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1.
Pharmacol Res Perspect ; 8(1): e00560, 2020 02.
Article in English | MEDLINE | ID: mdl-31990455

ABSTRACT

M1 muscarinic acetylcholine receptor (M1 R) activation can be a new therapeutic approach for the treatment of cognitive deficits associated with cholinergic hypofunction. However, M1 R activation causes gastrointestinal (GI) side effects in animals. We previously found that an M1 R positive allosteric modulator (PAM) with lower cooperativity (α-value) has a limited impact on ileum contraction and can produce a wider margin between cognitive improvement and GI side effects. In fact, TAK-071, a novel M1 R PAM with low cooperativity (α-value of 199), improved scopolamine-induced cognitive deficits with a wider margin against GI side effects than a high cooperative M1 R PAM, T-662 (α-value of 1786), in rats. Here, we describe the pharmacological characteristics of a novel low cooperative M1 R PAM T-495 (α-value of 170), using the clinically tested higher cooperative M1 R PAM MK-7622 (α-value of 511) as a control. In rats, T-495 caused diarrhea at a 100-fold higher dose than that required for the improvement of scopolamine-induced memory deficits. Contrastingly, MK-7622 showed memory improvement and induction of diarrhea at an equal dose. Combination of T-495, but not of MK-7622, and donepezil at each sub-effective dose improved scopolamine-induced memory deficits. Additionally, in mice with reduced acetylcholine levels in the forebrain via overexpression of A53T α-synuclein (ie, a mouse model of dementia with Lewy bodies and Parkinson's disease with dementia), T-495, like donepezil, reversed the memory deficits in the contextual fear conditioning test and Y-maze task. Thus, low cooperative M1 R PAMs are promising agents for the treatment of memory deficits associated with cholinergic dysfunction.


Subject(s)
Allosteric Regulation/drug effects , Cholinergic Agents/administration & dosage , Diarrhea/chemically induced , Memory Disorders/drug therapy , Receptor, Muscarinic M1/antagonists & inhibitors , Animals , CHO Cells , Cholinergic Agents/adverse effects , Cholinergic Agents/chemistry , Cholinergic Agents/pharmacology , Cricetulus , Disease Models, Animal , Dose-Response Relationship, Drug , Male , Memory Disorders/chemically induced , Mice , Rats , Scopolamine/adverse effects
2.
Neuroscience ; 414: 60-76, 2019 08 21.
Article in English | MEDLINE | ID: mdl-31299348

ABSTRACT

Activation of the M1 muscarinic acetylcholine receptor (M1R) may be an effective therapeutic approach for Alzheimer's disease (AD), dementia with Lewy bodies, and schizophrenia. Previously, the M1R/M4R agonist xanomeline was shown to improve cognitive function and exert antipsychotic effects in patients with AD and schizophrenia. However, its clinical development was discontinued because of its cholinomimetic side effects. We compared in vivo pharmacological profiles of a novel M1R-selective positive allosteric modulator, TAK-071, and xanomeline in rodents. Xanomeline suppressed both methamphetamine- and MK-801-induced hyperlocomotion in mice, whereas TAK-071 suppressed only MK-801-induced hyperlocomotion. In a previous study, we showed that TAK-071 improved scopolamine-induced cognitive deficits in a rat novel object recognition task (NORT) with 33-fold margins versus cholinergic side effects (diarrhea). Xanomeline also improved scopolamine-induced cognitive impairments in a NORT; however, it had no margin versus cholinergic side effects (e.g., diarrhea, salivation, and hypoactivity) in rats. These side effects were observed even in M1R knockout mice. Evaluation of c-Fos expression as a marker of neural activation revealed that xanomeline increased the number of c-Fos-positive cells in several cortical areas, the hippocampal formation, amygdala, and nucleus accumbens. Other than in the orbital cortex and claustrum, TAK-071 induced similar c-Fos expression patterns. When donepezil was co-administered to increase the levels of acetylcholine, the number of TAK-071-induced c-Fos-positive cells in these brain regions was increased. TAK-071, through induction of similar neural activation as that seen with xanomeline, may produce procognitive and antipsychotic effects with improved cholinergic side effects.


