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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.


Allosteric ruxolitinib resistant variants activate the enzymatic activity.(a) A coomassie blue stained gel showing partially purified full length JAK2 and its variants. (b) Steady state enzyme kinetics showing the activation of ruxolitinib resistant mutants except L983F. Variant R683T is highly active, while Y931C showed increased Vmax and and reduced Km(ATP). The Apparent values for Km(ATP) and Vmax are shown on the right side of the graph for each mutant. (c) Dose response analysis against ruxolitinib showing resistance to inhibition. IC50 values for each mutant (as an indirect measure for drug affinity) are indicated on the right side of the graph.
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f6: Allosteric ruxolitinib resistant variants activate the enzymatic activity.(a) A coomassie blue stained gel showing partially purified full length JAK2 and its variants. (b) Steady state enzyme kinetics showing the activation of ruxolitinib resistant mutants except L983F. Variant R683T is highly active, while Y931C showed increased Vmax and and reduced Km(ATP). The Apparent values for Km(ATP) and Vmax are shown on the right side of the graph for each mutant. (c) Dose response analysis against ruxolitinib showing resistance to inhibition. IC50 values for each mutant (as an indirect measure for drug affinity) are indicated on the right side of the graph.

Mentions: To understand the mechanisms underlying how mutations from the FERM, SH2 and pseudokinase domain confer resistance, we performed steady state kinetics to determine the Km for ATP and Vmax. One principal mechanism for developing resistance against ATP-competitive inhibitors is to select for kinase activating mutations. In some instances these activating mutations may change the Km for ATP providing additional support to compete out inhibitor binding; a common mechanism exploited by gatekeeper mutations to confer resistance. To determine the kinetic parameters we expressed full length JAK2-V617F and seven ruxolitinib resistant variants in insect cells, proteins were purified using Strep-Tactin resins (Fig. 6a). Kinetic experiments were carried out at saturating concentration of STAT5 peptide (50 uM) with varying concentrations of ATP to determine the Km (ATP)app and Vmax (Fig. 6b). These analyses revealed that allosteric mutations have higher enzymatic activity as anticipated from the cell based transformation assay (Supplementary Fig. 3A,B). Resistant variant R683T (pseudokinase domain) displayed a 5-fold increase in Vmax, while variants R277K (FERM domain), G417P (SH2 domain), D544H (SH2-pseudokinase linker) and Y931C (Kinase domain) displayed ~2 fold increase Vmax compared to JAK2-V617F. Interestingly variants L551P (pseudokinase domain) and Y931C (kinase domain) have reduced Km (ATP)app . However, resistant variant L983F did not show any significant change in Km (ATP)app and Vmax. Whereas, a kinetic analysis of purified kinase domain displayed ~20 fold (Y931C), 4 fold (E930G) and 2 fold (L983F) increase in Vmax with significant changes in Km (ATP)app by Y931C and L983F (Supplementary Fig. 16) . The ATP Km value for full length JAK2-V617F and kinase domain are in close agreement as recently reported41. Dose response analysis against ruxolitinib, as an indirect measure of affinity, using these full-length proteins showed resistance to ruxolitinib, however, with differences in IC50 values reflecting their apparent differences in mediating resistance (Fig. 6c). Likewise, purified kinase domains having ruxolitinib resistant mutations conferred resistance to ruxolitinib but not to fedratinib (Supplementary Fig. 16C,D). However, dose response analysis against CYT-387 showed resistance for Y931C but both E930G and L983F were sensitive (Supplementary Fig. 16e).


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)

Allosteric ruxolitinib resistant variants activate the enzymatic activity.(a) A coomassie blue stained gel showing partially purified full length JAK2 and its variants. (b) Steady state enzyme kinetics showing the activation of ruxolitinib resistant mutants except L983F. Variant R683T is highly active, while Y931C showed increased Vmax and and reduced Km(ATP). The Apparent values for Km(ATP) and Vmax are shown on the right side of the graph for each mutant. (c) Dose response analysis against ruxolitinib showing resistance to inhibition. IC50 values for each mutant (as an indirect measure for drug affinity) are indicated on the right side of the graph.
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f6: Allosteric ruxolitinib resistant variants activate the enzymatic activity.(a) A coomassie blue stained gel showing partially purified full length JAK2 and its variants. (b) Steady state enzyme kinetics showing the activation of ruxolitinib resistant mutants except L983F. Variant R683T is highly active, while Y931C showed increased Vmax and and reduced Km(ATP). The Apparent values for Km(ATP) and Vmax are shown on the right side of the graph for each mutant. (c) Dose response analysis against ruxolitinib showing resistance to inhibition. IC50 values for each mutant (as an indirect measure for drug affinity) are indicated on the right side of the graph.
Mentions: To understand the mechanisms underlying how mutations from the FERM, SH2 and pseudokinase domain confer resistance, we performed steady state kinetics to determine the Km for ATP and Vmax. One principal mechanism for developing resistance against ATP-competitive inhibitors is to select for kinase activating mutations. In some instances these activating mutations may change the Km for ATP providing additional support to compete out inhibitor binding; a common mechanism exploited by gatekeeper mutations to confer resistance. To determine the kinetic parameters we expressed full length JAK2-V617F and seven ruxolitinib resistant variants in insect cells, proteins were purified using Strep-Tactin resins (Fig. 6a). Kinetic experiments were carried out at saturating concentration of STAT5 peptide (50 uM) with varying concentrations of ATP to determine the Km (ATP)app and Vmax (Fig. 6b). These analyses revealed that allosteric mutations have higher enzymatic activity as anticipated from the cell based transformation assay (Supplementary Fig. 3A,B). Resistant variant R683T (pseudokinase domain) displayed a 5-fold increase in Vmax, while variants R277K (FERM domain), G417P (SH2 domain), D544H (SH2-pseudokinase linker) and Y931C (Kinase domain) displayed ~2 fold increase Vmax compared to JAK2-V617F. Interestingly variants L551P (pseudokinase domain) and Y931C (kinase domain) have reduced Km (ATP)app . However, resistant variant L983F did not show any significant change in Km (ATP)app and Vmax. Whereas, a kinetic analysis of purified kinase domain displayed ~20 fold (Y931C), 4 fold (E930G) and 2 fold (L983F) increase in Vmax with significant changes in Km (ATP)app by Y931C and L983F (Supplementary Fig. 16) . The ATP Km value for full length JAK2-V617F and kinase domain are in close agreement as recently reported41. Dose response analysis against ruxolitinib, as an indirect measure of affinity, using these full-length proteins showed resistance to ruxolitinib, however, with differences in IC50 values reflecting their apparent differences in mediating resistance (Fig. 6c). Likewise, purified kinase domains having ruxolitinib resistant mutations conferred resistance to ruxolitinib but not to fedratinib (Supplementary Fig. 16C,D). However, dose response analysis against CYT-387 showed resistance for Y931C but both E930G and L983F were sensitive (Supplementary Fig. 16e).

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.