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The hydrophobicity maps of thrombin and p38 mitogen-activated protein (MAP) kinase are presented here. The approach has been previously validated on 10 protein-ligand complexes [6]. In five of these complexes the ligand was a natural peptide or protein, while in the other five it was an organic compound with hydrophobic moieties. In all complexes tested up to date, the hydrophobicity maps correctly predict the regions of the receptor which are occupied by the nonpolar groups of the ligand.

Thrombin

Thrombin is a trypsin-like serine protease which fulfills an essential role in both haemostasis and thrombosis [39]. In the blood coagulation cascade, thrombin is the final enzyme that cleaves fibrinogen to release fibrinopeptides A and B and generate fibrin, which can then polymerize to form a haemostatic plug. The S3 and S2 precleavage subpockets of the active site have a hydro-phobic character, whereas at the bottom of the S1 or recognition pocket the carboxyl group of Asp 189 is a salt bridge partner for basic side chains. Na-((2-naphthylsulfinyl)glycyl)-dl-p-amidinophenylalanylpiperidine (NAPAP) is an archetypal active site inhibitor ofthrombin (Figure la). It fills the S3 and S2 pockets with its naphthalene and piperidine groups, respectively. Moreover, it is anchored by its basic group (benzamidine) into S1 to form a salt bridge with Asp 189 [40].

The hydrophobicity map of the nonprime region of the thrombin active site is shown in Figure la. The side chains of Asp189 and NAPAP are also shown. S3 and part of S2 are identified as hydrophobic, while S1 shows a hydrophilic character. The naphthalene and piperidine groups of NAPAP are in contact with hydrophobic zones and bury 13 of the 100 most hydrophobic points (over a total of about 55 000) on the SAS of thrombin. The polar groups of NAPAP bind to the hydrophilic zones of the thrombin active site: the Asp 189 side chain and the Gly216 backbone polar groups. The energy loss of removing water from the hydrophilic zones is compensated upon binding by favorable electrostatic ligand-receptor interactions. The lower part of S1, despite being a narrow concave cavity, is identified as hydrophilic, since electrostatic desolvation of Asp189 dominates over the favorable vdW interactions between the probe sphere and the surrounding thrombin atoms. The curvature mapped on the MS (not shown) does not take into account thrombin electrostatic desolvation and suggests that the bottom of the S1 pocket is the most hydrophobic region, in contrast with the actual binding mode of NAPAP.

TyreoA S7

# in ^ y

116174 A

k

Gly216

SI

Thr106

-"Lys53

Glu71

Met109

Asp168

Figure 1. Hydrophobicity maps calculated with Equations 1, 2 and 8, and displayed with the program GRASP [23]. The molecular surface is displayed with colors ranging from green (hy-drophobic) through white (intermediate) to blue (hydrophilic) according to the hydrophobicity map value. (a) Thrombin-NAPAP complex [40]. The transparent MS of the thrombin active site is displayed together with the side chain of Asp189 and NAPAP in a cylinder model (carbon atoms are white, nitrogen blue, oxygen red, and sulfur yellow). (b) Complex of the p38 MAP kinase and the triarylimidazole SB203580 (PDB code 1A9U) [48]. The 70 most hydrophobic points on the ATP binding site surface are displayed in magenta. Yellow crosses mark the five hydrophobic regions discussed in the text.

p38 MAP kinase

MAP kinases are essential enzymes for intracellular signalling cascades because they phosphorylate several regulatory proteins. They are responsive to hormones, cytokines, environmental stresses and other extracellular stimuli, and are activated by a dual phosphorylation of a threonine and tyrosine in the TXY motif in the so-called phosphorylation lip. p38 MAP kinase (or CSBP2) plays a role in processes as diverse as transcriptional regulation, production ofinterleukins, andapoptosis ofneuronal cells [41-44]. Inhibitors of p38 activity could therefore be useful as a treatment strategy for inflammatory and neurodegenerative diseases. The CSAIDTM (cytokine suppressive anti-inflammatory drugs) class of anti-inflammatory compounds inhibits the synthesis of cytokines, such as interleukin-1 and tumor necrosis factor, by specific inhibition of the MAP kinase p38 [41,45,46]. They have a common chemical pattern: A central five-membered ring, either imidazole or pyrrole, substituted by a pyridine or a pyrimidine ring, a fluorinated or iodinated phenyl ring, and a third substituent at position 1 or 2 (Figures lb and 6). These low-molecular weight inhibitors and their analogs bind to the ATP-binding cleft of the inactivated form of p38 and are competitive with respect to ATP. They are potent inhibitors, with IC50 in the nanomolar range [45,47], and highly selective for p38 compared to the other MAP kinases.

In Figure lb the most hydrophobic points in the ATP binding site are displayed together with the triarylimidazole inhibitor SB203580 [48]. Five hydrophobic regions of concave shape are found by the computational approach described above. They are colored in green in Figure lb and their approximate center is marked by a yellow cross. Three regions are consistent with the available structural data of p38 MAP kinase/inhibitor complexes [48,49], whereas two regions are novel. The most hydrophobic pocket is located between the Thr106 and Lys53 side chains, and is occupied by the phenyl group of the diaryl- and triarylimidazole inhibitors. The hydrophobic pocket lined by the Thr106 and Met109 side chains is occupied by the pyridine or pyrimidine cycle. In the diarylimidazole inhibitors, the N-substituent of the central imidazole is in contact with the hydrophobic region close to the Val30 and Val38 side chains [48]. Surprisingly, the hydrophobic pockets below Glu71 and Met109 are empty in the available crystal structures of the MAP kinase p38/inhibitor complexes [48,49]. For the inhibitors known to bind at the ATP site, it is expected that additional nonpolar substituents directed towards the two unoccupied hydrophobic pockets will improve the binding affinity.

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