Caspases

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 

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 alkylated 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 

4 Finding and Linking Fragments in One Step Tethering with Extenders 315 

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 

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BH3 domain) of the BH3-only proteins binds to other Bcl-2 family members thereby influencing their conformation. This interaction facilitates the release of cytochrome C and other mitochondrial proteins from the intermembrane space of mitochondria. Despite much effort the exact biochemical mechanism which governs this release is not yet fully understood. The release of cytochrome C facilitates the formation of the apoptosome, the second platform for apoptosis initiation besides the DISC. At the apoptosome which is also a multi-protein complex the initiator caspase-9 is activated. At this point the two pathways converge. 

Active caspases 8, 9 and 10 can convert caspase-3, the most abundant effector caspase from its pro-form to its active cleaved form. Cleavage of a number of different substrates by caspase-3 and also by caspase-6 and -7 which are two other executioner caspases besides caspase-3 then results in the typical morphology which is characteristic of apoptosis. Yet, the activation of caspase-3 and also of caspase-9 can be counteracted by IAPs, so called inhibitor of apoptosis proteins. However, concomitantly with cytochrome C also other proteins are released from mitochondria, including Smac/DIABLO. Smac/DIABLO and potentially other factors can interact with IAPs and thereby neutralize their caspase-inhibitory activity. This releases the breaks on the cell death program and allows apoptosis to ensue. 

There is also crosstalk between the two pathways above the mitochondria. The BH3-only protein BID is cleaved by caspase-8 and -10 which yields truncated BID (tBED), the active pro-apoptotic fragment of BID. Thereby, even in cells in which the direct apoptosis pathway which result from death receptor crosslinking is blocked, e.g. by high expression levels ofthex-linked IAP (XIAP), the activity of tBED on mitochondria can result in the activation of caspase-3 because the IAP-imposed block on full caspase-3 activation and caspase-9 activity at the apoptosome is released by Smac/ DIABLO. 

Apoptotic executioner caspases (caspase-3, -6, -7) constitute a subgroup of the caspase family. These proteases are the workhorses of the apoptotic process as they are responsible for cleaving many down-stream substrates important for cellular morphology, organelle homeostasis, cell cycle arrest, and regulation of transcription and translation. 

Apoptotic initiator caspases (caspase-2, -8, -9 and -10) constitute a subgroup of the caspase family. These caspases are the first to become proteolytically active in the apoptotic cascade. Their activation takes place in multiprotein complexes initiated by pro-apoptotic stimuli, such as TNFa, a-Fas, staurosporine. Once activated, they can process their substrates, which include the apoptotic executioner caspases. 

Apoptosis occurs as a result of a cascade ofproteolysis that culminates in the destruction of the cell. Apoptotic proteolysis is catalyzed by the caspase family of... 

So far ten catalytically active caspases have been reported in mouse (caspase-1, -2, -3, -6, -7, -8, -9, -11, -12,-14) and eleven in human (caspase-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -14) (Fig. 1). Caspases are expressed as inactive proenzymes that contain an amino-terminal prodomain of variable length followed by two domains with conserved sequences a large subunit ( 20 kDa, p20) and a small carboxy-terminal subunit ( 10 kDa, plO). Caspases can be divided according to absence (-3, -6, -7, -14) or presence (-1, -2, -8, -9, -10, -11, -12) of an extended prodomain containing protein-protein interaction motifs belonging to the death domain (DD) superfamily, in particular the death effector domains (DED) and the caspase activation and recruitment domains (CARD). 

Caspases. Figure 1 Schematic representation of caspase domain architecture. Illustration of all identified Homo sapiens and Mus musculus caspases. The prodomain, and the large (p20) and small (p10) subunits are proportionally depicted. 

Caspases. Figure 2 Caspase activating complexes. Schematic representation of all described long prodomain caspase activation complexes. Each complex contains essentially three functionally different building blocks a sensor/platform, an adaptor and an effector in the form of a particular caspase. Some instigating ligands, possible outcomes and regulatory proteins are indicated. 

Too strong, too weak, or aberrant activation of one or more caspases is a hallmark of many human ailments. In Table 1 we provide a list of experimental diseases in... 

Caspases. Table 1 Diseases associated with inappropriate caspase activation. Contribution of apoptosis (A) and inflammation (I) are indicated... 

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