Caspase3

One of the biggest challenges in fragment-based drug discovery is not finding fragments but linking them. In the case of TS (above), we used structure-based

9.4 Finding and Linking Fragments in One Step: Tethering with Extenders 313

9.4 Finding and Linking Fragments in One Step: Tethering with Extenders 313

Select

Complementary Fragment

Extender

Fig. 9.6 Tethering with extenders. An extender is used to modify a residue in the protein; the extender has some inherent affinity for the protein and also contains a thiol that can be used for Tethering. When a complementary fragment is identified, this can be linked with binding elements from the extender to generate a potent inhibitor. (LG = leaving group).

Select

Complementary Fragment

Link

Extender

Fig. 9.6 Tethering with extenders. An extender is used to modify a residue in the protein; the extender has some inherent affinity for the protein and also contains a thiol that can be used for Tethering. When a complementary fragment is identified, this can be linked with binding elements from the extender to generate a potent inhibitor. (LG = leaving group).

drug design to improve the potency of a fragment identified from Tethering. However, the true combinatorial power of fragment-based approaches only becomes apparent when two fragments are linked together to generate a potent inhibitor. For a general method to link fragments, we invented Tethering with extenders [24].

Tethering with extenders (Fig. 9.6) takes a fragment that binds a protein at a desired site, and modifies the fragment so that it becomes a platform for Tethering. This fragment needs only to have modest affinity and could come from a previous experiment using Tethering or other sources. The fragment is modified to contain an electrophile that reacts with a cysteine on the target protein, plus a potentially masked thiol residue. The resulting modified fragment is called an "extender." After the extender forms a covalent complex with the protein target, the thioester (if present) can be deprotected to reveal the thiol for Tethering. Fragments identified through these screens can be identified through the mass spec-trometry and deconvolution process used for Tethering without extenders.

The two-dimensional connectivity between the extender and any fragments identified from subsequent screens will be known, even if the exact placement of both fragments is not. With this knowledge, binding elements from the extender can be easily connected to newly discovered fragments. In theory, the resulting molecule should have two separate binding elements and bind the target molecule more tightly than either fragment alone.

We tested this strategy on the enzyme caspase-3, a cysteine-aspartyl protease that is one of the central "executioners" of apoptosis. Excess apoptosis is attributed to a variety of diseases, from stroke to Alzheimer's Disease to sepsis, making caspase-3 a popular drug target [25]. The enzyme also made an ideal starting point for constructing extenders. It is well characterized both structurally and mechanistically and contains an active site cysteine residue that is irreversibly al-kylated by small molecule inhibitors.

The first extender we constructed is shown in Fig. 9.7. Mass spectrometry showed we could modify caspase-3 cleanly and quantitatively with this molecule, even though the large subunit of the enzyme contains four other cysteine residues. We could also fully deprotect the thioester to reveal a free thiol. Screens

Fig. 9.7 Tethering with extenders on caspase-3. The extender (3) covalently modifies the protein and can then be deprotected to reveal a thiol for Tethering. One of the strongest hits is the salicylic acid derivative shown.

against a library of about 7000 disulfide-containing fragments yielded one strong hit, a sulfamoyl salicylic acid (Fig. 9.7). By simply replacing the disulfide bond with two methylenes and replacing the irreversible warhead with a reversible aldehyde, we created an inhibitor with K = 2.8 mM. By rigidifying the linker, we boosted the affinity to 200 nM. Further medicinal chemistry allowed us to obtain 20 nM inhibitors (Fig. 9.8) [24-26].

To ensure the technique could be generalized, we constructed a second extender to explore a slightly different area of the protein. We modified caspase-3 with this extender, deprotected the thioester, and screened the conjugate against our fragment library. We did not rediscover the salicylic acid hit from our first extender screen, but we did identify several other hits, including a thiophene sulfone. When this fragment was linked to the extender, the resulting inhibitor

9.4 Finding and Linking Fragments in One Step: Tethering with Extenders 315

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