Download Development of a High-Throughput Screening

Development of a High-Throughput Screening Assay to Identify Inhibitors of the de novo Purine Biosynthetic Pathway
David Whalley , Keith Ansell , Peter Coombs , Craig Southern , Chido Mpamhanga , Michelle Newman , Zaynab Isseljee , Debra Taylor , Andy Merritt ,
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Steve Firestine , Nils Visser and Daniel Peeper
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1. MRC Technology Centre for Therapeutics Discovery, 1-3 Burtonhole Lane, Mill Hill, London, NW7 1AD
2. College of Pharmacy and Health Sciences, Wayne State Univeristy, 42 W. Warren Ave., Detroit, MI, 48202
3. Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX
[email protected]
1. Introduction
3. Assay Development
4. Pilot Screen
We set out to develop a 384 well plate format high-throughput screening assay for the identification of inhibitors
of the synthetase activity. In order to maximise assay sensitivity we chose to detect the accumulation of ADP
rather than Pi generated by the reaction. The objective was to establish conditions for a robust assay amenable
to automation that could be used to screen ~100K small molecules from the MRCT compound collection.
Figure 12: Pilot screen assay procedure (10µl final reaction volume)
Structure & Function
• The de novo purine biosynthetic pathway is composed of 10 enzymes which catalyse the
conversion of phosphoribosyl pyrophosphate (PPRP) to inosine monophsphate (IMP) (Figure
1).
• Human PAICS possesses AIR carboxylase (AIRc) and SAICAR synthetase (SAICARs) activities. The
AIR carboxylase first processes substrates AIR and CO2 to generate CAIR. The SAICAR synthetase then
catalyses the conversion of CAIR, aspartate and ATP to SAICAR, ADP and inorganic phosphate (Pi)
(Figure 2).
• The bifunctional enzyme Phosphoribosylaminoimidazole carboxylase/
phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) catalyses two
consecutive steps (six and seven) of the de novo purine biosynthetic pathway and plays a
pivotal role in the generation of adenine and guanine.
• PAICS may be an attractive target for cancer, since transformed cells depend heavily on the
de novo purine biosynthetic pathway in order to sustain a large nucleotide pool for increased
RNA and DNA expression, and frequently lack the salvage pathway used for nucleotide
synthesis by normal cells.
• PAICS exists as a homo-octamer, with each subunit being composed of distinct AIRc and SAICARs
domains. One octameric AIRc complex and four dimers of SAICARs domains are assembled to give a
compact octamer (Figure 3).
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5μl PAICS (50mM Tris-HCI/EDTA pH 8.0)
Steady-State Kinetic Parameters
The steady state kinetic parameters for CAIR, ATP and aspartic acid were determined, using the Biomol Green™
assay. The kinetic constants for the SAICAR synthetase activity of E. coli-expressed full length human PAICS were
compared to those for the Gallus gallus enzyme3 and are summarised in Table 1.
1μl 100μM compound (1:10
intermediate dilution of 1mM in 100% DMSO)
30 mins pre-incubation at RT
Figure 3: Crystal structure of human PAICS1
2μl CAIR (25mM MgCl +50mM KHCO )
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• In the context of metastatic breast cancer, over-expression of PAICS has been observed in
various human breast cancer cell lines compared to non-tumorogenic mammary epithelial
cells, and in breast cancer patients, higher expression levels are associated with poor
prognosis.
A
With respect to CAIR and ATP the Km values indicate efficient utilisation of the substrates by both human and the
Gallus gallus enzymes. In contrast aspartic acid is utilised more efficiently by the human enzyme as shown by an
8-fold decrease in the Km
B
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1h equilibration at RT
2μl ATP/aspartic acid mix (50mM Tris-HCI/EDTA
pH8.0, 10mM DTT, 0.01% BSA, 0.01% Brij-35)
Optimisation of the Transcreener ADP2 Fluorescence Intensity Assay for High Throughput
Screening of PAICS
Figure 1: The de novo purine biosynthetic pathway in vertebrates
The Transcreener ADP2 Fluorescence Intensity assay from Bellbrook Labs was selected as the primary assay for
the PAICS high throughput screen. This is a homogeneous, competitive displacement fluorescence intensity assay
which uses direct immunodetection of ADP (Figure 9). The Transcreener assay does not reply on the use of
multiple coupling enzymes and can be tuned to produce good signal windows, suitable for HTS, with low percent
conversion of low levels of ATP.
