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1.
J Med Chem ; 65(21): 14366-14390, 2022 11 10.
Article in English | MEDLINE | ID: mdl-36261130

ABSTRACT

The branched-chain amino acid transaminases (BCATs) are enzymes that catalyze the first reaction of catabolism of the essential branched-chain amino acids to branched-chain keto acids to form glutamate. They are known to play a key role in different cancer types. Here, we report a new structural class of BCAT1/2 inhibitors, (trifluoromethyl)pyrimidinediones, identified by a high-throughput screening campaign and subsequent optimization guided by a series of X-ray crystal structures. Our potent dual BCAT1/2 inhibitor BAY-069 displays high cellular activity and very good selectivity. Along with a negative control (BAY-771), BAY-069 was donated as a chemical probe to the Structural Genomics Consortium.


Subject(s)
Amino Acids, Branched-Chain , Transaminases , Transaminases/metabolism , Amino Acids, Branched-Chain/metabolism , Keto Acids/metabolism
2.
Acta Crystallogr D Struct Biol ; 77(Pt 2): 237-248, 2021 Feb 01.
Article in English | MEDLINE | ID: mdl-33559612

ABSTRACT

Wild-type human glutathione peroxidase 4 (GPX4) was co-expressed with SBP2 (selenocysteine insertion sequence-binding protein 2) in human HEK cells to achieve efficient production of this selenocysteine-containing enzyme on a preparative scale for structural biology. The protein was purified and crystallized, and the crystal structure of the wild-type form of GPX4 was determined at 1.0 Šresolution. The overall fold and the active site are conserved compared with previously determined crystal structures of mutated forms of GPX4. A mass-spectrometry-based approach was developed to monitor the reaction of the active-site selenocysteine Sec46 with covalent inhibitors. This, together with the introduction of a surface mutant (Cys66Ser), enabled the crystal structure determination of GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer]. The mass-spectrometry-based approach described here opens the path to further co-complex crystal structures of this potential cancer drug target in complex with covalent inhibitors.


Subject(s)
Enzyme Inhibitors , Phospholipid Hydroperoxide Glutathione Peroxidase , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , HEK293 Cells , Humans , Phospholipid Hydroperoxide Glutathione Peroxidase/chemistry , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Protein Binding , Protein Conformation
3.
Nat Chem Biol ; 16(5): 497-506, 2020 05.
Article in English | MEDLINE | ID: mdl-32231343

ABSTRACT

We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.


Subject(s)
Enzyme Inhibitors/pharmacology , Nitriles/chemistry , Nitriles/pharmacology , Phospholipid Hydroperoxide Glutathione Peroxidase/antagonists & inhibitors , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Animals , Cell Line, Tumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Ferroptosis/drug effects , Humans , Lipid Peroxidation/drug effects , Mice, SCID , Molecular Probes/chemistry , Molecular Targeted Therapy , Oxides/chemistry , Phospholipid Hydroperoxide Glutathione Peroxidase/chemistry , Prodrugs/chemistry , Rats, Wistar , Selenocysteine/chemistry , Selenocysteine/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity Relationship
4.
Leukemia ; 33(10): 2403-2415, 2019 10.
Article in English | MEDLINE | ID: mdl-30940908

ABSTRACT

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Differentiation/drug effects , Enzyme Inhibitors/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line, Tumor , Dihydroorotate Dehydrogenase , Female , HL-60 Cells , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Promyelocytic, Acute/drug therapy , Leukemia, Promyelocytic, Acute/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Pyrimidines/metabolism , THP-1 Cells , Translocation, Genetic/drug effects
5.
ChemMedChem ; 12(10): 728-737, 2017 05 22.
Article in English | MEDLINE | ID: mdl-28488817

ABSTRACT

Adenosine is known to be released under a variety of physiological and pathophysiological conditions to facilitate the protection and regeneration of injured ischemic tissues. The activation of myocardial adenosine A1 receptors (A1 Rs) has been shown to inhibit myocardial pathologies associated with ischemia and reperfusion injury, suggesting several options for new cardiovascular therapies. When full A1 R agonists are used, the desired protective and regenerative cardiovascular effects are usually overshadowed by unintended pharmacological effects such as induction of bradycardia, atrioventricular (AV) blocks, and sedation. These unwanted effects can be overcome by using partial A1 R agonists. Starting from previously reported capadenoson we evaluated options to tailor A1 R agonists to a specific partiality range, thereby optimizing the therapeutic window. This led to the identification of the potent and selective agonist neladenoson, which shows the desired partial response on the A1 R, resulting in cardioprotection without sedative effects or cardiac AV blocks. To circumvent solubility and formulation issues for neladenoson, a prodrug approach was pursued. The dipeptide ester neladenoson bialanate hydrochloride showed significantly improved solubility and exposure after oral administration. Neladenoson bialanate hydrochloride is currently being evaluated in clinical trials for the treatment of heart failure.