Subject(s)
Motor Activity/drug effects , Muscarinic Agonists/pharmacology , Pyridines/pharmacology , Recognition, Psychology/drug effects , Thiadiazoles/pharmacology , Allosteric Regulation , Animals , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cognition/drug effects , Cognitive Dysfunction/chemically induced , Cognitive Dysfunction/drug therapy , Hippocampus/drug effects , Hippocampus/metabolism , Mice , Muscarinic Agonists/therapeutic use , Proto-Oncogene Proteins c-fos/metabolism , Pyridines/therapeutic use , Rats , Receptor, Muscarinic M1/agonists , Receptor, Muscarinic M4/agonists , Scopolamine , Thiadiazoles/therapeutic use
3.
Neuropsychopharmacology ; 44(5): 950-960, 2019 04.
Article in English | MEDLINE | ID: mdl-30089885

ABSTRACT

The muscarinic M1 receptor (M1R) is a promising target for treating cognitive impairment associated with cholinergic deficits in disorders such as Alzheimer's disease and schizophrenia. We previously reported that cooperativity (α-value) was key to lowering the risk of diarrhea by M1R positive allosteric modulators (M1 PAMs). Based on this, we discovered a low α-value M1 PAM, TAK-071 (α-value: 199), and characterized TAK-071 using T-662 as a reference M1 PAM with high α-value of 1786. Both TAK-071 and T-662 were potent and highly selective M1 PAMs, with inflection points of 2.7 and 0.62 nM, respectively. However, T-662 but not TAK-071 augmented isolated ileum motility. TAK-071 and T-662 increased hippocampal inositol monophosphate production through M1R activation and improved scopolamine-induced cognitive deficits in rats at 0.3 and 0.1 mg/kg, respectively. TAK-071 and T-662 also induced diarrhea at 10 and 0.1 mg/kg, respectively, in rats. Thus, taking into consideration the fourfold lower brain penetration ratio of T-662, TAK-071 had a wider margin between cognitive improvement and diarrhea induction than T-662. Activation of M1R increases neural excitability via membrane depolarization, reduced afterhyperpolarization, and generation of afterdepolarization in prefrontal cortical pyramidal neurons. T-662 induced all three processes, whereas TAK-071 selectively induced afterdepolarization. Combining sub-effective doses of TAK-071, but not T-662, with an acetylcholinesterase inhibitor, significantly ameliorated scopolamine-induced cognitive deficits in rats. TAK-071 may therefore provide therapeutic opportunities for cognitive dysfunction related to cholinergic deficits or reduced M1R expression, while minimizing peripheral cholinergic side effects.


Subject(s)
Cholinergic Agents/pharmacology , Cognitive Dysfunction/drug therapy , Diarrhea/chemically induced , Electrophysiological Phenomena/drug effects , Hippocampus/drug effects , Receptor, Muscarinic M1/drug effects , Animals , Cholinergic Agents/adverse effects , Cognitive Dysfunction/chemically induced , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Muscarinic Antagonists/pharmacology , Rats , Rats, Long-Evans , Rats, Sprague-Dawley , Scopolamine/pharmacology
4.
J Pharmacol Exp Ther ; 364(1): 28-37, 2018 01.
Article in English | MEDLINE | ID: mdl-29025977

ABSTRACT

Activation of muscarinic M1 receptor (M1R) is a promising approach for improving cognitive impairment in Alzheimer's disease. However, an M1R-selective positive allosteric modulator (PAM), benzyl quinolone carboxylic acid (BQCA), at 30 mg/kg, induced diarrhea in wild-type mice, but not in M1R knockout mice. Moreover, BQCA (0.1-1000 nM) augmented electric field stimulation (EFS)-induced ileum contraction in an in vitro Magnus assay. Thus, we decided to establish a drug-screening strategy to discover novel M1 PAMs producing potent cognitive improvement with minimized gastrointestinal (GI) dysfunction. We assessed PAM parameters of various M1 PAMs with ≥100-fold selectivity over other muscarinic receptor subtypes by using in vitro binding and functional analysis. Evaluation of these M1 PAMs in the Magnus assay revealed a significant correlation between percentage of ileum contractions at 1 µM and their α-value, a PAM parameter associated with the binding cooperativity between acetylcholine and M1 PAM. M1 PAMs with lower α-value showed lower impact on EFS-induced ileum contraction. Next, we characterized in vivo profiles of two M1 PAMs: compound A (log α = 1.18) and compound B (log α = 3.30). Compound A, at 30 mg/kg, significantly improved scopolamine-induced cognitive deficits without prominent signs of diarrhea at up to 1000 mg/kg in mice. In contrast, compound B, at 10 mg/kg, showed both significant improvement of scopolamine-induced cognitive deficits and severe diarrhea. Thus, fine adjustment of the α-values could be a key to discovering M1 PAMs yielding potent cognitive improvement with a lower risk of GI effects.


