P Pocket

The hydroxyl group of Serinel95 acts as a nucleophile, targeting the scis-sile amide bond of the substrate. There are three principal binding pockets at the cleaving site the specificity (S) pocket, the proximal (P) pocket and the distal (D) pocket modelled from the crystal structure [12,17,18]. 

It is known from X-ray crystallographic studies that Tyr-Pro-Trp loop, unique to thrombin, helps to form two hydrophobic moieties in which two hydrophobic moieties of the inhibitors are bound. The hydrophobic P-pocket, proximal to the catalytic center, corresponds to the S2 subsite and binds the inhibitor through the basic group (piperidide, piperizide, etc.) The P-pocket distal to the catalytic center is also hydrophobic and binds the aryl moieties of the inhibitors. The basic amino acid chain is placed in the P1 position with its carbonyl moiety binding in the oxyanion hole. 

Figure 3.9. x-Variable distribution for the 10 grid probes in the P pocket after BUW. Blue dots indicate energies in thrombin, red dots in trypsin, and green dots in factor Xa. 

Another type III peptidomimetic inhibitor was derived from the crystal structure of a bicyclic [3.1.3] inhibitor (170) complexed to thrombin (97) (Fig. 15.41). The X-ray structure revealed that one of the carbonyls was oriented towards the hydrophobic P-pocket (S2). The desolvation necessary to place a carbonyl in a hydrophobic pocket is unfavorable and various alkyl groups were used as possible replacements. This led to the potent (iCj = 13 nM) and selective (>760 for thrombin over tiypsin) inhibitor (98). 

Hoffmann-Roder, A., Schweizer, E., Egger, J., et al. (2006) Mapping the fluorophilicity of a hydrophobic pocket synthesis and biological evaluation of tricyclic thrombin inhibitors directing fluorinated alkyl groups into the P pocket. ChemMedChem, 1(11), 1205-1215. 

Figure 3. Cartoon of the binding mode of fibrinogen 2 in thrombin with the arginine side chain in the recognition pocket, the valine side chain in the P-pocket and the leucine and phenylalanine side chains in the D-pocket.

There is a small volume deep in the P pocket that must be occupied by a hydrophobic group. 

The reference inhibitors 8a have carbonyl oxygens at both R1/R2 and R3/R4 and bind with the lower carbonyl oxygen accepting a hydrogen bond from the -NH of Gly216 (as modeled) and the upper carbonyl oxygen in the P pocket... 

Another inhibitor series has been used to estimate the value of P pocket interactions - the Boehringer Mannheim di aryl sulfonamides [39] (also reported by 3-Di-mensional Pharmaceuticals [40-43]) (Fig. 7.11). 

The diaryl sulfonamide inhibitors were discovered by a screening exercise aimed at finding less basic thrombin inhibitors [44]. A crystal structure of the complex of thrombin with the R=CH3 compound 9b (BM14.1248) shows the phenyl group in the D pocket, the central tolyl group in the P pocket, and the 4-aminopyridine in the SI pocket (but not interacting directly with Aspl89) [45] [Iwt],... 

In a similar way, using 4-TAPAP as template [35], a methyl group deep in the P pocket was shown to produce an affinity gain of 17x with no change to the position of inhibitor binding [46]. 

We conclude from the above examples that up to 2 kcal mol of binding energy may be obtained by placing a methyl or similar small hydrophobic group correctly in the P pocket Something similar must be true of the D pocket, although examples with X-ray validation are missing. 

We further observe that it is possible to obtain low nanomolar inhibition without making the canonical benzamidine hydrogen bonds (Fig. 7.9). For a detailed discussion of non-canonical needles and recognition pocket and P pocket flexibility, the reader should consult ]41, 42, 45]. 

Heuristic models based on the binding of peptidomimetic inhibitors pointed to hydrophobic interactions in the D and P pockets and optimal hydrogen bonding to Gly216 and Aspl89 as being vital for good inhibition. 

P pocket. In contrast to the results for the S1 pocket, several probes have particularly high interaction energies in one of... 

Plate 12 Pseudofield plot showing MIF differences between thrombin (blue) and trypsin (red) for the GRID DRY probe within the P pocket. Energetically unfavourable fields are shown in a yellow contour. Reference inhibitor is NAPAP. °... 

The boundaries of the model represent the space available for the accommodation of the substrate. The important binding regions which determine the selectivity of the reaction are two hydrophobic pockets (Hl and Hs, with L = large and S = small) and two pockets of more polar character (Pp and Pb, with F = front and B = back). The best fit of a substrate is determined by positioning the ester group to be hydrolyzed close to the hydrolytically active serine residue and then arranging the remaining moieties in the H and P pockets. 

Figure 19.18 shows (a) the structure of the protein of EEAI in the FYVE domain, (b) the phosphate group in Ptdlns(3)P recognition by arginines, and (c) interactions in the Ptdlns(3)P pocket. 

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