Subject(s)
Adenosine A1 Receptor Agonists/pharmacology , Dipeptides/pharmacology , Heart Diseases/drug therapy , Prodrugs/pharmacology , Pyridines/pharmacology , Receptor, Adenosine A1/metabolism , Adenosine A1 Receptor Agonists/administration & dosage , Adenosine A1 Receptor Agonists/chemistry , Administration, Oral , Animals , Chronic Disease , Dipeptides/administration & dosage , Dipeptides/chemistry , Disease Models, Animal , Dose-Response Relationship, Drug , Humans , Injections, Intravenous , Molecular Structure , Prodrugs/administration & dosage , Prodrugs/chemistry , Pyridines/administration & dosage , Pyridines/chemistry , Rats , Solubility , Structure-Activity Relationship
6.
Acta Neuropathol ; 133(4): 629-644, 2017 04.
Article in English | MEDLINE | ID: mdl-28124097

ABSTRACT

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.


Subject(s)
Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , Astrocytoma/drug therapy , Benzimidazoles/pharmacology , Brain Neoplasms/drug therapy , Isocitrate Dehydrogenase/antagonists & inhibitors , Isocitrate Dehydrogenase/genetics , Aniline Compounds/chemistry , Aniline Compounds/pharmacokinetics , Aniline Compounds/toxicity , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/toxicity , Astrocytoma/enzymology , Astrocytoma/genetics , Benzimidazoles/chemistry , Benzimidazoles/pharmacokinetics , Benzimidazoles/toxicity , Brain Neoplasms/enzymology , Brain Neoplasms/genetics , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/toxicity , Escherichia coli , Female , Glutarates/metabolism , HEK293 Cells , Humans , Isocitrate Dehydrogenase/metabolism , Mice, Inbred BALB C , Mice, Nude , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarcoma/drug therapy , Sarcoma/enzymology , Sarcoma/genetics , Sf9 Cells , Xenograft Model Antitumor Assays
7.
Int J Hyperthermia ; 33(2): 178-190, 2017 Mar.
Article in English | MEDLINE | ID: mdl-27592502

ABSTRACT

PURPOSE: Doxorubicin (DOX)-loaded phosphatidyldiglycerol-based thermosensitive liposomes (DPPG2-TSL-DOX) combined with local hyperthermia (HT) was evaluated in cats with locally advanced spontaneous fibrosarcomas (soft tissue sarcoma [STS]). The study was designed to evaluate the safety and pharmacokinetic profile of the drug. Results from four dose-levels are reported. METHODS: Eleven client-owned cats with advanced STS were enrolled. Five cats received escalating doses of 0.1-0.4 mg/kg DOX (group I), three received 0.4 mg/kg constantly (group II) and three 0.6 mg/kg (group III) IV over 15 min. HT with a target temperature of 41.5 °C was started 15 min before drug application and continued for a total of 60 min. Six HT treatments were applied every other week using a radiofrequency applicator. Tumour growth was monitored by magnetic resonance imaging (MRI) and for dose level III also with 18F-FDG PET. RESULTS: Treatment was generally well tolerated and reasons for premature study termination in four cats were not associated with drug-induced toxicity. No DPPG2-TSL-DOX based hypersensitivity reaction was observed. One cat showed simultaneous partial response (PR) in MRI and positron emission tomography (PET) whereas one cat showed stable disease in MRI and PR in PET (both cats in dose level III). Pharmacokinetic measurements demonstrated DOX release triggered by HT. CONCLUSION: DPPG2-TSL-DOX + HT is a promising treatment option for advanced feline STS by means of targeted drug delivery. As MTD was not reached further investigation is warranted to determine if higher doses would result in even better tumour responses.

