Caspase-8 Variant G Regulates Rheumatoid Arthritis Fibroblast-Like Synoviocyte Aggressive Behavior.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase-8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs. METHODS: RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5-8. Caspase-8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase-8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase-8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase-8B and G were determined. RESULTS: Caspase-8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase-8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase-8) and induced by PDGF. The crystal structures of caspase-8 variant G and B were identical except for a unique unstructured 59 amino acid N-terminal domain in variant G. Selective knockdown of caspase-8G was solely responsible for the effects of caspase-8 on calpain activity and cell invasion in FLS. CONCLUSION: Blocking caspase-8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase-8 inhibition.

First-in-class pan caspase inhibitor developed for the treatment of liver disease.

A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of alpha-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.

Intrapleural low-molecular-weight urokinase or tissue plasminogen activator versus single-chain urokinase in tetracycline-induced pleural loculation in rabbits.

The authors compared the ability of a single dose of the proenzyme single-chain urokinase (scuPA), low-molecular-weight urokinase, tissue plasminogen activator (tPA), or a mutant site-inactive scuPA to resolve intrapleural loculations at 72 to 96 hours after tetracycline-induced pleural injury in rabbits. Both scuPA and tPA reversed loculations at 96 hours after injury P < or = .001, whereas low-molecular-weight urokinase and the scuPA mutant were ineffective. scuPA and tPA generated inhibitor complexes, induced fibrinolytic activity, and quenched plasminogen activator-1 activity in pleural fluids. The authors conclude that scuPA reverses loculations as effectively as tPA at clinically applied intrapleural doses, whereas low-molecular-weight urokinase was ineffective.

Structure-activity relationships within a series of caspase inhibitors. Part 2: Heterocyclic warheads.

Various heterocyclic hetero-methyl ketones of the 1-naphthyloxyacetyl-Val-Asp backbone have been prepared. A study of their structure-activity relationship (SAR) related to caspase-1, -3, -6, and -8 is reported. Their efficacy in a cellular model of cell death is also discussed. Potent broad-spectrum caspase inhibitors have been identified.

Structural and functional analysis of caspase active sites.

Amino acid sequences of caspases 1, 3, 7, and 8 were aligned with their published three-dimensional (3D) structures. The resultant alignment was used as a template to compare the primary structures of caspases 2, 4-6, and 9-11 to build 3D homology models. The structural models were subsequently refined and validated using structure-activity relationship data obtained from an array of substrate-like inhibitors. All caspases were shown to have identical S1 and catalytic dyad architecture but diverse S2-S4 structures. S2 pockets of these 11 caspases can be briefly categorized into two groups: Csp3, -6, and -7 as one and Csp1, -2, -4, -5, -8, -9, -10, and -11 as the other. S2 pockets of Csp3, -6, and -7 are smaller than those of the other eight caspases, and are limited to binding small P2 residues such as Ala and Val. At the S3 site, the presence of a conserved Arg in all caspases suggests that Glu would be a universally preferred P3 residue. Csp8 and Csp9 have an additional Arg in this pocket that can further enhance the binding of a P3 Glu, whereas Csp2 has a Glu adjacent to the conserved Arg. As such, Csp2 is the only caspase that can accommodate both positively and negatively charged P3. At S4, Csp1, -4, -5, and -11 are closely related with respect to their structures and binder preferences; all have a large hydrophobic pocket and prefer large hydrophobic residues such as Trp. S4 of Csp2, -3, and -7 represents an opposite group with a conformation that is highly specific in binding an Asp. The S4 structures of Csp6, -8, -9, and -10 appear to be hybrids of the two extremes, and have little specificity for any P4. Information revealed from this work provides a guide for designing potent caspase inhibitors with desirable specificity.

Acyl dipeptides as reversible caspase inhibitors. Part 1: initial lead optimization.

Parallel synthesis was used to explore the SAR of a peptidomimetic caspase inhibitor. The most potent compound had nanomolar activity against caspases 1, 3, 6, 7, and 8.

Acyl dipeptides as reversible caspase inhibitors. Part 2: further optimization.

A new structural class of broad spectrum caspase inhibitors was optimized for its activity against caspases 1, 3, 6, 7, and 8. The most potent compound had low nanomolar broad spectrum activity, in particular, single digit nanomolar inhibitory activity against caspase 8.