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180 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Overk and Bolton
ER = Estrogen receptor
ERE = Estrogen response element
ERE-luc = ERE-luciferase
FBS = Fetal bovine serum
HRT = Hormone replacement therapy
HAPs = Hydroxyapatite slurry
NAPRALERT = Natural Products Alert
NEAAs = Non-essential amino acids
PE = Petroleum ether
SERMs = Selective estrogen receptor modulators
SSRIs = Selective serotonin reuptake inhibitors
ACKNOWLEDGEMENTS
This work was supported, in part, by grant P50 AT00155 provided jointly by the
National Center for Complementary and Alternative Medicine (NCCAM), the
Office of Dietary Supplements (ODS), the Office for Research on Women’s
Health (ORWH), and the National Institute of General Medicine NIGMS) of the
National Institutes of Health (NIH). C.R.O. is grateful for a Ruther L. Kirschstein
NCCAM Predoctoral fellowship F31 AT 24232. The contents of this paper are
solely the responsibility of the authors and do not necessarily represent the official
views of NIH.
CONFLICT OF INTEREST
The authors state that there is no conflict of interest.
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Botanical Extracts Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 181
DISCLOSURE
This chapter has been updated from its original publication: Overk, C. R., Yao, P.,
Chen, S., Deng, S., Imai, A., Main, M., Schinkovitz, A., Farnsworth, N. R., Pauli,
G. F., and Bolton, J. L. (2008) High-content screening and mechanism-based
evaluation of estrogenic botanical extracts. Comb. Chem. High Throughput
Screen. 11, 283-293.
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Advances in Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 1, 187-218 187
CHAPTER 7
A Homogeneous Platform to Discover Inhibitors of the GoLoco
Motif/G-alpha Interaction
Adam J. Kimple
1
, Adam Yasgar
2
, Mark Hughes
3
, Ajit Jadhav
2
,
Francis S. Willard
1,§
, Robin E . Muller
1
, Christopher P. Austin
2
, James Inglese
2
,
Gordon C. Ibeanu
3
, David P. Siderovski
1
and Anton Simeonov
*,2
1
Departm en t of Pharmacology , Lineberger Compreh ensi ve Cancer Center, and UNC
Neuroscience Cen ter , The Univ ers ity of Nor th Car olin a at Chape l Hill , Chapel Hi l l, NC
27599- 73 65 , USA;
2
NIH Chemical Genomics Center , Nat io na l Center for Adva nc ing
Translat io na l Scienc es, National Institu tes of Health, Bethesda , MD 20892-3370 , USA;
3
BRITE Ins ti tute , N ort h Car ol in a Central Un iver s it y , Durh am , NC 27 70 7, US A
Abstract: The GoLoco motif is a short G-binding polypeptide sequence. It is often found
in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in
proteins that regulate mitotic spindle orientation and force generation during cell division,
such as GPSM2/LGN. Here, we describe a high-throughput fluorescence polarization (FP)
assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the
GoLoco motif i nte racti on with the G-protein alpha subunit G
i1
. The assay exhibits
considerable stability over time and is tolerant to DMSO up to 5%. The Z´-factors for
robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were
determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate
format and produced a Z´-factor of 0.80. To determine the screening factor window (Z-
factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity
Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay
would perform well when developed for 1,536-well format and scaled up to larger libraries.
We then miniaturized to a 4 μL final volume a pair of FP assays utilizing fluorescein-
(green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated
run, the Sigma-Aldrich LOPAC
1280
collection was screened three times with every library
compound being tested over a range of concent rati ons following the quantitative high-
throughput screening (qHTS) paradigm; excellent assay performance was noted with
average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
Keywords: Fluorescence anisotropy, fluorescence polarization, GoLoco motif,
heterotrimeric G-proteins, high-throughput screening.
*Address correspondence to Anton Simeonov: NIH Chemical Genomics Center, National Center for
Advancing Translational Sciences, 9800 Medical Center Drive, MSC 3370, Bethesda, MD 20892-3370 USA.
Tel: 1-301-217-5721; Email: asimeono@mail.nih.gov
§
Present address: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
188 Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 Kimple et al.
