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240 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Johnston et al.
We conducted a pilot screening assay in 96-well plates using baculovirus-infected
Sf9 cells to investigate the applicability of the M2-mAChR assay for high-
throughput screening. The screen was performed on the Library of
Pharmacologically Active Compounds (LOPAC; Sigma-Aldrich), with
compounds applied at a final concentration of 1 μM. Twelve documented
cholinergic agonists were detected with responses greater than 50% of the signal
observed with a saturating dose (10 nM) of oxotremorine. Representative
examples including the agonists arecoline, dioxolane, carbachol and the
antagonist atropine are annotated in Fig. (9). The Z-factor of the assay was
calculated as 0.62, indicative of a robust and sensitive screening assay [58]. The
compound TMB-8 (3,4,5-trimethoxybenzoic acid 8-[diethylamino]octyl ester)
was also identified as an M2-mAChR agonist in our pilot screen. The peptide-
detection assay was then used to directly compare TMB-8 and carbachol for M2-
mAChR activation (Fig. 10A). In terms of G-protein agonism, the two compounds
Fig. (9). A pilot, peptide-based screening assay for activators of the M2-muscarinic acetylcholine
receptor (M2-mAChR). Intact Sf9 cells were used as the source membrane material. Sf9 cells
infected with baculoviruses encoding the M2-mAChR receptor, G
i1
, and G
1
2
subunits were
washed with phosphate-buffered saline and applied to DNA-Bind 96-well plates at a density of
500,000 cells per well. Compounds from the LOPAC library (Sigma-Aldrich) were applied at a
final concentration of 1 μM. Compound additions and washing steps were performed with an
automated liquid handling device (Tecan, Durham, NC). Results are depicted graphically, with a
‘hit’ being defined as a response greater than 50% of the signal observed with a saturating dose of
oxotremorine (10 nM).
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G-Protein Activation Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 241
Fig. (10). Peptide-based measurements of M2-mAChR activation confirming agonist activity of
TMB-8. (A) TMB-8 is an equipotent partial agonist of the M2-mAChR as compared to carbachol.
(B) Dose-dependent atropine antagonism of carbachol- and TMB-8-agonist activity (10 μM of
either agonist). Assays were performed essentially as described in Fig. (8A). (C) Nucleotid e (an d
concentration) dependence of the peptide-based measurement of agonist activity on the M2-
mAChR by carbachol and TMB-8.
242 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Johnston et al.
were similar in potency, while the traditional agonist carabachol had a two-fold
greater efficacy. Antagonist competition experiments were consistent with this
data (Fig. 10B). As a positive control, we examined the guanine nucleotide
selectivity of the assay and demonstrated that G-protein activation could only be
detected in the presence of micromolar GTPS but not GDP (Fig. 10C). TMB-8
has previously been characterized as a muscarinic antagonist [59] and an allosteric
modulator of the M2 muscarinic acetylcholine receptor [60]. TMB-8 slows the
rate of binding and the dissociation of pre-bound acetylcholine from the M2
muscarinic receptor [61]. Our data are interesting in that they demonstrate that
TMB-8 has canonical agonist-like properties at the M2-mAChR with regard to G-
protein activation (Fig. 10A). However, we have not conducted the mechanistic
studies necessary to confirm or refute TMB-8 as an allosteric modulator of the
M2-mAChR. In this regard, it must be noted that TMB-8 has multiple
pharmacologic effects and targets, having been originally characterized as a
calcium anatagonist [62,63]. Therefore our discovery via this pilot, peptide-based
GPCR screen that TMB-8 is an agonist at the M2 muscarinic acetylcholine
receptor is a novel pharmacological finding.
CONCLUDING REMARKS
G-protein subunit binding peptides, identified by various screening methods, have
presented themselves as powerful research tools for studying target proteins
within vital signal transduction pathways. As described herein, these peptides
have numerous applications including:
(1) Serving as direct modulators of target protein activity.
(2) Providing structural insights into previously undefined mechanisms of target
protein function.
(3) Supplying a template for designing biosensors capable of reporting the
spatiotemporal dynamics of target protein signaling.