90mins incubation at RT
10μl ADP Detection Reagent
1h incubation at RT
Detect fluorescence Ex. 590nm Em. 617nm
Figure 9: The Transcreener ADP2 FI assay principle
Figure 13: Pilot screen of Sequoia & NINDS libraries and 1K diverse compounds
PAICS
Figure 2: The reaction catalysed by human PAICS
The Transcreener ADP2 FI Assay measures the progress of any enzyme that produces ADP. Displacement of the
tracer by ADP causes an increase in fluorescence at excitation 590nm and emission 617nm. The assay uses a redshifted tracer, which reduces compound interference.
A) Ribbon representation of the overall structure of the PAICS octamer. CO2 binding sites and substrate tunneling system
in the AIRc domain are indicated.
B) Ribbon representation of monomer structure of PAICS. ATP and CAIR are shown bound to the SAICARs domain.
Table 2 shows the statistical outcomes of the pilot screens. High controls are complete reaction mix
with 10% DMSO. Low controls are complete reaction mix with 10% DMSO –PAICS. The pilot screen
statistics show that the automated assay is robust and amenable to HTS.
Figure 10: Determining the optimal incubation time and enzyme concentration for HTS
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2. Biochemical Characterisation of Full-Length Human PAICS
Determination of AIR Carboxylase Activity
The enzyme catalyzed equilibrium between AIR and CAIR lies in favour of the reverse, non-physiological direction
(CAIR → AIR). To determine the carboxylase function of PAICS, a UV absorbance assay was utilised to measure
the PAICS-mediated decarboxylation of CAIR (Figure 4).
Effect of MgCl2 on AIR/CAIR Equilibrium
The AIR/CAIR equilibrium may be shifted in favour of the forward direction by the introduction of appropriate
concentrations of MgCl2 and/or KHCO3. The presence of MgCl2 causes the specific activity of the decarboxylation reaction
to decrease due to the formation of a CAIR-MgCl2 complex which is not a substrate for the PAICS AIR carboxylase.
Figure 4: The spectral changes
for the conversion of CAIR
to AIR allow for continuous
monitoring at 260nm. A time-
Figure 5: Determination
of AIR/CAIR equilibration
conditions.
B
The change in the OD at 260nm
for 300μM CAIR and 2nM PAICS
at various MgCl2 and KHCO3
concentrations was monitored over
4h. The optimal MgCl2 and KHCO3
concentrations were found to be
25mM and 50mM, respectively. AIR/
CAIR equilibrium was achieved after
~1h under these conditions.
dependent decrease in the optical
density at 260nm was observed
which can be attributed to the CAIR
decarboxylase activity of PAICS. Each
line represents a 1min increment.
The correlation between two separate screens of known drugs (Sequoia and NINDS libraries) and
1000 diverse small molecules was determined. There was good agreement between the two runs
with 2.2% of the compounds screened giving percent residual enzyme activity values 3 standard
deviations below the high control mean (80%). 84% of hits below the 3SD mean for run 1 were
confirmed in run 2. These screen hits spanned a broad range of potency. A number of these
compounds were known frequent hitters so were disregarded. The compounds highlighted in green
were selected for concentration response analysis.
Figure 14: Concentration response analysis of pilot screen hits
Determination of SAICAR Synthetase Activity
As the AIR carboxylase-catalysed reaction for CAIR to AIR is faster than the ATP- and aspartic acid-dependent conversion of CAIR to SAICAR, the CAIR to AIR reaction was allowed to reach equilibrium in the presence of 25mM MgCl2 and
50mM KHCO3 for 1h prior to reaction initiation by the addition of ATP and aspartic acid.
Figure 6: The AIR/CAIR equilibrium ratio
A
A) The spectrum for 300µM CAIR prior
to reaction initiation by the addition
of 2nM PAICS with 25mM MgCl2 and
50mM KHCO3. Each line represents a
1min increment.
B
B) The spectrum for 300µM CAIR after reaction initiation
and 1h equilibration. A halving in the OD indicates a
reduction in the [CAIR] by 50%. Prior to equilibration 50%
of the initial CAIR concentration is decarboxylated to AIR.
The AIR/CAIR equilibrium ratio is 2.0. Each line represents
a 1min increment.
A) The change in fluorescence at various timepoints when PAICS is titrated. The enzyme reactions were run at
the Km for ATP and aspartic acid and 5µM equilibrated CAIR. A PAICS concentration 2.5nM or below gave a linear
response over 2h.
