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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.


Ruxolitinib-resistant variants confer cross resistance to AZD1480, CYT-387 and lestaurtinib—but not to fedratinib.IC50 values for AZD1480 (a), CYT-387 (b), lestaurtinib (c) and fedratinib (d) were normalized to 1 with JAK2-V617F and plotted on a semilogarithmic scale. Structural domains, FERM, SH2 and pseudokinase, are indicated to the left side of each graph. The most frequent and highly resistant mutation, L983F, is sensitive to CYT-387 and fedratinib. Ruxolitinib-resistant variants are fully sensitive or mildly resistant to TG10138, when cells were grown with or without IL-3, respectively. (e) Structural model of AZD1480 bound to ATP binding site, showing steric clash with phenylalanine substituted for Leu 983. (f) Structural model of CYT-387 bound to ATP binding site, showing no direct clash with bulkier phenylalanine substitution for Leu 983, explains why this variant is still sensitive to CYT-387. (g) Structural model of lestaurtinib bound to the ATP-binding site, showing steric clash with phenylalanine substituted for leucine 983.
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f4: Ruxolitinib-resistant variants confer cross resistance to AZD1480, CYT-387 and lestaurtinib—but not to fedratinib.IC50 values for AZD1480 (a), CYT-387 (b), lestaurtinib (c) and fedratinib (d) were normalized to 1 with JAK2-V617F and plotted on a semilogarithmic scale. Structural domains, FERM, SH2 and pseudokinase, are indicated to the left side of each graph. The most frequent and highly resistant mutation, L983F, is sensitive to CYT-387 and fedratinib. Ruxolitinib-resistant variants are fully sensitive or mildly resistant to TG10138, when cells were grown with or without IL-3, respectively. (e) Structural model of AZD1480 bound to ATP binding site, showing steric clash with phenylalanine substituted for Leu 983. (f) Structural model of CYT-387 bound to ATP binding site, showing no direct clash with bulkier phenylalanine substitution for Leu 983, explains why this variant is still sensitive to CYT-387. (g) Structural model of lestaurtinib bound to the ATP-binding site, showing steric clash with phenylalanine substituted for leucine 983.

Mentions: Because each inhibitor binds a unique conformational state, engaging different sets of amino acid residues37, we assumed that some ruxolitinib-resistant variants might be sensitive to other JAK2 inhibitors. We therefore carried out dose-response analyses with ruxolitinib-resistant variants and four other JAK2 inhibitors that are in clinical trials (AZD1840, CYT-387, lestaurtinib and fedratinib). Of these, many mutants displayed cross-resistance to AZD1840, CYT-387 and Lestaurtinib—but not to fedratinib (Fig. 4a–d). As expected, drug response varied with mutation type and cytokine receptor activation, enforcing the notion that each inhibitor targets distinct kinase conformations. For instance, Y931C is fully sensitive to lestaurtinib in the absence of IL-3, but showed 5-fold resistance in the presence of IL-3 (Fig. 4c). Likewise, while pseudokinase domain variants showed greater resistance to lestaurtinib in the absence of IL-3, and greater resistance to CYT-387 in the presence of IL-3; FERM domain variants also demonstrated greater resistance to CYT-387 in the presence of IL-3 (Fig. 4). Finally, variant L983F showed resistance to AZD1480 and lestaurtinib, but retained full sensitivity to CYT-387. Structural modeling and docking revealed that AZD1480, lestaurtinib and CYT-387 are type-I ATP-site inhibitors (Fig. 4e–g and supplementary Figs 9–11). Substitution of Phe for Leu 983 caused steric hindrance to AZD1480 and lestaurtinib, but did not affect CYT-387 binding (Fig. 4e–g) providing an explanation for the sensitivity of L983F variant to CYT-387. These data suggest that each inhibitor targets a distinct kinase conformation, and alteration in conformational dynamics by allosteric mutations destabilize drug binding state, thus conferring resistance38. Once the ternary structure of JAK2 is solved, our mutational data will be instrumental in understanding how inter and intramolecular interactions modulate kinase conformation and activation.


