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Crystal structures of PRK1 in complex with the clinical compounds lestaurtinib and tofacitinib reveal ligand induced conformational changes.

Chamberlain P, Delker S, Pagarigan B, Mahmoudi A, Jackson P, Abbasian M, Muir J, Raheja N, Cathers B - PLoS ONE (2014)

Bottom Line: The C-tail fully encircles the catalytic domain placing a phenylalanine in the ATP-binding site.Our inhibitor structures include examples of molecules which both interact with, and displace the C-tail from the active site.This information may assist in the design of inhibitors targeting both PRK and other members of the AGC kinase family.

View Article: PubMed Central - PubMed

Affiliation: Celgene Corporation, San Diego, California, United States of America; Department of Biochemistry and Structural Biology, Celgene Corporation, San Diego, California, United States of America.

ABSTRACT
Protein kinase C related kinase 1 (PRK1) is a component of Rho-GTPase, androgen receptor, histone demethylase and histone deacetylase signaling pathways implicated in prostate and ovarian cancer. Herein we describe the crystal structure of PRK1 in apo form, and also in complex with a panel of literature inhibitors including the clinical candidates lestaurtinib and tofacitinib, as well as the staurosporine analog Ro-31-8220. PRK1 is a member of the AGC-kinase class, and as such exhibits the characteristic regulatory sequence at the C-terminus of the catalytic domain--the 'C-tail'. The C-tail fully encircles the catalytic domain placing a phenylalanine in the ATP-binding site. Our inhibitor structures include examples of molecules which both interact with, and displace the C-tail from the active site. This information may assist in the design of inhibitors targeting both PRK and other members of the AGC kinase family.

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Disordering of the PRK1 C-tail in the lestaurtinib crystal structure.(a) PRK1:Ro-31-8220 is shown with the protein in blue, with the inhibitor in green. The C-tail is shown as a surface in gray; the region disordered in the lestaurtinib structure is shown in yellow on the Ro-31-8220 structure. (b) PRK1:Lestaurtinib is shown colored orange with the C-tail shown as a surface in grey, with a gap visible due to disorder of the C-tail proximal to the inhibitor.
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pone-0103638-g004: Disordering of the PRK1 C-tail in the lestaurtinib crystal structure.(a) PRK1:Ro-31-8220 is shown with the protein in blue, with the inhibitor in green. The C-tail is shown as a surface in gray; the region disordered in the lestaurtinib structure is shown in yellow on the Ro-31-8220 structure. (b) PRK1:Lestaurtinib is shown colored orange with the C-tail shown as a surface in grey, with a gap visible due to disorder of the C-tail proximal to the inhibitor.

Mentions: Staurosporine, lestaurtinib and Ro-31-8220 inhibit PRK1 with IC50s of 2 nM, 27 nM and 9 nM, respectively (Table 2). The structures of the two staurosporine analogues in complex with PRK1 are consistent with the known literature binding modes for this ligand class, with two hydrogen bonds between the ligand and the kinase hinge. A further hydrogen bond is made by Ro-31-8220 to Asp708 in the ribose pocket (Figure 3). In the structure with Ro-31-8220, the 2 indole rings are able to rotate to achieve a compact structure able to fit into the PRK1 binding site as it occurs in the apo structure. However, the indole ring in lestaurtinib is constrained to a more planar structure (Figure 1) which cannot be accomodated in the PRK1 binding site without conformational changes. As shown in the structural superposition between lestaurtinib and Ro-31-8220 (Figure 3), lestaurtinib clashes with the C-tail residue Phe910 where Ro-31-8220 is able to rotate the indole rings into a conformation in which there is no steric clash. In the bound conformation, Ro-31-8220 is able to form hydrophobic interactions with Phe910. In contrast, a substantial disordering of the C-tail occurs upon binding of lestaurtinib, with 12–14 residues from the active site tether region of the C-tail no longer visible in the electron density (Figure 4). Despite the fact that lestaurtinib appears to displace a substantial region of the protein from the ATP-binding site, lestaurtinib is only ∼3-fold less potent than Ro-31-8220 (Table 2). To determine the effect of Phe910 displacement on ligand potency, we mutated Phe910 to an alanine. Phe910Ala mutant PRK1 exhibited no significant difference in activity to WT and exhibited a similar Km for ATP (data not shown). As shown in Table 2, when assayed under the same conditions there are no significant differences in IC50 values between the mutant and the WT protein.


