Kaushik Datta

Discovery and characterization of a novel inhibitor of matrix metalloprotease-13 that reduces cartilage damage in vivo without joint fibroplasia side effects.

Matrix metalloproteinase-13 (MMP13) is a Zn2+-dependent protease that catalyzes the cleavage of type II collagen, the main structural protein in articular cartilage. Excess MMP13 activity causes cartilage degradation in osteoarthritis, making this protease an attractive therapeutic target. However, clinically tested MMP inhibitors have been associated with a painful, joint-stiffening musculoskeletal side effect that may be due to their lack of selectivity. In our efforts to develop a disease-modifying osteoarthritis drug, we have discovered MMP13 inhibitors that differ greatly from previous MMP inhibitors; they do not bind to the catalytic zinc ion, they are noncompetitive with respect to substrate binding, and they show extreme selectivity for inhibiting MMP13. By structure-based drug design, we generated an orally active MMP13 inhibitor that effectively reduces cartilage damage in vivo and does not induce joint fibroplasias in a rat model of musculoskeletal syndrome side effects. Thus, highly selective inhibition of MMP13 in patients may overcome the major safety and efficacy challenges that have limited previously tested non-selective MMP inhibitors. MMP13 inhibitors such as the ones described here will help further define the role of this protease in arthritis and other diseases and may soon lead to drugs that safely halt cartilage damage in patients.

Inhibition of peroxisome-proliferator-activated receptor (PPAR)α by MK886

Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371--375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-alpha, -beta and -gamma activity was assessed using reporter assay systems (peroxisome-proliferator response element--luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10--20 microM MK886 inhibited Wy14,643 activation of PPAR alpha by approximately 80%. Similar inhibition of PPAR alpha by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPAR beta and PPAR gamma. MK886 inhibited PPAR alpha by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPAR alpha protein, and a dose-response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand-receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPAR alpha-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPAR alpha and PPAR gamma agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPAR alpha, but may also indicate that PPAR alpha is not directly involved in MK886-induced apoptosis. Although numerous PPAR activators have been identified, the results show that MK886 can inhibit PPAR alpha, making it the first compound identified to have such an effect.

Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis.

Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-x(L)-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be approximately 30% lower in bcl-x(L) cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome beta-subunit, as determined by Western blot analyses, were similar in control and bcl-x(L) cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-x(L) cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-7-amino-4-methylcou marin (Ac-DEVD-AMC), was twice that observed in bcl-x(L) cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-x(L) cells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-x(L) cells by 12 h, although the maximal increases found in bcl-x(L) cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-x(L) provide some protection against proteasome inhibitor-induced apoptosis.

A Relationship between 5-Lipoxygenase-activating Protein and bcl-xL Expression in Murine Pro-B Lymphocytic FL5.12 Cells

Inhibitors of 5-lipoxygenase-activating protein (FLAP) have been found to induce apoptosis. The current study examined the expression of FLAP and bcl family proteins and the induction of apoptosis in interleukin-3-dependent control andbcl-x L -overexpressing FL5.12 cell lines after treatment with MK886, a specific FLAP inhibitor. FL5.12 cells contained a substantial amount of FLAP protein and mRNA but surprisingly had no measurable 5-lipoxygenase protein or 5-, 12-, or 15-lipoxygenase activity. The basal level of FLAP protein in cells overexpressingbcl-x L was 70% less than in controls. FLAP disappeared 4 h after withdrawal of interleukin-3 inbcl-x L cells but not in control cells, which underwent apoptosis. A dose- and time-response study revealed that 5 nmol of MK886/106 cells was sufficient to induce apoptosis both in control and bcl-x L cells, respectively, but to different degrees. bcl-xL and bcl-2 proteins, but not bax or FLAP, were decreased by 4 h after 5 nmol of MK886/106 cells in both cell lines, although the higher levels of bcl-xL in overexpressors took longer to disappear. This early loss of bcl-xL and bcl-2 was not attributable to generalized proteolysis, as shown by Coomassie Blue staining and by the maintenance of bax. Caspase-3 was activated 2 h after MK886 treatment in control cells but not inbcl-x L cells. Inhibition of caspase-3 decreased MK886-induced apoptosis by 50% in control cells. Inhibition of this caspase after MK886 treatment was unable to prevent the loss of bcl-xL in control cells but did provide partial protection for the loss of the transfected form, but not the endogenous form, in overexpressing cells. These data indicate that MK886 induces extensive apoptosis that is partially caspase-3 dependent and may be related to a rapid loss of bcl-xL. Although caspase-3 inhibitors had no effect on the loss of bcl-xL, other caspases or protease systems may still be involved. The absence of 5-lipoxygenase in cells containing FLAP, the lower level of FLAP in bcl-xL cells, the apoptosis-inducing activity of MK886, and the rapid loss of bcl-xL and bcl-2 proteins after treatment with MK886 strongly indicate that FLAP has activities unrelated to lipoxygenase and suggest a possible functional or regulatory link between these proteins, which share similar subcellular localizations.

