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Structure of PKA with Staurosporine

Amongst the potentially therapeutic inhibitors of natural origin are the in-dolocarbazoles, such as staurosporine, and the bisindolylmaleimides. Stau-rosporine, a potent but nonselective protein kinase inhibitor, is a microbial alkaloid from Streptomyces sp. It inhibits PKA with a Ki of about 10 nM, along with many other kinases. Staurosporine is rather unselective against some kinases such as CK1 and CK2, with inhibitory kinetics in the micromolar range, a fact that has been connected to the existence of relatively bulky sidechains in the ATP pocket of these kinases (Meggio et al. 1995). Again, its widespread use in signalling research and as a lead compound for potential therapeutics was triggered by its efficient inhibitory activity

Pka Residues
Fig. 11 Induced fit movements of enzyme residues in the vicinity of staurosporine upon binding of staurosporine to PKA (Prade et al. 1997). Residues rendered as stick models are from the 1STC structure, residues from 1CDK are represented as lines

against PKC. Staurosporine shows similar binding modes with different protein kinases. Cocrystal structures were solved from staurosporine in complex with inactive CDK2 (PDB-code 1AQ1) (Lawrie et al. 1997) and with active PKA (1STC) (Prade et al. 1997; Toledo and Lydon 1997), with CSK (1BYG) (Lamers et al. 1999), Chk1 (1NVR) (Zhao et al. 2002), MapKap kinase 2 (1NXK) (Underwood et al. 2003), Gsk3b (1Q3D) (Bertrand et al. 2003), Lck (1QPj) (Zhu et al. 1999), and with the AGC kinase PDK1 (Koman-der et al. 2003). The molecule binds to PKA with two hinge region H-bonds, one to Val123 amide and the other to the Glu121 carbonyl group. Both hydrogen bonds are conserved adenine contacts, and established in all other staurosporine structures in an identical manner. Also comparable to ATP, the staurosporine methylamino group makes contacts to both the carbonyl of Glu170 (seen also with all H-inhibitors except H-1152P) and to the side-chain of Glu127. The latter contact was also made by fasudil, but not by other H-inhibitors cocrystallized with PKA. In addition to these four hydrogen bonds, staurosporine makes a large number of VDW contacts with the enzyme, possibly because the kinase undergoes induced fit movements upon binding of the inhibitor. In order to accommodate the large staurosporine, most sidechains in the contact area of the inhibitor, the peptide backbone in the region of Val104 and Thr183, and the C-terminal stretch including residue Phe327, move by up to almost 5 A and expand the ATP pocket (Fig. 11). Phe327, however, makes favourable edge-to-plane contacts with one of the staurosporine indoles. None of the other kinases that have been cocrystal-lized with staurosporine, including PDK1, possesses a residue corresponding to Phe327, which is conserved amongst most AGC kinases. Another aromat-

ic residue that undergoes a conformational change to contact staurosporine is Phe54 at the tip of the glycine-rich loop. Phe54 bends inwards towards the inhibitor and makes favourable VDW contacts with the methyl group at the staurosporine sugar ring. A similar contact is observed in Chkl, where a ty-rosine residue in the homologous position interacts with the methyl group of the staurosporine sugar. Although Cdk2, Csk, Gsk3b and Lck have a Tyr or Phe residue at the corresponding position at the tip of the glycine-rich loop, none of them makes a similar contact with this aromat.

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