Crystal structures of PRK1 in complex with the clinical compounds lestaurtinib and tofacitinib reveal ligand induced conformational changes.

Chamberlain P, Delker S, Pagarigan B, Mahmoudi A, Jackson P, Abbasian M, Muir J, Raheja N, Cathers B - PLoS ONE (2014)

Disordering of the PRK1 C-tail in the lestaurtinib crystal structure.(a) PRK1:Ro-31-8220 is shown with the protein in blue, with the inhibitor in green. The C-tail is shown as a surface in gray; the region disordered in the lestaurtinib structure is shown in yellow on the Ro-31-8220 structure. (b) PRK1:Lestaurtinib is shown colored orange with the C-tail shown as a surface in grey, with a gap visible due to disorder of the C-tail proximal to the inhibitor.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4128815&req=5

pone-0103638-g004: Disordering of the PRK1 C-tail in the lestaurtinib crystal structure.(a) PRK1:Ro-31-8220 is shown with the protein in blue, with the inhibitor in green. The C-tail is shown as a surface in gray; the region disordered in the lestaurtinib structure is shown in yellow on the Ro-31-8220 structure. (b) PRK1:Lestaurtinib is shown colored orange with the C-tail shown as a surface in grey, with a gap visible due to disorder of the C-tail proximal to the inhibitor.
Mentions: Staurosporine, lestaurtinib and Ro-31-8220 inhibit PRK1 with IC50s of 2 nM, 27 nM and 9 nM, respectively (Table 2). The structures of the two staurosporine analogues in complex with PRK1 are consistent with the known literature binding modes for this ligand class, with two hydrogen bonds between the ligand and the kinase hinge. A further hydrogen bond is made by Ro-31-8220 to Asp708 in the ribose pocket (Figure 3). In the structure with Ro-31-8220, the 2 indole rings are able to rotate to achieve a compact structure able to fit into the PRK1 binding site as it occurs in the apo structure. However, the indole ring in lestaurtinib is constrained to a more planar structure (Figure 1) which cannot be accomodated in the PRK1 binding site without conformational changes. As shown in the structural superposition between lestaurtinib and Ro-31-8220 (Figure 3), lestaurtinib clashes with the C-tail residue Phe910 where Ro-31-8220 is able to rotate the indole rings into a conformation in which there is no steric clash. In the bound conformation, Ro-31-8220 is able to form hydrophobic interactions with Phe910. In contrast, a substantial disordering of the C-tail occurs upon binding of lestaurtinib, with 12–14 residues from the active site tether region of the C-tail no longer visible in the electron density (Figure 4). Despite the fact that lestaurtinib appears to displace a substantial region of the protein from the ATP-binding site, lestaurtinib is only ∼3-fold less potent than Ro-31-8220 (Table 2). To determine the effect of Phe910 displacement on ligand potency, we mutated Phe910 to an alanine. Phe910Ala mutant PRK1 exhibited no significant difference in activity to WT and exhibited a similar Km for ATP (data not shown). As shown in Table 2, when assayed under the same conditions there are no significant differences in IC50 values between the mutant and the WT protein.

Bottom Line: The C-tail fully encircles the catalytic domain placing a phenylalanine in the ATP-binding site.Our inhibitor structures include examples of molecules which both interact with, and displace the C-tail from the active site.This information may assist in the design of inhibitors targeting both PRK and other members of the AGC kinase family.

View Article: PubMed Central - PubMed

Affiliation: Celgene Corporation, San Diego, California, United States of America; Department of Biochemistry and Structural Biology, Celgene Corporation, San Diego, California, United States of America.

ABSTRACT
Protein kinase C related kinase 1 (PRK1) is a component of Rho-GTPase, androgen receptor, histone demethylase and histone deacetylase signaling pathways implicated in prostate and ovarian cancer. Herein we describe the crystal structure of PRK1 in apo form, and also in complex with a panel of literature inhibitors including the clinical candidates lestaurtinib and tofacitinib, as well as the staurosporine analog Ro-31-8220. PRK1 is a member of the AGC-kinase class, and as such exhibits the characteristic regulatory sequence at the C-terminus of the catalytic domain--the 'C-tail'. The C-tail fully encircles the catalytic domain placing a phenylalanine in the ATP-binding site. Our inhibitor structures include examples of molecules which both interact with, and displace the C-tail from the active site. This information may assist in the design of inhibitors targeting both PRK and other members of the AGC kinase family.

Show MeSH
Related in: MedlinePlus