8.
BMC Med Educ ; 15: 189, 2015 Oct 29.
Article in English | MEDLINE | ID: mdl-26511721

ABSTRACT

BACKGROUND: Inter-professional teamwork is key for patient safety and team training is an effective strategy to improve patient outcome. In-situ simulation is a relatively new strategy with emerging efficacy, but best practices for the design, delivery and implementation have yet to be evaluated. Our aim is to describe and evaluate the implementation of an inter-professional in-situ simulated team and resuscitation training in a teaching hospital with a programmatic approach. METHODS: We designed and implemented a team and resuscitation training program according to Kern's six steps approach for curriculum development. General and specific needs assessments were conducted as independent cross-sectional surveys. Teamwork, technical skills and detection of latent safety threats were defined as specific objectives. Inter-professional in-situ simulation was used as educational strategy. The training was embedded within the workdays of participants and implemented in our highest acuity wards (emergency department, intensive care unit, intermediate care unit). Self-perceived impact and self-efficacy were sampled with an anonymous evaluation questionnaire after every simulated training session. Assessment of team performance was done with the team-based self-assessment tool TeamMonitor applying Van der Vleuten's conceptual framework of longitudinal evaluation after experienced real events. Latent safety threats were reported during training sessions and after experienced real events. RESULTS: The general and specific needs assessments clearly identified the problems, revealed specific training needs and assisted with stakeholder engagement. Ninety-five interdisciplinary staff members of the Children's Hospital participated in 20 in-situ simulated training sessions within 2 years. Participant feedback showed a high effect and acceptance of training with reference to self-perceived impact and self-efficacy. Thirty-five team members experiencing 8 real critical events assessed team performance with TeamMonitor. Team performance assessment with TeamMonitor was feasible and identified specific areas to target future team training sessions. Training sessions as well as experienced real events revealed important latent safety threats that directed system changes. CONCLUSIONS: The programmatic approach of Kern's six steps for curriculum development helped to overcome barriers of design, implementation and assessment of an in-situ team and resuscitation training program. This approach may help improve effectiveness and impact of an in-situ simulated training program.


Subject(s)
Interprofessional Relations , Patient Safety , Resuscitation/education , Curriculum , Humans , Patient Care Team , Simulation Training
9.
Circ Heart Fail ; 6(3): 563-71, 2013 May.
Article in English | MEDLINE | ID: mdl-23564604

ABSTRACT

BACKGROUND: Adenosine elicits cardioprotection through A1-receptor activation. Therapy with adenosine A1-receptor agonists, however, is limited by undesirable actions of full agonism, such as bradycardia. This study examined the effects of capadenoson (CAP), a partial adenosine A1-receptor agonist, on left ventricular (LV) function and remodeling in dogs with heart failure. METHODS AND RESULTS: Twelve dogs with microembolization-induced heart failure were randomized to 12 weeks oral therapy with CAP (7.5 mg BID; n=6) or to no therapy (control; n=6). LV end-diastolic and end-systolic volumes, ejection fraction, plasma norepinephrine, and n-terminal pro-brain natriuretic peptide were measured before (pre) and 1 and 12 weeks after therapy (post). LV tissue obtained at post was used to assess volume fraction of interstitial fibrosis, sarcoplasmic reticulum calcium ATPase-2a activity, expression of mitochondria uncoupling proteins (UCP) and glucose transporters (GLUT). In controls, end-diastolic and end-systolic volumes increased and ejection fraction decreased significantly from pre to post (ejection fraction, 30±2 versus 27±1%; P<0.05). In CAP-treated dogs, end-diastolic volume was unchanged; ejection fraction increased significantly after 1 week (36±2 versus 27±2%; P<0.05) with a further increase at post (39±2%; P<0.05), whereas end-systolic volume decreased. CAP significantly decreased volume fraction of interstitial fibrosis, normalized sarcoplasmic reticulum calcium ATPase-2a activity and expression of UCP-2 and UCP-3, and GLUT-1 and GLUT-2 and significantly decreased plasma norepinephrine and n-terminal pro-brain natriuretic peptide. CONCLUSIONS: In heart failure dogs, CAP improves LV function and prevents progressive remodeling. Improvement of LV systolic function occurs early after initiating therapy. The results support development of partial adenosine A1-receptor agonists for the treatment of chronic heart failure.


