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1.
Cancer Res ; 78(23): 6691-6702, 2018 12 01.
Article in English | MEDLINE | ID: mdl-30297535

ABSTRACT

: PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity. SIGNIFICANCE: These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.


Subject(s)
ADP Ribose Transferases/antagonists & inhibitors , Breast Neoplasms/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Spindle Apparatus/drug effects , Spindle Apparatus/metabolism , Animals , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Checkpoint Kinase 1/metabolism , Disease Models, Animal , Female , Humans , Mice , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Signal Transduction/drug effects , Substrate Specificity , Xenograft Model Antitumor Assays
2.
Synapse ; 65(1): 64-8, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20506301

ABSTRACT

We previously reported the absence of high-affinity binding of the group II metabotropic glutamate receptor agonists LY 354,740 and LY 379,268 to the D2L dopamine receptor. A rebuttal to our findings has since been reported (see Introduction section); this study represents our response. Analysis by LCMS of LY 354,740 and LY 379,268 used in this study revealed the correct molecular mass for these compounds. Both LY 354,740 and LY 379,268 exhibited potent agonist activity for mGluR2 in the ³5S-GTPγS assay. Functionally, neither compound displayed antagonist activity in the GTPγS assay with recombinant D2. At concentrations up to 10 µM, both compounds failed to displace [³H]-raclopride, [³H]-PHNO, or [³H]-domperidone in filter-binding assays under isotonic (120 mM NaCl or N-methyl glucamine) or low-ionic strength (no NaCl or N-methyl glucamine) conditions. Some displacement of [³H]-domperidone (20-40%) was observed at 30 µM of LY 354,740 under low-ionic strength and under isotonic conditions in the absence of NaCl. No displacement of [³H]-domperidone was detected in a two site model at lower (<100 nM) concentrations of either compound. Moreover, no D2 activity was observed for LY 354,740 or LY 379,268 in the CellKey™ (cellular dielectric spectroscopy) assay. In this communication, we discuss the possible reasons for differences in our study and the previously published work and implications of these studies for mechanisms of antipsychotic action.


Subject(s)
Amino Acids/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bridged Bicyclo Compounds/pharmacology , Receptors, Dopamine D2/metabolism , Receptors, Metabotropic Glutamate/agonists , Animals , Binding, Competitive , CHO Cells , Cricetinae , Cricetulus , Domperidone/pharmacology , Excitatory Amino Acid Agonists/pharmacology , Mass Spectrometry , Raclopride/pharmacology , Radioligand Assay
3.
Synapse ; 64(8): 624-33, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20340171

ABSTRACT

Positron emission tomography (PET) antagonist ligands such as [(11)C]-raclopride are commonly used to study dopamine D2 receptor (D2) binding of antipsychotics. It has been suggested that agonist radioligands bind preferentially to the high-affinity state of D2 receptor and may provide a more relevant means of assessing D2 occupancy. The main objective of this study was to determine if D2 receptor occupancy (RO) could be differentiated with agonist and antagonist radioligands in vivo. Agonist radioligands [(3)H]-MNPA and [(3)H]-(+)-PHNO were synthesized and compared to antagonist [(3)H]-raclopride in the in vitro binding and in vivo occupancy studies. In vivo, unanesthetized rats were pretreated with quinpirole (full agonist), aripiprazole (partial agonist), or haloperidol (antagonist) prior to administration of the agonist or antagonist radioligand. All three pretreatment compounds showed equivalent dose-dependent D2 receptor occupancy in the rat striatum with each radioligand. The in vivo receptor occupancy results suggested that the binding of quinpirole, aripiprazole, and haloperidol to the high or low affinity state of the D2 receptor could not be differentiated using radiolabeled agonists or antagonists, presumably due to a predominance of high affinity states of the D2 receptor in vivo. This hypothesis was supported in part by the in vitro binding results. Our in vitro results show that [(3)H]-MNPA binds to D2S transfected CHO cell membranes at a single high affinity site. Displacement of [(3)H]-(+)-PHNO binding by quinpirole and elimination of most [(3)H]-(+)-PHNO binding by the guanine nucleotide GppNHp in striatal membranes suggest that the majority of D2 in striatal tissue is G-protein coupled. Together, these findings suggest that D2 agonist radioligands produce in vivo receptor occupancy comparable to [(3)H]-raclopride.


Subject(s)
Apomorphine/analogs & derivatives , Brain/drug effects , Brain/metabolism , Oxazines/metabolism , Raclopride/metabolism , Receptors, Dopamine D2/metabolism , Animals , Apomorphine/metabolism , Binding Sites/drug effects , Binding Sites/physiology , Binding, Competitive/drug effects , Binding, Competitive/physiology , CHO Cells , Cricetinae , Cricetulus , Dopamine Agonists/metabolism , Dopamine Antagonists/metabolism , Dopamine D2 Receptor Antagonists , Male , Rats , Rats, Long-Evans , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/metabolism
4.
Biochem Pharmacol ; 78(7): 880-8, 2009 Oct 01.
Article in English | MEDLINE | ID: mdl-19615981

ABSTRACT

AZD0328, a novel spirofuropyridine neuronal nicotinic receptor partial agonist, was used to investigate the role of alpha7 neuronal nicotinic receptor (NNR) activation in the modulation of midbrain dopamine neuron function, cortical dopamine release and on two behavioral tasks known to be dependent on optimal levels of cortical dopamine. In vivo recordings from area 10 (ventral tegmental area) in rat brain showed an increased firing of putative dopamine neurons in response to low (0.00138 mg/kg) doses of AZD0328. Bursting patterns of dopamine neuron activity remained largely unchanged by application of AZD0328. In vivo microdialysis in awake rats showed an increase in extracellular prefrontal cortical dopamine in response to low doses of AZD0328. Compound-stimulated dopamine release showed an inverted dose effect relation that was maximal at the lowest dose tested (0.00178 mg/kg). Peak extracellular dopamine levels were reached 2h after dosing with AZD0328. Acquisition of operant responding with delayed reinforcement in rats was dose dependently enhanced by AZD0328 with a plateau effect measured at 0.003 mg/kg. This effect was blocked by pre-treatment of animals with the selective alpha7 antagonist methyllycaconitine. AZD0328 improved novel object recognition in mice over a broad range of doses (0.00178-1.78 mg/kg) and the compound effect was found to be absent in homozygous alpha7 KO animals. Together, these data indicate that selective interaction with alpha7 NNRs by AZD0328 selectively enhances midbrain dopaminergic neuronal activity causing an enhancement of cortical dopamine levels; these neurochemical changes likely, underlie the positive behavioral responses observed in two different animal models. Our results suggest selective alpha7 NNR agonists may have significant therapeutic utility in neurologic and psychiatric indications where cognitive deficits and dopamine neuron dysfunction co-exist.


Subject(s)
Attention/drug effects , Cerebral Cortex/drug effects , Dopamine/metabolism , Furans/pharmacology , Learning/drug effects , Nicotinic Agonists/pharmacology , Quinuclidines/pharmacology , Receptors, Nicotinic/physiology , Action Potentials/drug effects , Animals , Cell Line , Cerebral Cortex/metabolism , Conditioning, Operant/drug effects , Female , Humans , Male , Neurons/physiology , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Radioligand Assay , Rats , Rats, Sprague-Dawley , Recognition, Psychology/drug effects , Reinforcement, Psychology , Ventral Tegmental Area/drug effects , Ventral Tegmental Area/physiology , Xenopus laevis , alpha7 Nicotinic Acetylcholine Receptor
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