Development of a High-Throughput Screening Assay to

Identify Inhibitors of the de novo Purine Biosynthetic

Pathway

David Whalley1, Keith Ansell1, Peter Coombs1, Craig Southern1, Chido Mpamhanga1, Michelle Newman1, Zaynab Isseljee1, Debra Taylor1, Andy Merritt1,Steve Firestine2, Nils Visser3 and Daniel Peeper3

1. MRC Technology Centre for Therapeutics Discovery,

2. College of Pharmacy and Health Sciences, Wayne State Univeristy

3. Netherlands Cancer Institute

Derived from SLAS2013 Poster

Introduction

• The de novo purine biosynthetic pathway has been studied

extensively for over 50 years and in vertebrates is composed of nine enzymes which catalyse the conversion of phosphoribosyl pyrophosphate (PPRP) to inosine monophsphate (IMP).

• 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 has been suggested to be an attractive drug target for cancer since rapidly dividing

cancer cells may be more dependent on de novo purine biosynthesis than normal cells.

Moreover, PAICS expression has been associated with several malignancies.

Assay Development

• We set out to develop a 384 well plate format high throughput screening assay for identification of inhibitors of the synthetase activity based on the detection of ADP 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.

Steady-State Kinetic Parameters

To establish balanced assay conditions for high throughput screening, 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.

Table 1: Steady-State Kinetic Parameters for SAICAR Synthetase

Substrate Km (human) Km (Gallus gallus)

CAIR 3.2µM 3µM ATP 30µM 10µM

Aspartic Acid 180µM 1.4mM

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

Optimisation of the Transcreener ADP2

Fluorescence Intensity Assay

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. 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.

The assay can be read

in the kinetic or end-

point mode. An

increase in signal is

observed with ADP

production.

Optimal incubation time for HTS

Change in fluorescence at various timepoints when PAICS is titrated. The enzyme reactions were run at the Km for ATP and aspartic acid of 30µM and 180µM, respectively, and 5µM equilibrated CAIR. PAICS concentrations 2.5nM and below gave the most linear response over 2h, indicated by the dotted line.

Optimal [Enzyme] For HTS

PAICS titration after 2h incubation. The area between the two solid red lines represents 3%-20% ADP conversion which is the recommended concentration of enzyme to generate acceptable Z’ for primary screening. The EC50 value of 2.5nM PAICS, indicated by the dotted line, was selected for HTS.

Transcreener HTS assays are optimized

to detect product formed under initial

enzyme velocity conditions.

Relationship between the conversion

of ATP and CAIR

Correlation between assay methods. The amount of ATP and CAIR converted by 2.5nM PAICS after 38 minutes, 1h and 2h was calculated. The enzyme reactions were run at the Km for ATP and aspartic acid of 30µM and 180µM, respectively, and 5µM equilibrated CAIR. The graph shows that a total 31pmol (10%) ATP was converted and 28pmol (56%) AIR/CAIR was consumed after 2h. This demonstrates a 1:1 molar ratio between CAIR and ATP and is consistent with observations reported in the literature, using the absorbance technique.

Pilot Screen: Assay Procedure & Stats

Table 2: The statistical outcomes of the pilot screen (mean values)

Z’ S:B High

Controls

High Control

%CV

Low Controls

Low Controls

%CV Screen 1 0.69 3.0 48343 4.4 16027 7.3 Screen 2 0.78 4.9 46678 4.5 9465 6.4

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.

The pilot screen statistics show that the automated assay is robust and amenable to HTS.

Good correlation between 2 Screens

Pilot screen of Sequoia & NINDS libraries and 1K diverse compounds (2400 cmpds). There was a good correlation 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%). 71% 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 light blue were selected for concentration response analysis

Dose-response curves for 3 HTS hits

IC50 Determinations- Of the 14 primary hits, 9 compounds demonstrated IC50 values spanning a broad range of potency, and the rank order of potency of these compounds was in alignment with their respective single point results at 10µM. The dose-dependency curves were generated with the EC50

concentration of PAICS with all substrates at Km.

Summary 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