(4) Presenting a model for screening small molecule libraries to identify
inhibitors of target G-protein subunit activity and,
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G-Protein Activation Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 243
(5) Serving as the starting material for novel, non-radioactive assays for
measuring agonist and antagonist activity on GPCR/heterotrimer
complexes.
Recently, screening methods including phage-display and mRNA-display
techniques have successfully identified such peptides targeting both G and G
subunits [12,16,21,23,35,39,40]. These peptides have already given valuable
insights into various aspects of G-protein signaling and have been elegantly
applied in various scenarios just described. Continued efforts to identify novel
peptide sequences to additional G-protein subfamilies should afford further
discoveries into heterotrimer nucleotide cycling as well as future assay platforms
for receptor-mediated G-protein signaling.
ABBREVIATIONS
GDI = Guanine nucleotide dissociation inhibitor
GDP = Guanosine diphosphate
GEF = Guanine nucleotide exchange factor
GPCR = G protein-coupled receptor
GTP = Guanosine triphosphate
KB = Karo Bio
ACKNOWLEDGEMENTS
Work described in this chapter was funded in part by grants R01 GM074268 (to
D.P.S.) and F32 GM076944 (to C.A.J.) from the U.S. National Institute of
General Medical Sciences.
CONFLICT OF INTEREST
The authors state that there is no conflict of interest.
244 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Johnston et al.
DISCLOSURE
Primary data, its analysis, and literature surveys described in this chapter were
updated from a previously published article in Combinatorial Chemistry & High
Throughput Screening, Volume 11, Issue 5, 2008, pp. 370 to 381.
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248 Advances in Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 1, 248-258
Rathnam Chaguturu (Ed.)
All rights reserved-© 2013 Bentham Science Publishers
INDEX
A
Acceptor fluorophore 62-3
Accumulation partial order ranking (APOR) 3, 5, 12, 27, 30
Acetylcholine receptor 239-40
Activated T-cells response element 94
Activating heterotrimeric G-proteins 220
Activation-state-selective peptides 231
Activation-state-selective phage peptides 238
Adenosine receptors 116-17
Adenylyl cyclase (AC) 87, 90, 108, 114, 220, 224, 233
Aequorin assays 90
Agnus-castus 168, 173, 176
Alkaline phosphatase (AP) 94, 147, 161, 165, 173-4, 177
Alkaline phosphatase assays 168-9, 173
Allosteric modulator 242
ALP assays 168, 174, 177-8
Aluminum fluoride (AlF4) 108, 198-200, 224, 227-8, 230-2, 234-5
7-Amino-trifluoromethyl coumarin (AFC) 75
Amplified luminescent proximity homogeneous assay 90
Angelica sinensis 162-3, 169, 176-7
Antagonist alkaline phosphatase assay 177
Antagonist ALP assay 178
Antagonist assays 174, 177
Antagonist atropine 238-40
Anti-cAMP antibody 68, 70, 90
Antibody-based assays 144
Antibody engineering 130, 133-4
Antiestrogenic activity 159, 165, 168, 171, 176-7
Antiestrogenic ALP assay 178
Antiestrogenic assay 171
Antiestrogenic Ishikawa assay 177
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Index Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 249
Applied DMR assays 106
Arbitrary Compounds 29
Arginine residue 188-9
Arrestin recruitment assays 93
Asclepias tuberosa 162-3, 169
Assayable apoptosis markers 75
Assaying heterotrimeric G-protein activation 237
Atom-centered fragments (ACF) 39
B
Bacterial IgG-binding receptors 142
Bacterial receptor 135, 143
Baculoviruses 239
BDP2 17, 23
Beta-arrestin 72
Beta-galactosidase 66, 71-2, 76
Beta-galactosidase enzyme 70, 72
Beta-lactamase 66, 71, 76
Beta vulgaris 162-3, 169
Bi-polar 67
Binding calcium 67, 69
Binding peptides 136, 223, 231, 233, 236-9
Binding polypeptide sequence 187
Bio-inspired mimic compounds 130
Biological ligands 142
Biological targets 36-7, 40, 42, 46, 48, 133
Bioluminescence resonance energy transfer (BRET) 71, 91
Biomimetic ligands 141-2
Biosensor assays 228
Biosensors 95-8, 100, 103, 109, 195, 219, 225, 236
Biotinylated KB-series peptides 227-8
Bovine serum 109, 165
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