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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.


Related in: MedlinePlus

Random mutagenesis and in vitro drug-resistant screening reveal lack of genetic resistance against fedratinib.(a) Frequency of resistance of BaF3 cells transformed with randomly mutagenized JAK2-V617F. For each experiment, 1.0 × 107 BaF3 cells were transduced with randomly mutagenized JAK2-V617F virus. After 16 to 20 hours, cells were exposed to different concentrations of drug inhibitors, for two weeks. The number of clones obtained per 5 × 106 cells from two independent experiments was averaged and plotted. (b) Frequency of resistant mutations from the FERM domain. (c) Frequency of resistant mutations from the SH2 and pseudokinase (JH2) domains. (d) Frequency of resistant mutations from the kinase-linker and kinase (JH1) domains.
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f1: Random mutagenesis and in vitro drug-resistant screening reveal lack of genetic resistance against fedratinib.(a) Frequency of resistance of BaF3 cells transformed with randomly mutagenized JAK2-V617F. For each experiment, 1.0 × 107 BaF3 cells were transduced with randomly mutagenized JAK2-V617F virus. After 16 to 20 hours, cells were exposed to different concentrations of drug inhibitors, for two weeks. The number of clones obtained per 5 × 106 cells from two independent experiments was averaged and plotted. (b) Frequency of resistant mutations from the FERM domain. (c) Frequency of resistant mutations from the SH2 and pseudokinase (JH2) domains. (d) Frequency of resistant mutations from the kinase-linker and kinase (JH1) domains.

Mentions: We performed a ruxolitinib resistant screen using BaF3-MPL cells that showed emergence of resistant clones (data not shown). Although these clones conferred robust resistance to ruxolitinib, sequencing did not reveal mutations. In vitro characterization of these clones showed both higher IC50 and increased resistance to ruxolitinib, thus suggesting that the BaF3-MPL cells expressing JAK2-V617F are not addicted to JAK2 because MPL overexpression seemingly bypasses the JAK2 dependent survival. Therefore, we performed screening using parental BaF3 cells transduced with randomly mutagenized JAK2-V617F and two clinically relevant JAK2 inhibitors: ruxolitinib and fedratinib. Ruxolitinib-resistant clones emerged at 1, 2 and 5 μM inhibitor—representing 10−, 25− and 50-fold increases in IC50 values for JAK2-V617F (~100 nM), respectively (Fig. 1a). In contrast, selection against fedratinib at concentrations 2-fold above IC50 (~0.9 μM) did not result in any resistant clones (Fig. 1a, lower panel). From the 190 ruxolitinib-resistant colonies, we identified 211 distinct amino-acid substitutions affecting 149 residues. Amino-acid substitutions at 58% of the positions were identified more than once (Fig. 1b–d).


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)

Random mutagenesis and in vitro drug-resistant screening reveal lack of genetic resistance against fedratinib.(a) Frequency of resistance of BaF3 cells transformed with randomly mutagenized JAK2-V617F. For each experiment, 1.0 × 107 BaF3 cells were transduced with randomly mutagenized JAK2-V617F virus. After 16 to 20 hours, cells were exposed to different concentrations of drug inhibitors, for two weeks. The number of clones obtained per 5 × 106 cells from two independent experiments was averaged and plotted. (b) Frequency of resistant mutations from the FERM domain. (c) Frequency of resistant mutations from the SH2 and pseudokinase (JH2) domains. (d) Frequency of resistant mutations from the kinase-linker and kinase (JH1) domains.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588578&req=5

f1: Random mutagenesis and in vitro drug-resistant screening reveal lack of genetic resistance against fedratinib.(a) Frequency of resistance of BaF3 cells transformed with randomly mutagenized JAK2-V617F. For each experiment, 1.0 × 107 BaF3 cells were transduced with randomly mutagenized JAK2-V617F virus. After 16 to 20 hours, cells were exposed to different concentrations of drug inhibitors, for two weeks. The number of clones obtained per 5 × 106 cells from two independent experiments was averaged and plotted. (b) Frequency of resistant mutations from the FERM domain. (c) Frequency of resistant mutations from the SH2 and pseudokinase (JH2) domains. (d) Frequency of resistant mutations from the kinase-linker and kinase (JH1) domains.
Mentions: We performed a ruxolitinib resistant screen using BaF3-MPL cells that showed emergence of resistant clones (data not shown). Although these clones conferred robust resistance to ruxolitinib, sequencing did not reveal mutations. In vitro characterization of these clones showed both higher IC50 and increased resistance to ruxolitinib, thus suggesting that the BaF3-MPL cells expressing JAK2-V617F are not addicted to JAK2 because MPL overexpression seemingly bypasses the JAK2 dependent survival. Therefore, we performed screening using parental BaF3 cells transduced with randomly mutagenized JAK2-V617F and two clinically relevant JAK2 inhibitors: ruxolitinib and fedratinib. Ruxolitinib-resistant clones emerged at 1, 2 and 5 μM inhibitor—representing 10−, 25− and 50-fold increases in IC50 values for JAK2-V617F (~100 nM), respectively (Fig. 1a). In contrast, selection against fedratinib at concentrations 2-fold above IC50 (~0.9 μM) did not result in any resistant clones (Fig. 1a, lower panel). From the 190 ruxolitinib-resistant colonies, we identified 211 distinct amino-acid substitutions affecting 149 residues. Amino-acid substitutions at 58% of the positions were identified more than once (Fig. 1b–d).

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.


Related in: MedlinePlus