B) PAICS titration after 2h incubation. The area between the two solid red lines represents 3%-20% ADP conversion
which is ideal for primary screening with the Transcreener assay. The EC50 value of 2.5nM PAICS, indicated by the
dotted line, was selected for HTS.
Figure 11: Relationship between the conversion of ATP and CAIR
Detection of Inorganic Phosphate
To determine the synthetase function of PAICS a malachite green-based assay was employed to measure free inorganic phosphate (Pi) generated by the ATP-dependent aspartylation of CAIR. Biomol Green™ from Enzo Life Sciences is based
on the classic cationic malachite green dye. This was then compared to a kinetic fluorescence intensity assay for the detection of inorganic phosphate in real time (Figure 7).
Figure 7: Rhodamine phosphate biosensor (RH-PBP)
A
B
Figure 8: SAICAR synthetase- and time-dependent inorganic phosphate accumulation
The amount of ATP and CAIR converted by 2.5nM PAICS over 2h under optimised conditions was calculated. The
graph shows that a total 31pmol (10%) ATP and 28pmol (56%) AIR/CAIR was converted after 2h. This demonstrates
a 1:1 molar ratio between CAIR and ATP and is consistent with observations reported in the literature3.
A. Biomol Green™ Assay
Figure 14 shows examples of dose-response curves for three of the pilot screen hits generated using
the EC50 concentration of PAICS with all substrates at Km.
All 14 selected hits from the pilot screen, apart from one, displayed a dose response relationship.
Of the 13 confirmed hits, 3 displayed solubility issues and an accurate IC50 could not be determined
and 3 gave a dose response relationship in the technology interference counterscreen so were
disregarded.
The 9 remaining compounds gave IC50s spanning a broad range of potency and the rank order of
B. RH-PBP Assay
5. Summary
A) Crystal structure of RH-PBP. The phosphate biosensor is a rhodamine-labelled Phosphate Binding Protein (RHPBP), developed by M. Webb at the MRC National Institute for Medical Research, UK. The RH-PBP is labelled at
specific sites with two rhodamine moieties, which are stacked in the apo conformation. On binding Pi the moieties
move apart, giving an increase in fluorescence2.
B) Fluorescence excitation and emission spectra for RH-PBP. Excitation and emission wavelengths are 555nm and
575nm, respectively. The phosphate biosensor facilitates the kinetic characterisation of Pi-generating proteins.
A) The change in the OD at 620nm at various timepoints after the addition of 5mM aspartic acid and 50µM ATP to
20nM PAICS and 150µM CAIR post-equilibration in the Biomol Green™ assay. A time-dependent increase in OD620
was observed, which can be attributed to release of Pi by the SAICAR synthetase activity of PAICS.
B) The SAICAR synthetase activity of PAICS was confirmed using the RH-PBP biosensor to detect Pi. After the addition
of 5mM aspartic acid and 50µM ATP to 20nM PAICS and 150µM CAIR post-equilibration a time-dependent linear
increase in fluorescence was observed in real time.
• PAICS is a key enzyme in the de novo purine biosynthesis pathway that could represent an attractive target for cancer.
• We have biochemically characterised full-length human PAICS using a “toolbox” of various assays in order to derive conditions suitable for high-throughput screening.
• A robust, homogeneous fluorescence intensity competitive displacement assay has been developed for the SAICAR synthetase (SAICARs) activity of PAICS based on detection of ADP
produced by the reaction.
• A pilot screen of 2,400 compounds consisting of known drugs and ~1000 diverse compounds has be used to validate the assay and has yielded 14 compounds for initial follow up.
• 93% of the selected pilot screen single point hits gave a dose-response relationship and IC50s spanning a broad range of potencies in follow-up studies
• We plan to carry out a full in-house screening campaign of 100K compounds from the MRCT Diversity Set in combination with in silico and fragment screening approaches to identify
further chemical starting points for lead generation.
References
1. Li, Shu-Xing; Tong, Yong-Ping; Xie, Xiao-Cong; Wang, Qi-Hai; Zhou, Hui-Na; Han, Yi; Zhang, Zhan-Yu; Gao, Wei; Li, Sheng-Guang; Zhang, Xuejun C.; Bi, Ru-Chang, Journal of Molecular Biology 2007, 366, 1603-1614
2. Okoh, M. P.; Hunter, J. L.; Corrie, J. E. T.; Webb, M. R. Biochemistry 2006, 45, 14764-14771
3. Firestine, S. M.; Davisson, V. J. Biochemistry 1994, 33, 11917-11926