Subject(s)
Cognition Disorders/drug therapy , Diarrhea/prevention & control , Drug Discovery/methods , Ileum/drug effects , Nootropic Agents/therapeutic use , Receptor, Muscarinic M1/agonists , Allosteric Regulation/drug effects , Allosteric Regulation/physiology , Animals , CHO Cells , Cognition Disorders/physiopathology , Cricetinae , Cricetulus , Diarrhea/chemically induced , Diarrhea/physiopathology , Dose-Response Relationship, Drug , Humans , Ileum/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Nootropic Agents/adverse effects , Nootropic Agents/pharmacology , Organ Culture Techniques , Quinolines/adverse effects , Quinolines/pharmacology , Quinolines/therapeutic use , Random Allocation , Receptor, Muscarinic M1/physiology , Structure-Activity Relationship
5.
FEBS J ; 276(8): 2416-29, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19348026

ABSTRACT

CYP175A1 from Thermus thermophilus is a thermophilic cytochrome P450 and has great potential for industrial applications. However, a native electron transport system for CYP175A1 has not been identified. Here, an electron transport system for CYP175A1 was isolated from T. thermophilus HB27 by multistep chromatography, and identified as comprising ferredoxin (Fdx; locus in the genome, TTC1809) and ferredoxin-NAD(P)+reductase (FNR; locus in the genome, TTC0096) by N-terminal amino acid sequence analysis and MALDI-TOF-MS, respectively. Although TTC0096, which encodes the FNR, is annotated as a thioredoxin reductase in the T. thermophilus HB27 genome database, TTC0096 lacks an active-site dithiol/disulfide group, which is required to exchange reducing equivalents with thioredoxin. The FNR reduced ferricyanide, an artificial electron donor, in the presence of NADH and NADPH, but preferred NADPH as a cofactor (Km for NADH = 2440 +/- 546 microM; Km for NADPH = 4.1 +/- 0.2 microM). Furthermore, the FNR reduced cytochrome c in the presence of NADPH and Fdx. The Tm value of the FNR was 99 degrees C at pH 7.4. With an electron transport system consisting of Fdx and FNR, CYP175A1 efficiently catalyzed the hydroxylation of beta-carotene at the 3-position and 3'-position at 65 degrees C, and the Km and Vmax values for beta-carotene hydroxylation were 14.3 +/- 1.6 microM and 18.3 +/- 0.6 nmol beta-cryptoxanthin min(-1) nmol(-1) CYP175A1, respectively. This is the first report of a native electron transport system for CYP175A1.


Subject(s)
Bacterial Proteins/chemistry , Cytochrome P-450 Enzyme System/chemistry , Thermus thermophilus/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Cytochrome P-450 Enzyme System/isolation & purification , Electron Transport , Ferredoxins/chemistry , Ferredoxins/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Molecular Sequence Data , Mutagenesis, Site-Directed , Phylogeny , Sequence Alignment , Temperature , beta Carotene/chemistry , beta Carotene/metabolism
6.
Biochem Biophys Res Commun ; 384(1): 61-5, 2009 Jun 19.
Article in English | MEDLINE | ID: mdl-19389383

ABSTRACT

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates beta-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP(+) reductase (FNR): H(2)N-CYP175A1-Fdx-FNR-COOH (175FR) and H(2)N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V(max) value for beta-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k(m) values of these enzymes were similar. 175RF retained 50% residual activity even at 80 degrees C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.


Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Hot Temperature , Protein Engineering , Recombinant Fusion Proteins/biosynthesis , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/genetics , Enzyme Stability , Hydrogen-Ion Concentration , Protein Structure, Tertiary/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics
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