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. 2012 Feb 15;3(2):129-40.
doi: 10.1021/cn200109w. Epub 2011 Nov 14.

Characterization of Non-Nitrocatechol Pan and Isoform Specific catechol-O-methyltransferase Inhibitors and Substrates

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Free PMC article

Characterization of Non-Nitrocatechol Pan and Isoform Specific catechol-O-methyltransferase Inhibitors and Substrates

Ronald G Robinson et al. ACS Chem Neurosci. .
Free PMC article

Abstract

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

Keywords: Catechol-O-methyltransferase; dihydroxyphenylacetic acid; fluorescence polarization and hepatotoxicity; high throughput screen; homovanillic acid.

Figures

Figure 1
Figure 1
(A) 3.5 μg recombinant proteins were resolved on a 4–12% Bis-Tris gel and stained with Instant Blue. From left to right: SeeBlue Plus2 Pre-Stained standard (molecular weight in kDa, Invitrogen), human MB-COMT (M51A, V158) with 3′ EK and 6-HIS, recombinant human S-COMT (V158) with 3′ EK and 6-HIS, recombinant rat MB-COMT (M44A) with 3′ 10-HIS and recombinant rat S-COMT with 3′ 10-HIS. (B) ∼150 ng recombinant proteins were resolved on 4–12% Bis-Tris gels and subjected to Western blotting with a primary antibody to COMT from Millipore (AB5873).
Figure 2
Figure 2
Modified fluorescence polarization based time course assays were used to identify novel compounds as COMT substrates. A 5 μL mix comprising 40 μM compound stocks, 8 μM SAM, and 40 mM MgCl2 in assay buffer was placed into assay wells (black 96-well round-bottom polystyrene plates from Costar; catalog # 3792). Enzyme reactions were initiated at room temperature by adding 35 μL of one of the COMT forms (1–6 ng of protein) also diluted into assay buffer. The final concentrations of the test compounds and SAM were 5 and 1 μM, respectively. Additional assays replacing the compounds with a final 2 μM concentration of dopamine were also included as a positive COMT substrate reference. The enzyme assays were quenched at various time points with 5 μL of 250 mM EDTA and subsequently 20 μL of a preformed complex containing SAC TAMRA tracer and anti-SAH antibody was added (described in the Methods section). The extent of SAH production was used to evaluate compounds as COMT substrates. Increasing levels of SAH inversely lowered polarization measurements of the SAC TAMRA/SAH antibody complex by displacing the fluorescence label from the antibody. Graphs A through D show time course assay results using 2 μM dopamine or 5 μM novel compounds as substrates, respectively, for human MB-COMT, rat MB-COMT, human S-COMT, and rat S-COMT.
Figure 3
Figure 3
Modified fluorescence polarization (FP) based time course assays to evaluate tolcapone and methylated derivatives as COMT substrates. (A–D) 1 μM tolcapone or 2 μM dopamine provided as substrates for human MB-COMT, rat MB-COMT, human S-COMT, or rat S-COMT. (E) 1 μM 4-methoxy-tolcapone or 1 μM 3-methoxy-tolcapone provided as substrates for rat S-COMT.
Figure 4
Figure 4
Representative Western blot demonstrating the ratio of MB to S-COMT in blood (lanes 1–2), brain (lanes 3–4), or liver (lanes 5–6) fractions utilized in the COMT ex vivo assay from male Wistar rat. To demonstrate similar immunoreactivity for rat MB versus S-COMT, equal amounts of recombinant rat MB (lane 7) or S-COMT (lane 8) were also resolved. Immunoblotting with COMT antibody (Santa Cruz-135872, dilution 1:200) revealed immunoreactivity for MB (indicated by upper arrow) and S-COMT (lower arrow).
Figure 5
Figure 5
Effect of tolcapone, entacapone, or novel COMT inhibitors on rat heart mitochondrial membrane potential (MMP). Values listed next to compounds are concentrations that resulted in a 50% reduction of the mitochondrial membrane potential based on a vehicle treatment control representing maximum membrane potential and 3 mM carbonylcyanide m-chlorophenyl hydrazone (CCCP) treatment representing minimum membrane potential.

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