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)

Ruxolitinib-resistant variants confer cross resistance to AZD1480, CYT-387 and lestaurtinib—but not to fedratinib.IC50 values for AZD1480 (a), CYT-387 (b), lestaurtinib (c) and fedratinib (d) were normalized to 1 with JAK2-V617F and plotted on a semilogarithmic scale. Structural domains, FERM, SH2 and pseudokinase, are indicated to the left side of each graph. The most frequent and highly resistant mutation, L983F, is sensitive to CYT-387 and fedratinib. Ruxolitinib-resistant variants are fully sensitive or mildly resistant to TG10138, when cells were grown with or without IL-3, respectively. (e) Structural model of AZD1480 bound to ATP binding site, showing steric clash with phenylalanine substituted for Leu 983. (f) Structural model of CYT-387 bound to ATP binding site, showing no direct clash with bulkier phenylalanine substitution for Leu 983, explains why this variant is still sensitive to CYT-387. (g) Structural model of lestaurtinib bound to the ATP-binding site, showing steric clash with phenylalanine substituted for leucine 983.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Ruxolitinib-resistant variants confer cross resistance to AZD1480, CYT-387 and lestaurtinib—but not to fedratinib.IC50 values for AZD1480 (a), CYT-387 (b), lestaurtinib (c) and fedratinib (d) were normalized to 1 with JAK2-V617F and plotted on a semilogarithmic scale. Structural domains, FERM, SH2 and pseudokinase, are indicated to the left side of each graph. The most frequent and highly resistant mutation, L983F, is sensitive to CYT-387 and fedratinib. Ruxolitinib-resistant variants are fully sensitive or mildly resistant to TG10138, when cells were grown with or without IL-3, respectively. (e) Structural model of AZD1480 bound to ATP binding site, showing steric clash with phenylalanine substituted for Leu 983. (f) Structural model of CYT-387 bound to ATP binding site, showing no direct clash with bulkier phenylalanine substitution for Leu 983, explains why this variant is still sensitive to CYT-387. (g) Structural model of lestaurtinib bound to the ATP-binding site, showing steric clash with phenylalanine substituted for leucine 983.
Mentions: Because each inhibitor binds a unique conformational state, engaging different sets of amino acid residues37, we assumed that some ruxolitinib-resistant variants might be sensitive to other JAK2 inhibitors. We therefore carried out dose-response analyses with ruxolitinib-resistant variants and four other JAK2 inhibitors that are in clinical trials (AZD1840, CYT-387, lestaurtinib and fedratinib). Of these, many mutants displayed cross-resistance to AZD1840, CYT-387 and Lestaurtinib—but not to fedratinib (Fig. 4a–d). As expected, drug response varied with mutation type and cytokine receptor activation, enforcing the notion that each inhibitor targets distinct kinase conformations. For instance, Y931C is fully sensitive to lestaurtinib in the absence of IL-3, but showed 5-fold resistance in the presence of IL-3 (Fig. 4c). Likewise, while pseudokinase domain variants showed greater resistance to lestaurtinib in the absence of IL-3, and greater resistance to CYT-387 in the presence of IL-3; FERM domain variants also demonstrated greater resistance to CYT-387 in the presence of IL-3 (Fig. 4). Finally, variant L983F showed resistance to AZD1480 and lestaurtinib, but retained full sensitivity to CYT-387. Structural modeling and docking revealed that AZD1480, lestaurtinib and CYT-387 are type-I ATP-site inhibitors (Fig. 4e–g and supplementary Figs 9–11). Substitution of Phe for Leu 983 caused steric hindrance to AZD1480 and lestaurtinib, but did not affect CYT-387 binding (Fig. 4e–g) providing an explanation for the sensitivity of L983F variant to CYT-387. These data suggest that each inhibitor targets a distinct kinase conformation, and alteration in conformational dynamics by allosteric mutations destabilize drug binding state, thus conferring resistance38. Once the ternary structure of JAK2 is solved, our mutational data will be instrumental in understanding how inter and intramolecular interactions modulate kinase conformation and activation.

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.