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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.


Mapping of ruxolitinib-resistant mutations on JAK2 structure.(a) Primary structure of JAK2 kinase showing structural subdomains. (b) FERM domain model, developed by homology modeling using FAK coordinates (PDB: 2J0J and 2J0L), showing different subdomains and distribution of resistant variants. Subdomains A and C have the highest number of mutations. In contrast to FERM domain of FAK, we identified two extra β-sheets in JAK2 between subdomains B and C (cyan arrow). (c) Homology model of the SH2 domain based on SRC and ABL structures (1OPK and 2SRC), showing resistant mutations in the α-helices. (d) Pseudokinase structure (PDB: 4FVR) showing the oncogenic mutation V617F (green) and many resistant variants clustered in the catalytic site—around the ATP-binding site (red), P-loop (green), and helix-C (pink) (e). JAK2 kinase domain structure (PDB: 2B7A) docked with ruxolitinib showing the distribution of resistant mutations. Many mutations are clustered around the ATP binding and catalytic sites, and in the C-lobe. The catalytic site is comprised of a catalytic loop (red), hinge region (green) and activation loop (yellow). α-C of mutant residues are presented as circles.
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f2: Mapping of ruxolitinib-resistant mutations on JAK2 structure.(a) Primary structure of JAK2 kinase showing structural subdomains. (b) FERM domain model, developed by homology modeling using FAK coordinates (PDB: 2J0J and 2J0L), showing different subdomains and distribution of resistant variants. Subdomains A and C have the highest number of mutations. In contrast to FERM domain of FAK, we identified two extra β-sheets in JAK2 between subdomains B and C (cyan arrow). (c) Homology model of the SH2 domain based on SRC and ABL structures (1OPK and 2SRC), showing resistant mutations in the α-helices. (d) Pseudokinase structure (PDB: 4FVR) showing the oncogenic mutation V617F (green) and many resistant variants clustered in the catalytic site—around the ATP-binding site (red), P-loop (green), and helix-C (pink) (e). JAK2 kinase domain structure (PDB: 2B7A) docked with ruxolitinib showing the distribution of resistant mutations. Many mutations are clustered around the ATP binding and catalytic sites, and in the C-lobe. The catalytic site is comprised of a catalytic loop (red), hinge region (green) and activation loop (yellow). α-C of mutant residues are presented as circles.

Mentions: We next mapped resistant mutations onto crystal structures of JAK2 kinase domains25, pseudokinase domains26, and homology-based structural models of the FERM and SH2 domains, developed using SWISS-MODEL27 and the structures of FAK (PDB:2J0J, 2J0L and 2J0M), SRC (PDB:2SRC) and ABL kinases (PDB: 1OPJ, 1OPL, 2G2H and 2G2I) (Fig. 2). We identified four structural hot spots in the kinase catalytic domain—specifically in the active site and allosteric sites A, B and C (Supplementary Fig. 1). Interestingly, these hot spots are similar to imatinib-resistant mutations identified from both drug-resistant screens and relapsed patients2829. These mutations are, therefore, likely to be involved in allosteric regulation of kinase conformation (probably by inter- and intra-domain interactions modulating auto and co-inhibitory state) and drug-binding state.


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)

Mapping of ruxolitinib-resistant mutations on JAK2 structure.(a) Primary structure of JAK2 kinase showing structural subdomains. (b) FERM domain model, developed by homology modeling using FAK coordinates (PDB: 2J0J and 2J0L), showing different subdomains and distribution of resistant variants. Subdomains A and C have the highest number of mutations. In contrast to FERM domain of FAK, we identified two extra β-sheets in JAK2 between subdomains B and C (cyan arrow). (c) Homology model of the SH2 domain based on SRC and ABL structures (1OPK and 2SRC), showing resistant mutations in the α-helices. (d) Pseudokinase structure (PDB: 4FVR) showing the oncogenic mutation V617F (green) and many resistant variants clustered in the catalytic site—around the ATP-binding site (red), P-loop (green), and helix-C (pink) (e). JAK2 kinase domain structure (PDB: 2B7A) docked with ruxolitinib showing the distribution of resistant mutations. Many mutations are clustered around the ATP binding and catalytic sites, and in the C-lobe. The catalytic site is comprised of a catalytic loop (red), hinge region (green) and activation loop (yellow). α-C of mutant residues are presented as circles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Mapping of ruxolitinib-resistant mutations on JAK2 structure.(a) Primary structure of JAK2 kinase showing structural subdomains. (b) FERM domain model, developed by homology modeling using FAK coordinates (PDB: 2J0J and 2J0L), showing different subdomains and distribution of resistant variants. Subdomains A and C have the highest number of mutations. In contrast to FERM domain of FAK, we identified two extra β-sheets in JAK2 between subdomains B and C (cyan arrow). (c) Homology model of the SH2 domain based on SRC and ABL structures (1OPK and 2SRC), showing resistant mutations in the α-helices. (d) Pseudokinase structure (PDB: 4FVR) showing the oncogenic mutation V617F (green) and many resistant variants clustered in the catalytic site—around the ATP-binding site (red), P-loop (green), and helix-C (pink) (e). JAK2 kinase domain structure (PDB: 2B7A) docked with ruxolitinib showing the distribution of resistant mutations. Many mutations are clustered around the ATP binding and catalytic sites, and in the C-lobe. The catalytic site is comprised of a catalytic loop (red), hinge region (green) and activation loop (yellow). α-C of mutant residues are presented as circles.
Mentions: We next mapped resistant mutations onto crystal structures of JAK2 kinase domains25, pseudokinase domains26, and homology-based structural models of the FERM and SH2 domains, developed using SWISS-MODEL27 and the structures of FAK (PDB:2J0J, 2J0L and 2J0M), SRC (PDB:2SRC) and ABL kinases (PDB: 1OPJ, 1OPL, 2G2H and 2G2I) (Fig. 2). We identified four structural hot spots in the kinase catalytic domain—specifically in the active site and allosteric sites A, B and C (Supplementary Fig. 1). Interestingly, these hot spots are similar to imatinib-resistant mutations identified from both drug-resistant screens and relapsed patients2829. These mutations are, therefore, likely to be involved in allosteric regulation of kinase conformation (probably by inter- and intra-domain interactions modulating auto and co-inhibitory state) and drug-binding state.

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.