upper panel) and total STAT5 (lower panel) in BaF3 cells expressing JAK2-V617/Y931C variants. Variants S1025C, Y1045W and V1075F are resistant to 5 μM fedratinib, and also conferred highest resistance in cell-proliferation assays.
Mentions: To further probe the mechanism of fedratinib binding to the substrate-binding site, we created substrate-site mutations in the background of JAK2-V617F-Y931C by introducing bulkier, hydrophobic amino acids proximal to the inhibitor-binding site. We used the JAK2-V617F-Y931C, because blocking one binding site is probably insufficient to observe resistance conferred by a secondary site. As expected, mutations I1018F/W and L1026F reduced kinase activity, while F1019L, W1020C and Y1021H resulted in inactive kinase (Supplementary Fig. 17). I1018F/W and L1026F exhibited increased IC50 values in a dose-dependent cell proliferation assay (Fig. 8a–e) and a phospho-STAT5 inhibition assay (Fig. 8k). Next, we performed a resistant screen using 100 million BaF3 cells expressing JAK2-V617F-Y931C; cells were treated with fedratinib at 2.0 μM—with the assumption that a lower drug concentration may select for clones conferring mild resistance, which could inform the secondary binding site. This screen produced one resistant clone, sequencing of which revealed a total of 13 different mutations—all in the substrate-binding pocket (Fig. 8f). Structural modeling suggested that these mutations affect fedratinib binding either by direct steric hindrance (S1025C, S1039F, G1041R, V1042F, I1074F, and V1075F) or by affecting stability of the conserved IFWY motif of the activation loop in the substrate-binding site (W1038C, Y1045W/*, P1058F, F1061W, I1079L, and L1082R) (Fig. 8g–i). To validate these findings, we designed the mutations (S1025C, W1038C, S1039F, Y1045W/*, F1061W, V1075F) in the context of JAK2V617F-Y931C. As expected, all 13 variants displayed weaker kinase activity, compared to parental, JAK2-V617F/Y931C kinase (Supplementary Fig. 17). While 4 variants (S1025C, Y1045W, F1061W and V1075F) conferred strong biochemical and cellular resistance (Fig. 8J,K), others (W1038C, S1039F and Y1045*) conferred subtle changes in cellular IC50 but no change in STAT5 inhibition. This suggests either these variants are false positives or express a catalytically inactive kinase that acts as a sink for inhibitor, which is reflected in moderate change in drug response.

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.