The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure-activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.

The cysteine protease CPP32 has been expressed in a soluble form in Escherichia coli and purified to >95% purity. The three-dimensional structure of human CPP32 in complex with the irreversible tetrapeptide inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone was determined by x-ray crystallography at a resolution of 2.3 Å. The asymmetric unit contains a (p17/p12) 2 tetramer, in agreement with the tetrameric structure of the protein in solution as determined by dynamic light scattering and size exclusion chromatography. The overall topology of CPP32 is very similar to that of interleukin-1␤-converting enzyme (ICE); however, differences exist at the N terminus of the p17 subunit, where the first helix found in ICE is missing in CPP32. A deletion/insertion pattern is responsible for the striking differences observed in the loops around the active site. In addition, the P1 carbonyl of the ketone inhibitor is pointing into the oxyanion hole and forms a hydrogen bond with the peptidic nitrogen of Gly-122, resulting in a different state compared with the tetrahedral intermediate observed in the structure of ICE and CPP32 in complex with an aldehyde inhibitor. The topology of the interface formed by the two p17/p12 heterodimers of CPP32 is different from that of ICE. This results in different orientations of CPP32 heterodimers compared with ICE heterodimers, which could affect substrate recognition. This structural information will be invaluable for the design of small synthetic inhibitors of CPP32 as well as for the design of CPP32 mutants.Genetic and biochemical studies have established the importance of the CED3/ICE 1 proteases in programmed cell death or apoptosis (1-4). Of the known mammalian CED3/ICE proteases, CPP32 is the most similar in sequence homology and substrate specificity to the CED3 protease (2, 5), whose function is required for programmed cell death in the developing Caenorhabditis elegans hermaphrodite (6). CED3/ICE proteases are synthesized as single chain proenzymes that are cleaved proteolytically to produce catalytically active cysteine proteases with specificity for cleavage at Asp-X peptide bonds (7-9). In several apoptotic model systems, CPP32 is rapidly processed from its p32 proenzyme form to a p17/p12 catalytically active form (10 -12). Cleavage and activation of CPP32 are thought to be mediated by CED3/ICE proteases (12, 13), possibly including active CPP32 itself, MCH2, and/or MCH4 (14, 15). Several proteins that regulate cellular homeostasis and function, including poly(ADP-ribose) polymerase, the 70-kDa subunit of the U1 small ribonucleoprotein, the catalytic subunit of DNA-dependent protein kinase, and the Huntington's disease gene product (huntingtin), are thought to be target substrates for activated CPP32 (8, 16 -18). Thus, activation of CED3/ICE proteases, including CPP32, within pre-apoptotic cells is thought to lead to the proteolytic inactivation of proteins that are necessary for cell viability. In support of this hypothesis, CED3/ICE protease inhibitors have been shown to bl...

The human proto-oncogene bcl-2 and its Caenorhabditis elegans homologue ced-9 inhibit programmed cell death. In contrast, members of the human interleukin-1beta converting enzyme (ICE) family of cysteine proteases and their C. elegans homologue CED-3 promote the death program. Genetic experiments in C. elegans have shown that ced-9 is formally a negative regulator of ced-3 function, but neither those studies nor others have determined whether CED-9 or Bcl-2 proteins act biochemically upstream or downstream of CED-3/ICE proteases. CPP32, like all known members of the CED-3/ICE family, is synthesized as a proenzyme that is subsequently processed into an active protease with specificity for cleavage at Asp-X peptide bonds. In this report, we demonstrate that the CPP32 proenzyme is proteolytically processed and activated in Jurkat cells induced to die by Fas ligation. CPP32 activation is blocked by cell-permeable inhibitors of aspartate-directed, cysteine proteases, suggesting that pro-CPP32 is cleaved by active CPP32 or by other ICE family members. Heterologous expression of Bcl-2 in Jurkat cells prevents Fas-induced cell death as well as proteolytic processing and activation of CPP32. Thus, Bcl-2 acts at or upstream of the CPP32 activation step to inhibit apoptosis induced by Fas stimulation.

Neuronal apoptosis occurs during nervous system development and after pathological insults to the adult nervous system. Inhibition of CED3/ICE-related proteases has been shown to inhibit neuronal apoptosis in vitro and in vivo, indicating a role for these cysteine proteases in neuronal apoptosis. We have studied the activation of the CED3/ICE-related protease CPP32 in two in vitro models of mouse cerebellar granule neuronal cell death: K+/serum deprivation-induced apoptosis and glutamate-induced necrosis. Pretreatment of granule neurons with a selective, irreversible inhibitor of CED3/ICE family proteases, ZVAD-fluoromethylketone, specifically inhibited granule neuron apoptosis but not necrosis, indicating a selective role for CED3/ICE proteases in granule neuron apoptosis. Extracts prepared from apoptotic, but not necrotic, granule neurons contained a protease activity that cleaved the CPP32 substrate Ac-DEVD-aminomethylcoumarin. Induction of the protease activity was prevented by inhibitors of RNA or protein synthesis or by the CED3/ICE protease inhibitor. Affinity labeling of the protease activity with an irreversible CED3/ICE protease inhibitor, ZVK(biotin)D-fluoromethylketone, identified two putative protease subunits, p20 and p18, that were present in apoptotic but not necrotic granule neuron extracts. Western blotting with antibodies to the C terminus of the large subunit of mouse CPP32 (anti-CPP32) identified p20 and p18 as processed subunits of the CPP32 proenzyme. Anti-CPP32 specifically inhibited the DEVD-amc cleaving activity, verifying the presence of active CPP32 protease in the apoptotic granule neuron extracts. Western blotting demonstrated that the CPP32 proenzyme was expressed in granule neurons before induction of apoptosis. These results demonstrate that the CED3/ICE homolog CPP32 is processed and activated during cerebellar granule neuron apoptosis. CPP32 activation requires macromolecular synthesis and CED3/ICE protease activity. The lack of CPP32 activation during granule neuron necrosis suggests that proteolytic processing and activation of CED3/ICE proteases are specific biochemical markers of apoptosis.

ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ∼50–100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ∼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a ∼10–100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.