Subject(s)
Adenosine A1 Receptor Agonists/pharmacology , Aminopyridines/pharmacology , Heart Failure/physiopathology , Thiazoles/pharmacology , Ventricular Function, Left/drug effects , Ventricular Remodeling/drug effects , Adenosine A1 Receptor Agonists/therapeutic use , Animals , Dogs , Echocardiography, Doppler , Heart Failure/diagnostic imaging , Heart Failure/drug therapy , Hemodynamics/drug effects , Stroke Volume/drug effects , Ventricular Function, Left/physiology , Ventricular Pressure/drug effects , Ventricular Pressure/physiology , Ventricular Remodeling/physiology
10.
Am J Med ; 124(9): 841-51.e2, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21854893

ABSTRACT

BACKGROUND: Evidence from individual trials comparing Mediterranean to low-fat diets to modify cardiovascular risk factors remains preliminary. METHODS: We systematically searched MEDLINE, EMBASE, Biosis, Web of Science, and the Cochrane Central Register of Controlled Trials from their inception until January 2011, as well as contacted experts in the field, to identify randomized controlled trials comparing Mediterranean to low-fat diets in overweight/obese individuals, with a minimum follow-up of 6 months, reporting intention-to-treat data on cardiovascular risk factors. Two authors independently assessed trial eligibility and quality. RESULTS: We identified 6 trials, including 2650 individuals (50% women) fulfilling our inclusion criteria. Mean age of enrolled patients ranged from 35 to 68 years, mean body mass index from 29 to 35 kg/m(2). After 2 years of follow-up, individuals assigned to a Mediterranean diet had more favorable changes in weighted mean differences of body weight (-2.2 kg; 95% confidence interval [CI], -3.9 to -0.6), body mass index (-0.6 kg/m(2); 95% CI, -1 to -0.1), systolic blood pressure (-1.7 mm Hg; 95% CI, -3.3 to -0.05), diastolic blood pressure (-1.5 mm Hg; 95% CI, -2.1 to -0.8), fasting plasma glucose (-3.8 mg/dL, 95% CI, -7 to -0.6), total cholesterol (-7.4 mg/dL; 95% CI, -10.3 to -4.4), and high-sensitivity C-reactive protein (-1.0 mg/L; 95% CI, -1.5 to -0.5). The observed heterogeneity across individual trials could, by and large, be eliminated by restricting analyses to trials with balanced co-interventions or trials with restriction of daily calorie intake in both diet groups. CONCLUSION: Mediterranean diets appear to be more effective than low-fat diets in inducing clinically relevant long-term changes in cardiovascular risk factors and inflammatory markers.


Subject(s)
Cardiovascular Diseases/diet therapy , Diet, Fat-Restricted , Diet, Mediterranean , Obesity/diet therapy , Overweight/diet therapy , Adult , Aged , Body Mass Index , Cardiovascular Diseases/prevention & control , Diet, Reducing , Exercise , Female , Humans , Male , Middle Aged , Myocardial Infarction/diet therapy , Myocardial Infarction/prevention & control , Randomized Controlled Trials as Topic , Risk Factors , Sedentary Behavior , Waist Circumference
11.
PLoS One ; 6(3): e18048, 2011 Mar 28.
Article in English | MEDLINE | ID: mdl-21464936

ABSTRACT

The release of the neurotransmitter norepinephrine (NE) is modulated by presynaptic adenosine receptors. In the present study we investigated the effect of a partial activation of this feedback mechanism. We hypothesized that partial agonism would have differential effects on NE release in isolated hearts as well as on heart rate in vivo depending on the genetic background and baseline sympathetic activity. In isolated perfused hearts of Wistar and Spontaneously Hypertensive Rats (SHR), NE release was induced by electrical stimulation under control conditions (S1), and with capadenoson 6 · 10(-8) M (30 µg/l), 6 · 10(-7) M (300 µg/l) or 2-chloro-N(6)-cyclopentyladenosine (CCPA) 10(-6) M (S2). Under control conditions (S1), NE release was significantly higher in SHR hearts compared to Wistar (766+/-87 pmol/g vs. 173+/-18 pmol/g, p<0.01). Capadenoson led to a concentration-dependent decrease of the stimulation-induced NE release in SHR (S2/S1  =  0.90 ± 0.08 with capadenoson 6 · 10(-8) M, 0.54 ± 0.02 with 6 · 10(-7) M), but not in Wistar hearts (S2/S1  =  1.05 ± 0.12 with 6 · 10(-8) M, 1.03 ± 0.09 with 6 · 10(-7) M). CCPA reduced NE release to a similar degree in hearts from both strains. In vivo capadenoson did not alter resting heart rate in Wistar rats or SHR. Restraint stress induced a significantly greater increase of heart rate in SHR than in Wistar rats. Capadenoson blunted this stress-induced tachycardia by 45% in SHR, but not in Wistar rats. Using a [(35)S]GTPγS assay we demonstrated that capadenoson is a partial agonist compared to the full agonist CCPA (74+/-2% A(1)-receptor stimulation). These results suggest that partial adenosine A(1)-agonism dampens stress-induced tachycardia selectively in rats susceptible to strong increases in sympathetic activity, most likely due to a presynaptic attenuation of NE release.


