X-Ray Structures and Binding Mode of Balanol Derivatives

Novel azepane derivatives have been synthesized and the X-ray structures of the PKA-PKI-complexes solved (Breitenlechner et al. 2004). The chemical structures involve variations of different parts of the natural balanol: the phenol has been replaced by a pyridine ring, the substituents on the ben-zophenone have been modified and the ester linker between ring B and C has been extensively modified in order to optimize pharmacokinetic properties.

Three binding regions were defined in reference to the balanol-type interactions: the pyridine pocket, the azepane pocket and the benzophenone pocket (Fig. 8). These regions correspond closely to the adenine, ribose and phosphate binding sites (Breitenlechner et al. 2004).

The pyridine pocket contains the pyridine moiety and the amide connecting it to the azepane ring. This space is occupied by the adenine in the complex of AMP-PNP and PKA (Bossemeyer et al. 1993) (Fig. 8) and accepts in general aromatic groups. The glycine loop (Leu49, Val57), b-sheet 3 (Ala70), the hinge region (Met120, Tyr122, Val123, Glu127), the catalytic loop (Leu173), the beginning of the activation loop (Thr183), and Phe327 from the C-terminal stretch are involved in VDW contacts to the pyridine and amide portion of compound 1 (Fig. 9).

The hinge region residue Val123 makes an H-bond to the pyridine via its main chain amide. This H-bond to Val123 (or its homologue in other kinas-es) is nearly universal amongst protein kinase inhibitor complexes and is

Fig. 8 Binding of a balanol derivative to the ATP binding pocket of PKA (light grey) superimposed with the AMP-PNP structure (dark grey); [1CDK (Bossemeyer et al. 1993)]
Fig. 9 Binding of a balanol derivative to the ATP binding pocket of PKA in structural detail

apparently critical for tight-binding inhibitors. Two H-bonds exist from the amide group to two water molecules that bridge polar contacts between the amide NH and Glu127 and Leu49 and between the amides CO to Lys72 and Asp184.

The azepane pocket—stretching over the ribose subsite—contains the azepane and the ester part of the molecule. This pocket is bounded by residues from the glycine loop (Gly50, Thr51, Gly52, Val57), b-sheet 3 (Lys72), the catalytic loop (Glu170, Asn171) and Asp184 from the beginning of the activation loop. The protonated azepane nitrogen forms H-bonds to Asn171 and Asp184.

The benzophenone pocket is bounded on one side by the flexible glycine loop, which is displaced compared to the AMP-PNP PKA complex (see Fig. 8). Consequently, VDW contacts exist to glycine loop residues (Ser53, Phe54, Gly55, Arg56), and also to residue Leu74 from the b-sheet 3, to several residues from helix C (Gln84, His87, Thr88, Glu91), as well as to Gly186 and Phe187 from the activation loop.

The carbonyl oxygen of the benzophenone is involved in hydrogen bonds to the backbone amide nitrogen Phe54 at the tip of the glycine loop.

Glu91, which is essential for catalysis (Schneider 2002), forms a salt bridge to Lys72 in the active state as well as in the inhibitor complex with compound 1. In this complex, Glu91 can also form a hydrogen bond to the inhibitor hydroxyl group of the benzophenone (Breitenlechner et al. 2004).

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