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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

Fedratinib binds to substrate-site with higher affinity than ATP site.(a,b) Microscale thermophoresis (MST) analysis showing the dual binding of fedratinib to JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Fedratinib binding was carried out with label free proteins. (c,d) MST analysis shows a single binding site (Site I-ATP site) for ruxolitinib with JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Because, ruxolitinib showed auto-fluorescence that precluded label free binding analysis, we performed ruxolitinib binding with fluorescently labeled proteins. Note, we could not detect any secondary binding site for ruxolitinib. (e,f) MST analysis with JAK2-L983F showed only one binding site for fedratinib at site –II (substrate site). A phenylalanine substitution for Leu 983 causes steric hindrance to fedratinib and it no longer can bind to site-I, ATP site. In contrast, substrate site is not affected by this mutation and therefore binding of fedratinib to site-II is not affected showing similar kd values. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. (g) MST analysis with JAK2-L983F showing loss of ruxolitinib binding with mutant L983F. These experiments were performed twice in duplicates.
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f7: Fedratinib binds to substrate-site with higher affinity than ATP site.(a,b) Microscale thermophoresis (MST) analysis showing the dual binding of fedratinib to JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Fedratinib binding was carried out with label free proteins. (c,d) MST analysis shows a single binding site (Site I-ATP site) for ruxolitinib with JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Because, ruxolitinib showed auto-fluorescence that precluded label free binding analysis, we performed ruxolitinib binding with fluorescently labeled proteins. Note, we could not detect any secondary binding site for ruxolitinib. (e,f) MST analysis with JAK2-L983F showed only one binding site for fedratinib at site –II (substrate site). A phenylalanine substitution for Leu 983 causes steric hindrance to fedratinib and it no longer can bind to site-I, ATP site. In contrast, substrate site is not affected by this mutation and therefore binding of fedratinib to site-II is not affected showing similar kd values. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. (g) MST analysis with JAK2-L983F showing loss of ruxolitinib binding with mutant L983F. These experiments were performed twice in duplicates.

Mentions: To further characterize the dual binding of fedratinib we performed biophysical measurements using purified kinase domains of JAK2-WT and JAK2-L983F by Microscale Thermophoresis (MST). Thermophoresis measures the motion of molecules in microscopic temperature gradients that allows measuring the affinity constants and binding stoichiometry of a wide variety of interactions in the binding equilibrium. We reasoned that if fedratinib binds to two different sites i. e. the ATP and the substrate site, the use of an ATP site mutant for binding analysis will show only one binding site. As predicted, fedratinib showed two binding sites in JAK2-WT with binding affinities having Kd values of 8307 nM (Site –I/ATP site) and 21 nM (Site-II/substrate site), Fig. 7a,b. A similar analysis with ruxolitinib showed only one binding site (ATP site) having Kd of 804 nM, Fig. 7c,d. Perhaps more striking, ATP site variant JAK2-L983F showed only one binding site, site-II/substrate site, having a Kd value of 17 nM (Fig. 7e,f). In contrast, ruxolitinib no longer binds to JAK2-L983F (Fig. 7g). These data confirms in silico docking simulations, which predicted fedratinib binds to substrate site with higher affinity than ATP site.


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 substrate-site with higher affinity than ATP site.(a,b) Microscale thermophoresis (MST) analysis showing the dual binding of fedratinib to JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Fedratinib binding was carried out with label free proteins. (c,d) MST analysis shows a single binding site (Site I-ATP site) for ruxolitinib with JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Because, ruxolitinib showed auto-fluorescence that precluded label free binding analysis, we performed ruxolitinib binding with fluorescently labeled proteins. Note, we could not detect any secondary binding site for ruxolitinib. (e,f) MST analysis with JAK2-L983F showed only one binding site for fedratinib at site –II (substrate site). A phenylalanine substitution for Leu 983 causes steric hindrance to fedratinib and it no longer can bind to site-I, ATP site. In contrast, substrate site is not affected by this mutation and therefore binding of fedratinib to site-II is not affected showing similar kd values. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. (g) MST analysis with JAK2-L983F showing loss of ruxolitinib binding with mutant L983F. These experiments were performed twice in duplicates.
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Related In: Results  -  Collection

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f7: Fedratinib binds to substrate-site with higher affinity than ATP site.(a,b) Microscale thermophoresis (MST) analysis showing the dual binding of fedratinib to JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Fedratinib binding was carried out with label free proteins. (c,d) MST analysis shows a single binding site (Site I-ATP site) for ruxolitinib with JAK2-WT. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. Because, ruxolitinib showed auto-fluorescence that precluded label free binding analysis, we performed ruxolitinib binding with fluorescently labeled proteins. Note, we could not detect any secondary binding site for ruxolitinib. (e,f) MST analysis with JAK2-L983F showed only one binding site for fedratinib at site –II (substrate site). A phenylalanine substitution for Leu 983 causes steric hindrance to fedratinib and it no longer can bind to site-I, ATP site. In contrast, substrate site is not affected by this mutation and therefore binding of fedratinib to site-II is not affected showing similar kd values. Top panels are showing the fluorescence. Bottom panels are showing the bound fractions of inhibitors and proteins to calculate the dissociation constant (Kd), which is mentioned in the parenthesis. (g) MST analysis with JAK2-L983F showing loss of ruxolitinib binding with mutant L983F. These experiments were performed twice in duplicates.
Mentions: To further characterize the dual binding of fedratinib we performed biophysical measurements using purified kinase domains of JAK2-WT and JAK2-L983F by Microscale Thermophoresis (MST). Thermophoresis measures the motion of molecules in microscopic temperature gradients that allows measuring the affinity constants and binding stoichiometry of a wide variety of interactions in the binding equilibrium. We reasoned that if fedratinib binds to two different sites i. e. the ATP and the substrate site, the use of an ATP site mutant for binding analysis will show only one binding site. As predicted, fedratinib showed two binding sites in JAK2-WT with binding affinities having Kd values of 8307 nM (Site –I/ATP site) and 21 nM (Site-II/substrate site), Fig. 7a,b. A similar analysis with ruxolitinib showed only one binding site (ATP site) having Kd of 804 nM, Fig. 7c,d. Perhaps more striking, ATP site variant JAK2-L983F showed only one binding site, site-II/substrate site, having a Kd value of 17 nM (Fig. 7e,f). In contrast, ruxolitinib no longer binds to JAK2-L983F (Fig. 7g). These data confirms in silico docking simulations, which predicted fedratinib binds to substrate site with higher affinity than ATP site.

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