Subject(s)
Adenosine A1 Receptor Agonists/pharmacology , Heart Rate/drug effects , Norepinephrine/metabolism , Receptor, Adenosine A1/metabolism , Stress, Physiological/drug effects , Animals , Blood Pressure/drug effects , Female , In Vitro Techniques , Rats , Rats, Inbred SHR , Rats, Wistar , Restraint, Physical
12.
Curr Probl Dermatol ; 40: 20-35, 2011.
Article in English | MEDLINE | ID: mdl-21325837

ABSTRACT

Intranasal administration is an attractive option for local and systemic delivery of many therapeutic agents. The nasal mucosa is--compared to other mucosae--easily accessible. Intranasal drug administration is noninvasive, essentially painless and particularly suited for children. Application can be performed easily by patients or by physicians in emergency settings. Intranasal drug delivery offers a rapid onset of therapeutic effects (local or systemic). Nasal application circumvents gastrointestinal degradation and hepatic first-pass metabolism of the drug. The drug, the vehicle and the application device form an undividable triad. Its selection is therefore essential for the successful development of effective nasal products. This paper discusses the feasibility and potential of intranasal administration. A series of questions regarding (a) the intended use (therapeutic considerations), (b) the drug, (c) the vehicle and (d) the application device (pharmaceutical considerations) are addressed with a view to their impact on the development of products for nasal application. Current and future trends and perspectives are discussed.


Subject(s)
Administration, Intranasal , Drug Delivery Systems/methods , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , Blood-Brain Barrier , Cardiovascular Agents/administration & dosage , Drug Delivery Systems/instrumentation , Drug Delivery Systems/trends , Drug Discovery , Humans , Nasal Cavity/anatomy & histology , Nasal Cavity/physiology , Pharmaceutical Vehicles/administration & dosage , Vaccines/administration & dosage
13.
J Cell Biol ; 156(6): 1077-87, 2002 Mar 18.
Article in English | MEDLINE | ID: mdl-11901172

ABSTRACT

The molecular mechanisms of apoptosis are highly conserved throughout evolution. The homologs of genes essential for apoptosis in Caenorhabditis elegans and Drosophila melanogaster have been shown to be important for apoptosis in mammalian systems. Although a homologue for CED-4/apoptotic protease-activating factor (Apaf)-1 has been described in Drosophila, its exact function and the role of the mitochondrial pathway in its activation remain unclear. Here, we used the technique of RNA interference to dissect apoptotic signaling pathways in Drosophila cells. Inhibition of the Drosophila CED-4/Apaf-1-related killer (ARK) homologue resulted in pronounced inhibition of stress-induced apoptosis, whereas loss of ARK did not protect the cells from Reaper- or Grim-induced cell death. Reduction of DIAP1 induced rapid apoptosis in these cells, whereas the inhibition of DIAP2 expression did not but resulted in increased sensitivity to stress-induced apoptosis; apoptosis in both cases was prevented by inhibition of ARK expression. Cells in which cytochrome c expression was decreased underwent apoptosis induced by stress stimuli, Reaper or Grim. These results demonstrate the central role of ARK in stress-induced apoptosis, which appears to act independently of cytochrome c. Apoptosis induced by Reaper or Grim can proceed via a distinct pathway, independent of ARK.


Subject(s)
Apoptosis/genetics , Caenorhabditis elegans Proteins , Calcium-Binding Proteins/metabolism , Drosophila Proteins , Drosophila melanogaster/metabolism , Helminth Proteins/metabolism , Proteins/metabolism , Stress, Physiological/metabolism , Animals , Apoptotic Protease-Activating Factor 1 , Calcium-Binding Proteins/genetics , Caspase Inhibitors , Caspases/genetics , Caspases/metabolism , Cytochrome c Group/drug effects , Cytochrome c Group/genetics , Cytochrome c Group/metabolism , Down-Regulation/drug effects , Down-Regulation/physiology , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Enzyme Inhibitors/pharmacology , Helminth Proteins/genetics , Inhibitor of Apoptosis Proteins , Insect Proteins/genetics , Insect Proteins/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mitochondria/drug effects , Mitochondria/metabolism , Neuropeptides/genetics , Neuropeptides/metabolism , Peptides/genetics , Peptides/metabolism , Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Double-Stranded/genetics , RNA, Double-Stranded/pharmacology , Stress, Physiological/genetics , Stress, Physiological/physiopathology , Ultraviolet Rays
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