Sepsis induces lymphocyte apoptosis and prevention of lymphocyte death may improve the chances of surviving this disorder. We compared the efficacy of a selective caspase-3 inhibitor to a polycaspase inhibitor and to caspase-3-/- mice. Both inhibitors prevented lymphocyte apoptosis and improved survival. Caspase-3-/- mice shared a decreased, but not total, block of apoptosis. The polycaspase inhibitor caused a very substantial decrease in bacteremia. Caspase inhibitors did not benefit RAG-1-/- mice, which had a > tenfold increase in bacteremia compared to controls. Adoptive transfer of T cells that overexpressed the anti-apoptotic protein Bcl-2 increased survival. T cells stimulated with anti-CD3 and anti-CD28 produced increased interleukin 2 and interferon gamma by 6 h. Thus, caspase inhibitors enhance immunity by preventing lymphocyte apoptosis and lymphocytes act rapidly, within 24 h, to control infection.

We investigated the role of protein tyrosine phosphatase 1B (PTP1B) in mammary tumorigenesis using both genetic and pharmacological approaches. It has been previously shown that transgenic mice with a deletion mutation in the region of Erbb2 encoding its extracellular domain (referred to as NDL2 mice, for 'Neu deletion in extracellular domain 2') develop mammary tumors that progress to lung metastasis. However, deletion of PTP1B activity in the NDL2 transgenic mice either by breeding with Ptpn1-deficient mice or by treatment with a specific PTP1B inhibitor results in significant mammary tumor latency and resistance to lung metastasis. In contrast, specific overexpression of PTP1B in the mammary gland leads to spontaneous breast cancer development. The regulation of ErbB2-induced mammary tumorigenesis by PTB1B occurs through the attenuation of both the MAP kinase (MAPK) and Akt pathways. This report provides a rationale for the development of PTP1B as a new therapeutic target in breast cancer.

Contrary to the conventional wisdom of the peptide synthesis field, N,S-protected derivatives of cysteine can undergo substantial levels of racemization with widely-used reagents and protocols for stepwise incorporation. A systematic study of this problem has been carried out as a function of coupling conditions and beta-thiol protecting groups, i.e., S-acetamidomethyl (Acm), S-triphenylmethyl (trityl or Trt), S-2,4,6-trimethoxybenzyl (Tmob), and S-9H-xanthen-9-yl (Xan), taking advantage of a convenient and quantitative model system assay involving HPLC resolution of H-Gly-L-Cys-Phe-NH(2) from H-Gly-D-Cys-Phe-NH(2). For example, standard protocols for couplings mediated by phosphonium and aminium salts, e.g., (benzotriazolyloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), N-[[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl]methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), and (7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), typically involve 5-min preactivation times and are conducted in the presence of suitable additives such as 1-hydroxybenzotriazole (HOBt) or 7-aza-1-hydroxybenzotriazole (HOAt) plus a tertiary amine base such as N,N-diisopropylethylamine (DIEA) or N-methylmorpholine (NMM). Under such conditions, the levels of racemization in the model peptide, expressed as the ratio of D:L peptide formed, were in the entirely unacceptable range of 5-33%. However, these levels were in general reduced by a factor of 6- or 7-fold by avoiding the preactivation step. Additional strategies to reduce racemization involved change to a weaker base, with 2,4,6-trimethylpyridine (TMP, collidine) being substantially better than DIEA or NMM; 2-fold reduction in the amount of base; and change in solvent from neat N,N-dimethylformamide (DMF) to the less polar CH(2)Cl(2)-DMF (1:1). Coupling methods for the safe incorporation of cysteine with minimal racemization (<1% per step) in 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis include BOP (or HBTU or HATU)/HOBt (or HOAt)/TMP (4:4:4) without preactivation in CH(2)Cl(2)-DMF (1:1), DIPCDI/HOBt (or HOAt) (4:4) with 5-min preactivation, and preformed pentafluorophenyl (Pfp) esters in CH(2)Cl(2)-DMF (1:1).

Hypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model. Hypoxic-ischemic (H-I)1 encephalopathy in the prenatal and perinatal period is a major cause of morbidity and mortality and often results in cognitive impairment, seizures, and motor impairment leading to cerebral palsy (1, 2). Many studies of neonatal H-I brain injury have utilized the well characterized Levine model in which unilateral carotid ligation is followed by exposure to hypoxia in postnatal day (P) 7 rats (3-5). This model of H-I results in a reproducible pattern of hemispheric injury ipsilateral, but not contralateral, to the carotid ligation (5-7). There are prominent features of both apoptosis and necrosis when this model is performed in neonatal rats and mice (1, 8 -11). Inhibition of caspases utilizing a pan-caspase inhibitor partially protects against brain injury after neonatal H-I injury in this model (12), and similar inhibitors have been shown to partially protect against ischemic injury in adult models (13-16). Previously utilized peptide-based caspase inhibitors (e.g. Boc-D-fmk, z-VAD-fmk, z-DEVD-fmk) required relatively large doses in vivo for their protective effects, and at high concentrations, their effects are more likely to be less selective. Thus, although these studies suggest a role for caspases, the specific caspases and other proteases, which contribute to brain injury after neonatal H-I, have not been clarified.Caspases are a family of cysteine asp...

S,S'-Diethyl dithiomalonate 1 undergoes concomitant alkylation reaction and Michael addition with w-iodo-a,P-unsaturated ketones; the dithiomalonate group present in the cyclization products can be easily reduced by Raney nickel to the ethanol level.$ Iodo enone 10 was prepared from 3-(4-methoxyphenyl)propan-l-o1 according to the following scheme:Reagents and conditions: i, Na, NH3(1);S ii, TsCl, pyridine; iii, 0.1 mol dm-3 HC1; iv, NaI, acetone, reflux 0 The following scheme was used to prepare iodo enone 11. j L &c02..

Caspases are cysteine proteases that specifically cleave Asp-Xxx bonds. They are key agents in inflammation and apoptosis and are attractive targets for therapy against inflammation, neurodegeneration, ischemia, and cancer. Many caspase structures are known, but most involve either peptide or protein inhibitors, unattractive candidates for drug development. We present seven crystal structures of inhibited caspase-3 that illustrate several approaches to reducing the peptidyl characteristics of the inhibitors while maintaining their potency and selectivity. The inhibitors reduce the peptidyl nature of inhibitors while preserving binding potency by (1). exploiting a hydrophobic binding site C-terminal to the cleavage site, (2). replacing the negatively charged aspartyl residue at P4 with neutral groups, and (3). using a peptidomimetic 5,6,7-tricyclic system or a pyrazinone at P2-P3. In addition, we have found that two nicotinic acid aldehydes induce a significant conformational change in the S2 and S3 subsites of caspase-3, revealing an unexpected binding mode. These results advance the search for caspase-directed drugs by revealing how unacceptable molecular features can be removed without loss of potency.

No abstract

The activation of caspase-3 represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Upon induction of apoptosis, the large (p17) and small (p12) subunits, comprising active caspase-3, are generated via proteolytic processing of a latent proenzyme dimer. Two copies of each individual subunit are generated to form an active heterotetramer. The tetrameric form of caspase-3 cleaves specific protein substrates within the cell, thereby producing the apoptotic phenotype. In contrast to the proenzyme, once activated in HeLa cells, caspase-3 is difficult to detect due to its rapid degradation. Interestingly, however, enzyme stability and therefore detection of active caspase-3 by immunoblot analysis can be restored by treatment of cells with a peptide-based caspase-3 selective inhibitor, suggesting that the active form can be stabilized through protein-inhibitor interaction. The heteromeric active enzyme complex is necessary for its stabilization by inhibitors, as expression of the large subunit alone is not stabilized by the presence of inhibitors. Our results show for the first time, that synthetic caspase inhibitors not only block caspase activity, but may also increase the stability of otherwise rapidly degraded mature caspase complexes. Consistent with these findings, experiments with a catalytically inactive mutant of caspase-3 show that rapid turnover is dependent on the activity of the mature enzyme. Furthermore, turnover of otherwise stable active site mutants of capase-3 is rescued by the presence of the active enzyme suggesting that turnover can be mediated in trans.