Changes in ceramide and sphingomyelin following fludarabine treatment of human chronic B-cell leukemia cells

Fludarabine is used to treat chronic lymphocytic leukemia. Both in vitro and in vivo studies have indicated that apoptosis is an important mode of fludarabine-induced cell death. However, the apoptotic pathways activated are not known. The effects of apoptotic doses of fludarabine on sphingomyelin, ceramide and the production of reactive oxygen species were investigated in the chronic B-cell leukemia lines WSU and JVM-2. Apoptosis, as assessed by an increase in phosphatidylserine externalization, internucleosomal DNA fragmentation and caspase-3-like activity, was evident by 18 h after fludarabine in both cell lines. The general caspase inhibitor t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone (OMe, methyl ester) significantly inhibited apoptosis supporting a role for caspases in fludarabine-induced cell death. A 2.5- to threefold elevation in ceramide levels was observed 6 h after fludarabine treatment. Concomitantly, a decrease in sphingomyelin levels was observed. Fumonisin B1 (an inhibitor of ceramide synthase) pretreatment significantly prevented fludarabine-induced ceramide generation and apoptosis. Conversely, C6-ceramide induced apoptosis in both cell lines. No effect of fludarabine on indices of oxidative stress (dichlorofluorescin oxidation and glutathione disulfide formation) were detected, although partial protection from apoptosis, and prevention of ceramide generation and caspase-3 activation, were achieved with N-acetylcysteine. These findings are consistent with the involvement of caspases and ceramide in fludarabine-induced apoptosis in WSU and JVM-2 cells. Oxidative stress does not appear to be induced by fludarabine, although the protective effects of N-acetylcysteine suggest that thiol redox balance may play a role in the apoptotic pathway.

Mixed effects of 2,6-dithiopurine against cyclophosphamide mediated bladder and lung toxicity in mice

2,6-Dithiopurine (DTP) has been proposed as a possible chemopreventive agent because of its ability to react with electrophiles. Acrolein, an electrophilic metabolite of cyclophosphamide (CP) involved in the toxicities of this anticancer drug, can be scavenged by DTP. The present study examined the effect of DTP treatment on CP-mediated bladder and lung toxicity in male ICR mice. Mice fed a diet containing 4% DTP that were treated intraperitoneally (i.p.) with 350 mg/kg CP showed no significant bladder damage (measured as bladder blood content at 48 h) with respect to the group fed a control diet. DTP (50 and 100 mg/kg), given i.p. 0.5 and 7 h after the initial injection of CP, also prevented the bladder damage when compared with the group receiving CP alone. Surprisingly, although neither parenteral CP nor DTP alone caused any mortality at these doses, the combined treatment resulted in 67% mortality within 3 days. At 24 h after CP + DTP, blood urea nitrogen was elevated 6-fold and urine volumes decreased by 70%. Histopathological analyses revealed a diffuse myocardial degeneration and necrosis, severe granular degeneration in the liver, abundant cellularity and infiltrates in interalveolar spaces in the lung and swollen nephron epithelial cells with some necrosis. All mice survived treatment when the dose of CP was lowered to 250 and 25-75 mg/kg DTP was given i.p. 0.5 and 7 h after CP. These DTP regimens reduced the degree of CP-induced lung toxicity, measured by [3H]thymidine incorporation into lung DNA 7 days after CP, in a dose-dependent manner. DTP (75 mg/kg) also reduced CP-induced lung fibrosis estimated by lung hydroxyproline content 28 days after CP. Analyses of urine from mice given CP + DTP revealed large amounts of the metabolic product dithiouric acid, smaller amounts of the parent DTP and several smaller peaks. The major unique metabolite peak was collected and analyzed by mass spectrometry, but did not correspond to either acrolein-DTP or acrolein-dithiouric acid. Thus, either very small amounts of an acrolein adduct are generated, the adduct is broken down to an unidentified product, or the ability of DTP to prevent CP-induced lung and bladder damage is related to some other mechanism. The possibility that mercapturic acid metabolites of acrolein released the parent electrophile in the urine was not supported by the finding that probenecid did not prevent CP-induced bladder toxicity.