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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.


Resistant variants from the active site act by steric hindrance or disrupting active-site integrity.(a) A cartoon depiction of JAK2 active site bound with ruxolitinib (blue sticks) showing its interaction with Glu 930 and Asn 981 by hydrogen bonding (dashed line). Residue Tyr 931 interacts with Gln 853, Leu 855 and Gly 935 by hydrogen bonds with 5 water molecules (red circles). Residue Asp 994 (orange stick) from the DFG motif directly interacts with ruxolitinib and catalytic Lys 882. (b) Immunoblot analysis of BaF3 cells expressing JAK2-V617 variants Y931C and L983F, showing dose-dependent inhibition of Y931C by ruxolitinib (L983F is completely resistant). Upper panel showing increasing phospho-JAK2 levels with increasing drug concentration, possibly due to preferential binding and stabilization of the active conformation73. Lower panel showing phospho-STAT5 level. Blots were stripped and reprobed for total JAK2 and STAT5. (c) Structural model of ruxolitinib, showing steric clash with phenylalanine substituted for Leu 983.
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f3: Resistant variants from the active site act by steric hindrance or disrupting active-site integrity.(a) A cartoon depiction of JAK2 active site bound with ruxolitinib (blue sticks) showing its interaction with Glu 930 and Asn 981 by hydrogen bonding (dashed line). Residue Tyr 931 interacts with Gln 853, Leu 855 and Gly 935 by hydrogen bonds with 5 water molecules (red circles). Residue Asp 994 (orange stick) from the DFG motif directly interacts with ruxolitinib and catalytic Lys 882. (b) Immunoblot analysis of BaF3 cells expressing JAK2-V617 variants Y931C and L983F, showing dose-dependent inhibition of Y931C by ruxolitinib (L983F is completely resistant). Upper panel showing increasing phospho-JAK2 levels with increasing drug concentration, possibly due to preferential binding and stabilization of the active conformation73. Lower panel showing phospho-STAT5 level. Blots were stripped and reprobed for total JAK2 and STAT5. (c) Structural model of ruxolitinib, showing steric clash with phenylalanine substituted for Leu 983.

Mentions: Mutations within the ATP-binding site usually confer resistance by directly blocking drug binding, rather than by modulating conformational dynamics (the latter is a common mechanism used by allosteric mutations). Accordingly, many mutations clustered in the active site (i.e., Y966E, L983F, N986Y, E987D, and E1046K) were resistant, but did not exhibit any change in IC50 values when assayed in the presence or absence of IL-3 (Supplementary Fig. 2), or in JAK2-V617F malignant potential (Supplementary Fig. 4). This implies that these mutations were directly impacting drug binding, rather than modulating the kinase dynamics by altering inter- or intramolecular interactions. We next performed molecular-docking of ruxolitinib, by targeted docking to the ATP-site and blind docking to the whole kinase domain by SwissDock35 using the JAK2 coordinate (PDB:2B7A) . These analyses revealed that ruxolitinib anchors to the ATP binding cleft by three hydrogen bonds where it forms two hydrogen bonds in the hinge region with residues Glu 930, Leu 932 and one in catalytic site with Asn 981. Ruxolitinib requires DFG-in conformation for binding (Fig. 3a)—as seen for other type-1 kinase inhibitors. The hydrophobic rings of ruxolitinib make van der Walls interactions with the side chains of residues Val 911, Met 929, Leu 855, Lys 882, Leu932, Pro 933, Gly 935, Arg 980, Asn 981, Leu 983, and Asp 994 (Supplementary Fig. 5). Structural modeling of the active-site mutations suggests three principal mechanisms governing resistance: (i), loss of hydrogen-bond for anchoring, by substituting threonine for Asn 981, while variants E930G and L932F may disrupt hydrogen bond interactions perhaps by altering the dynamics of hinge region (Fig. 3a and Supplementary Fig. 5); (ii) steric hindrance (eg, V911L, L927F, G935R, and L983F) (Fig. 3c); and (iii) active-site destabilization, by mutations in the kinase hinge (Y931C, E930G, L932F, P933A, and E985K), the catalytic loop (N981I/T) and the DFG motif (D994N) (Supplementary Figs 5–7). Amongst these, Tyr 931 coordinates the structural integrity of the P-loop and active site, by interacting through four water molecules with Gln 853, Leu 855 and Gly 935 (Fig. 3a). In cellular transformation assays, cysteine or phenylalanine substitution at this position disrupted these interactions, resulting in full activation of the kinase much like BCR-ABL and TEL-JAK2 (Supplementary Fig. 7). Structural modeling of activation-loop variants (R971G, L1001W and E1006K), suggests that these mutations should destabilize the activation loop from the active kinase conformation (Supplementary Fig. 8A–C), suggesting that ruxolitinib preferably binds an active kinase conformation. The fact that phospho-JAK2 levels increase with increasing inhibitor concentration (Fig. 3b), also suggests that ruxolitinib binds the active state. If this model is correct, then activating variants of JAK2 should be hypersensitive to ruxolitinib inhibition, rather than conferring resistance. In contrast, we observed that activating variants R683T and Y931C conferred resistance to ruxolitinib (Fig. 3b, and Supplementary Fig. 2). Likewise, fully activated JAK2 (TEL-JAK2) exhibited 4-fold higher resistance than JAK2-V617F (Supplementary Fig. 8D), suggesting that ruxolitinib has reduced affinity for fully activated kinase and, instead, preferentially binds an intermediate active-state kinase conformation, much as intermediate kinase conformations of ABL and SRC that adopt DFG-in conformation, but without fully extended activation loops36.


