Ju

Extender with k O

COpH

Hit without disulfide 7 No detectable binding n-vy^ lnitial diaPh°re 4

n-vy^ lnitial diaPh°re 4

O H Rigidified molecule 5

O COoH

Rigidified, functionalized NÏhTT molecules ^OH Kj = 20 nM

fcOoH

cooh

H IX

,co2h

"oh

Diaphore with P2 element 8 K; = 100 nM

Fig. 9.8 Evolution of a fragment from Tethering with extenders to a potent caspase-3 inhibitor. Simple replacement of the disulfide linker with an alkyl linker resulted in a low micromolar inhibitor (4), and rigidification (5) and functionalization (6) of this linker led to increasingly potent inhibitors. The salicylic acid hit itself (7) had no detectable binding.

had Ki = 330 nM [24]. These examples illustrate the speed with which Tethering with extenders can lead to potent inhibitors. Moreover, these inhibitors are non-peptidic, and so more useful as drug leads.

We used crystallography to understand the binding mode of these fragments. The structure of the salicylic acid fragment bound through the disulfide is shown in Fig. 9.9a, superimposed upon the structure of a tetrapeptide-based inhibitor. Significantly, the two inhibitors occupy roughly the same volume, and make many of the same contacts, but do so using very different chemical moieties. Moreover, the S2 pocket in the salicylic acid structure is collapsed, while the S4 pocket expands to make room for the larger salicylic acid moiety. By introducing a substituent that bound in the S2 pocket, we boosted affinity by nearly two orders of magnitude (Fig. 9.8) [27].

All of these features contrast with the structure of the second extender-fragment complex, shown in Fig. 9.9b. Here, the extender forces itself into the S2 pocket, but the disulfide linker then curves back to place the thiophene sul-fone into the S4 pocket. The sulfone makes some of the same hydrogen bonds as the salicylic acid and the aspartyl residue in the tetrapeptide but with completely different chemistry. The flexibility of caspase-3 to accommodate different

Fig. 9.9 (a) Structure of the salicylic acid fragment covalently bound to caspase-3 (gray), superimposed on a tetrapeptide-based inhibitor (green). Note the collapse of the S2 pocket and the widening of the S4 pocket to accommodate the salicylic acid moiety. (b) Structure of a second fragment covalently bound to caspase-3 (blue) superimposed on the salicylic acid fragment. Here the S2 pocket is intact, and the linker takes an alternative path to arrive in the S4 pocket. Reprinted from [25] with permission. The molecular graphics in this and all other figures were done using the program PyMol (see DeLano, W. L. (2004) PyMOL, available at: http://pymol.sourceforge.net/).

Fig. 9.9 (a) Structure of the salicylic acid fragment covalently bound to caspase-3 (gray), superimposed on a tetrapeptide-based inhibitor (green). Note the collapse of the S2 pocket and the widening of the S4 pocket to accommodate the salicylic acid moiety. (b) Structure of a second fragment covalently bound to caspase-3 (blue) superimposed on the salicylic acid fragment. Here the S2 pocket is intact, and the linker takes an alternative path to arrive in the S4 pocket. Reprinted from [25] with permission. The molecular graphics in this and all other figures were done using the program PyMol (see DeLano, W. L. (2004) PyMOL, available at: http://pymol.sourceforge.net/).

inhibitors emphasizes the ability of Tethering to identify fragments that would not have been easy to predict using structure-based design.

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