Methyltransferases form a large class of enzymes, most of which use
S-adenosylmethionine as the methyl donor. In fact,
S-adenosylmethionine is second only to ATP in the variety of reactions for which ...it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of
S-adenosylmethionine to
S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol
O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.
The enzyme γ-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of γ-secretase) and signal peptide peptidase (SPP) are ...related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and γ-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid β-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, γ-secretase inhibitor radioligands were used to evaluate SPP and γ-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for γ-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of γ-secretase binding sites, making it essential to use selective radioligands for evaluation of γ-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family.
Several tetrahydroimidazopyrimidines were prepared using silver assisted cyclization as the key step. The binding affinities of compounds thus prepared were evaluated in vitro toward hCRF
1R. Initial ...lead compound
16 (
K
i
=
32
nM) demonstrated modest putative anxiolytic effects in the mouse canopy test. Further optimization using parallel synthesis provided compounds with
K
i’s <50
nM.
Several tetrahydroimidazopyrimidines were prepared using silver assisted cyclization as the key step. The binding affinities of compounds thus prepared were evaluated in vitro toward hCRF
1R. Initial lead compound
16 (
K
i
=
32
nM) demonstrated modest putative anxiolytic effects in the mouse canopy test. Further optimization using parallel synthesis provided compounds with
K
i’s <50
nM.
The synthesis and SAR of constrained dihydroimidazoimidazoles, and behavioral activity of compounds
7b (
K
i 42
nM) and
7k (
K
i 41
nM) in a mouse canopy model of anxiety, are reported.
...7-Aryl-6,7-dihydroimidazoimidazoles represent a novel series of high-affinity corticotropin-releasing factor 1 receptor antagonists. Here, we report their synthesis and SAR as well as behavioral activity of two exemplary compounds,
7b and
7k, in a mouse canopy model of anxiety.
The synthesis and SAR of tetraazacyclopenta
aindenes, pharmacokinetic properties and the anxiolytic activity of an orally dosed exemplary compound
9d (Ki 8.0
nM) are reported.
...8-Aryl-1,3a,7,8-tetraaza-cyclopenta
aindenes represent a novel series of high-affinity corticotropin-releasing factor-1 receptor (CRF1R) antagonists. Herein we report the synthesis and SAR around the tricyclic core and the anxiolytic activity of an orally dosed exemplary compound
9d (
K
i
=
8.0
nM) in a mouse canopy model.
2-Arylamino-4-trifluoromethyl-5-aminomethylthiazoles represent a novel series of high-affinity corticotropin releasing factor-1 receptor (CRF
1R) antagonists that are prepared in three steps in good ...overall yields. Herein, we report binding SAR as well as anxiolytic activity of an exemplary compound (
7a,
K
i=8.6 nM) in a mouse canopy model.
Graphic
Several tetrahydroimidazopyrimidines were prepared using silver assisted cyclization as the key step. The binding affinities of compounds thus prepared were evaluated in vitro toward hCRF sub(1)R. ...Initial lead compound 16 (K sub(i) = 32 nM) demonstrated modest putative anxiolytic effects in the mouse canopy test. Further optimization using parallel synthesis provided compounds with K sub(i)'s <50 nM. AB:
The enzyme γ-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic
subunit of γ-secretase) and signal peptide peptidase (SPP) are ...related transmembrane aspartyl proteases that cleave transmembrane
substrates. SPP and γ-secretase are pharmacologically similar in that they are targeted by many of the same small molecules,
including transition state analogs, non-transition state inhibitors, and amyloid β-peptide modulators. One difference between
presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP
requires no additional protein cofactors for activity. In this study, γ-secretase inhibitor radioligands were used to evaluate
SPP and γ-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition
state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to
those reported previously for γ-secretase. In the course of this study, cultured cells were found to contain an abundance
of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was
in excess of γ-secretase binding sites, making it essential to use selective radioligands for evaluation of γ-secretase binding
under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms
and structure in the SPP/presenilin protein family.
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