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)

Resistant variants from the active site act by steric hindrance or disrupting active-site integrity.(a) A cartoon depiction of JAK2 active site bound with ruxolitinib (blue sticks) showing its interaction with Glu 930 and Asn 981 by hydrogen bonding (dashed line). Residue Tyr 931 interacts with Gln 853, Leu 855 and Gly 935 by hydrogen bonds with 5 water molecules (red circles). Residue Asp 994 (orange stick) from the DFG motif directly interacts with ruxolitinib and catalytic Lys 882. (b) Immunoblot analysis of BaF3 cells expressing JAK2-V617 variants Y931C and L983F, showing dose-dependent inhibition of Y931C by ruxolitinib (L983F is completely resistant). Upper panel showing increasing phospho-JAK2 levels with increasing drug concentration, possibly due to preferential binding and stabilization of the active conformation73. Lower panel showing phospho-STAT5 level. Blots were stripped and reprobed for total JAK2 and STAT5. (c) Structural model of ruxolitinib, showing steric clash with phenylalanine substituted for Leu 983.
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f3: Resistant variants from the active site act by steric hindrance or disrupting active-site integrity.(a) A cartoon depiction of JAK2 active site bound with ruxolitinib (blue sticks) showing its interaction with Glu 930 and Asn 981 by hydrogen bonding (dashed line). Residue Tyr 931 interacts with Gln 853, Leu 855 and Gly 935 by hydrogen bonds with 5 water molecules (red circles). Residue Asp 994 (orange stick) from the DFG motif directly interacts with ruxolitinib and catalytic Lys 882. (b) Immunoblot analysis of BaF3 cells expressing JAK2-V617 variants Y931C and L983F, showing dose-dependent inhibition of Y931C by ruxolitinib (L983F is completely resistant). Upper panel showing increasing phospho-JAK2 levels with increasing drug concentration, possibly due to preferential binding and stabilization of the active conformation73. Lower panel showing phospho-STAT5 level. Blots were stripped and reprobed for total JAK2 and STAT5. (c) Structural model of ruxolitinib, showing steric clash with phenylalanine substituted for Leu 983.
Mentions: Mutations within the ATP-binding site usually confer resistance by directly blocking drug binding, rather than by modulating conformational dynamics (the latter is a common mechanism used by allosteric mutations). Accordingly, many mutations clustered in the active site (i.e., Y966E, L983F, N986Y, E987D, and E1046K) were resistant, but did not exhibit any change in IC50 values when assayed in the presence or absence of IL-3 (Supplementary Fig. 2), or in JAK2-V617F malignant potential (Supplementary Fig. 4). This implies that these mutations were directly impacting drug binding, rather than modulating the kinase dynamics by altering inter- or intramolecular interactions. We next performed molecular-docking of ruxolitinib, by targeted docking to the ATP-site and blind docking to the whole kinase domain by SwissDock35 using the JAK2 coordinate (PDB:2B7A) . These analyses revealed that ruxolitinib anchors to the ATP binding cleft by three hydrogen bonds where it forms two hydrogen bonds in the hinge region with residues Glu 930, Leu 932 and one in catalytic site with Asn 981. Ruxolitinib requires DFG-in conformation for binding (Fig. 3a)—as seen for other type-1 kinase inhibitors. The hydrophobic rings of ruxolitinib make van der Walls interactions with the side chains of residues Val 911, Met 929, Leu 855, Lys 882, Leu932, Pro 933, Gly 935, Arg 980, Asn 981, Leu 983, and Asp 994 (Supplementary Fig. 5). Structural modeling of the active-site mutations suggests three principal mechanisms governing resistance: (i), loss of hydrogen-bond for anchoring, by substituting threonine for Asn 981, while variants E930G and L932F may disrupt hydrogen bond interactions perhaps by altering the dynamics of hinge region (Fig. 3a and Supplementary Fig. 5); (ii) steric hindrance (eg, V911L, L927F, G935R, and L983F) (Fig. 3c); and (iii) active-site destabilization, by mutations in the kinase hinge (Y931C, E930G, L932F, P933A, and E985K), the catalytic loop (N981I/T) and the DFG motif (D994N) (Supplementary Figs 5–7). Amongst these, Tyr 931 coordinates the structural integrity of the P-loop and active site, by interacting through four water molecules with Gln 853, Leu 855 and Gly 935 (Fig. 3a). In cellular transformation assays, cysteine or phenylalanine substitution at this position disrupted these interactions, resulting in full activation of the kinase much like BCR-ABL and TEL-JAK2 (Supplementary Fig. 7). Structural modeling of activation-loop variants (R971G, L1001W and E1006K), suggests that these mutations should destabilize the activation loop from the active kinase conformation (Supplementary Fig. 8A–C), suggesting that ruxolitinib preferably binds an active kinase conformation. The fact that phospho-JAK2 levels increase with increasing inhibitor concentration (Fig. 3b), also suggests that ruxolitinib binds the active state. If this model is correct, then activating variants of JAK2 should be hypersensitive to ruxolitinib inhibition, rather than conferring resistance. In contrast, we observed that activating variants R683T and Y931C conferred resistance to ruxolitinib (Fig. 3b, and Supplementary Fig. 2). Likewise, fully activated JAK2 (TEL-JAK2) exhibited 4-fold higher resistance than JAK2-V617F (Supplementary Fig. 8D), suggesting that ruxolitinib has reduced affinity for fully activated kinase and, instead, preferentially binds an intermediate active-state kinase conformation, much as intermediate kinase conformations of ABL and SRC that adopt DFG-in conformation, but without fully extended activation loops36.

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.