INTRODUCTION
Many extracellular signals, including hormones, neurotransmitters, growth
factors, and sensory stimuli relay information intracellularly by activation of
plasma membrane-bound receptors. The largest class of such receptors is the
superfamily of seven transmembrane-domain G protein-coupled receptors
(GPCRs), so named because these cell-surface proteins were originally found to
couple extracellular stimuli into intracellular changes via activation of G-pr otein
heterotrimers (G) [1]. GPCRs represent a major therapeutic target giving rise
to the largest single fraction of the prescription drug market with annual sal es of
several billion dollars [2]; however, opportunities to develop therapeutics that
target the intracellular regulatory machinery controlling the kinetics and duration
of GPCR signal transduction have been relatively ignored by comparison.
A diverse family of G-interacting proteins has been shown to share a common
GoLoco (“G
i/o
-Loco” interaction) motif (Fig. 1) (reviewed in [3, 4]). GoLoco
motif-containing proteins generally bind to GDP-bound G subunits of the Gi
(adenylyl-cyclase inhibitory) class and act as GDP dissociation inhibitors (GDIs),
slowing the spontaneous exchange of GDP for GTP and preventing re-association
with G subunits [5-13]. Determination of the crystallographic structure of
G
i1
·GDP in complex with the GoLoco motif of RGS14 [7] revealed critical
determinants of G subunit specificity and GDI activity. The N-terminal alpha-
helix of the GoLoco motif peptide binds between switch II and the 3 helix of the
G
i1
Ras-like domain (Fig. 1B), grossly deforming the normal site of G
interaction [7]. The aspartate-glutamine-arginine triad, which defines the final
residues of the highly-conserved 19 amino-acid GoLoco motif signature (Fig.
1A), orients the arginine residue into the guanine nucleotide-binding pocket of
G, allowing contacts to be made between its basic -guanido group and the -
and -phosphates of GDP [7]. Mutation of this single arginine residue within the
Asp-Gln-Arg triad causes a loss of GDI activity [7, 8, 11].
A well-characterized physiological function of GoLoco motif proteins is in the
regulation of asymmetric cell division in worm, fruit fly, and mammalian
development (reviewed in [4, 14]). For example, GPSM2 (a quadruple GoLoco
motif-containing protein, previously known as LGN) binds to nuclear mitotic
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GoLoco Motif/G-alpha Advances in Combinatorial Chemistry & High Throughput Screening, Vol. 1 189
apparatus protein (NuMA) and regulates mitotic spindle assembly; altering
endogenous cellular levels of GPSM2, either via overexpression or RNA
interference-mediated knockdown, leads to aberrant chromosomal segregation
during mitosis [15]. Similar functions have also been ascribed to Dr osophila and
C. elegans homologs of GPSM2 (Pins and GPR-1/-2, respectively; refs. [16-20]).
Fig. (1). The GoLoco motif is a G
i
GDP -interacting po lypeptide found singly or in a rrays
in various proteins. (A) Domain architecture of representative GoLoco motif proteins and a
sequence alignment of the conserved core of the RGS12 and RGS14 GoLoco motifs. Domain
abbreviations: GPSM, G-protein signaling modulator; PDZ, PSD-95/Discs large/ZO-1 homology;
PTB, phosphotyrosine-binding domain; RGS, regulator of G-protein signaling box; RBD, Ras-
binding domain; RapGAP, Rap-specific GTPase-activating protein domain. (B) The crystal
structure of G
i1
(Ras-lik e domain in blue, all -h elic al do main in green, switch regions in cyan)
bound to the GoLoco motif of RGS14 (PDB ID 2OM2). The GoLoco motif peptide (tan) binds
across the Ras-like and all-helical domains of G
i1
, trapping GDP (magenta, with - and -
phosphates in orange) within its binding site. The bound magnesium ion is illustrated in lime
green.
Evidence is also emerging that GoLoco motif-containing proteins act as critical
components of cell-surface receptor-mediated signal transduction pathways.
GPSM2 over-expression has been found to affect both basal and GPCR-activated
potassium currents from GIRK channels [21], the latter effect similar to what we
previously observed via cellular microinjection of GoLoco motif peptides [22].
We have recently shown RGS12 to be a receptor-selective scaffold for
components of the mitogen-activated protein kinase (MAPK) cascade [23]. RNA
interference-mediated knockdown of RGS12 protein levels in primary mouse
dorsal root ganglion neurons blunts nerve growth factor-stimulated axonogenesis
[23]. Mutating the arginine residue within the Asp-Gln-Arg triad of the RGS12
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