H1152p

Further derivatization of fasudil led to H-1152P, with two additional methyl groups, one at the isoquinoline ring and the other at the homopiperazine ring (Tanaka et al. 1998). H-1152P inhibits Rho kinase in the low nanomolar range, with a 400-fold selectivity over PKA (Sasaki et al. 2002). Fasudil and H-1152P bind both with similar affinity to PKA in the same orientation in the adenosine pocket (Fig. 4). H-1152P, however, has only one H-bond contact to the enzyme, the one between the hinge region amide group to the iso-quinoline ring nitrogen. No H-bonds are formed with the nitrogen atom of the homopiperazine ring. The reason for this difference in binding of H-1152P and fasudil to PKA becomes obvious from an overlay of both inhibitor structures. In order to form the 'essential' H-bond to the hinge region, H-1152P can only bind, when the distal (as seen from the hinge) ho-mopiperazine ring is shifted toward the glycine-rich loop in order to avoid a steric clash between the extra methyl group at the homopiperazine ring, and the sidechain of Thr183.

Thr183 is one of the four residues which are in sidechain contact to ATP-side inhibitors and differ between PKA and Rho kinase. An alanine residue in this position, as in Rho kinase, would provide enough space for a closer

Fig. 4 Comparison of HA-1077 and H-1152P. Superimposed are the inhibitor binding sites of fasudil and H-1152P in PKA. A double arrow indicates a possible steric conflict between the selectivity defining methyl group C10 of H-1152P and the sidechain of Thr183, a residue which is alanine in Rho kinase. (Breitenlechner et al. 2003)

Fig. 4 Comparison of HA-1077 and H-1152P. Superimposed are the inhibitor binding sites of fasudil and H-1152P in PKA. A double arrow indicates a possible steric conflict between the selectivity defining methyl group C10 of H-1152P and the sidechain of Thr183, a residue which is alanine in Rho kinase. (Breitenlechner et al. 2003)

approach of the H-1152P homopiperazine ring towards the H-bond-forming groups of Glu170, and Gly127 (Fig. 4). Also, the exchange of the Leu49 residue to isoleucine could be an advantage for H-1152P, by increasing the number of VDW contacts and the hydrophobic interactions. The extra methyl group at the isoquinoline ring could benefit from the branching of the isoleucine sidechain at the Cb atom. The two other differences, Val123Met and Glu127Asp, are difficult to interpret in terms of selectivity for Rho kinase. The valine residue makes the largest number of contacts with fasudil, and the second largest number of contacts with H-1152P. As seen in protein ki-nase crystal structures with a methionine residue in the homologous position, the sidechain points away from the ATP binding pocket such that only Cb or Cg atoms can interact with an ATP-site ligand. The methionine therefore does not likely contribute to Rho kinase selectivity for HA-1077 or H-1152P, since its effect, if any, would be to remove interactions and lower the binding affinity (S. Bonn, S. Herrero, M. Gafiel, C.B. Breitenlechner, R. Engh, D. Bossemeyer, in preparation). An exchange of Glu127 to Asp is also expected to change little, because the shorter sidechain can still make the H-bond contacts to the homopiperazine nitrogen atoms. Overall, the contacts from the residues which differ between PKA and Rho kinase in the ATP binding site contribute most significantly to the interactions between the inhibitors and PKA.

Because sidechain interactions form the major kinase selectivity determinant of active conformations to ATP-site inhibitors, the conservation of Thr183, Val123, Leu49 and Glu127 should be predictive of H-1152P selectivity. None of these four residues is rare or exceptional (Table 2). However, this combination occurs in only six other kinases, none of which belong to the AGC group of protein kinases. Consideration of a fifth residue renders Rho kinases unique. AGC kinases, including PKA and Rho kinase, possess a C-terminal strand that inserts a phenylalanine sidechain in to the ATP binding site. The phenylalanine interacts in known AGC kinase structures with the purine base of ATP and most ATP-site ligands, and also with fasudil and H-1152P. The ATP-site ligand contacts to Phe327 are typically edge-edge aromat aromat contacts that affix the ligand to the adenine site. Thus, the

Table 2 Differences in Rho kinase and PKA and occurrence (according to Hanks and Quinn 1991; Manning et al. 2002)
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