Development of a Novel PDE10A Based Assay Jianming Lu, Louie Zhang, Kiho Han and Wenshan Hao Codex BioSolutions, Inc. 401 Professional Drive, Suite 160 Gaithersburg, MD 20879, USA Abstract Fig. 1 and ACTOne Biosensor Fig. 4 Endogenous PDE4B/4D Knock Down with shRNA The cyclic nucleotide (PDEs) are proteins that catalyze hydrolysis B of 3', 5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic A C PDE10A-PDE4B/4D KD guanosine monophosphate (cGMP), to their corresponding 5'-nucleotide monophos- 140 phates. These play important roles in controlling cellular concentrations of cy- T = 30 min 120 PDE10A-4B/4D KD (no Ro) PDE10A with Ro clic nucleotides and have central roles in a variety of intracellular signaling events. PDE10A without Ro Currently, most PDE assays are performed biochemically using pure substrates (cAMP 100 or cGMP) and purified recombinant PDE enzymes. There are no commercially available 80 cell-based screening assays for PDE inhibitors. As with other biochemical screenings on RFU 60 intracellular targets, false positives and false negatives may result from issues involving 40 cell membrane permeability, cellular metabolism, and the like. Cell-based assays would 20 provide better assessment of compound activity in a more physiological context. 0

Here we report the development of a novel PDE10A cell-based assay. The assay was 0.1 1 10 100 1000 10000 100000 based on the ACTOne™ technology which utilizes a modified cyclic nucleotide-gated PF-2545920 (nM) channel (CNG) to monitor intracellular cAMP/cGMP changes in live cells. The influx of (A) ACTOne PDE10A cells with endogenous PDE4B/4D knock down or ACTOne PDE10A cells (parental) were plated cations through the CNG channel can be quantified using ACTOne membrane potential overnight in 20 μl culture medium on a 384-well plate. The next day, cells were loaded with 20 μl/well of ACTOne (MP) dye. On that, we developed a PDE10A cell line by over-expressing the in the Membrane Potential Dye and incubated at room temperature for 2hr. Different concentrations of PF-2545920 solutions system. We obtained very specific response from a specific PDE10A inhibitor, PF- were prepared in the presence or absence of 10 M Ro 20-1724 and added into each well. The data was recorded on FlexStation 30 min after the compound addition. 2545920. To further improve the system, we knocked down the endogenous PDE4 ac- Total RNAs from PDE4B/4D shRNA knock down cells and the ACTOne PDE10A cells were extracted with Qiagen tivities by introducing shRNAs against PDE4D and PDE4B. With this improved system, RNeasy Mini Kit. RT-PCRs were performed with Invitrogen’s Super Script™ One Step RT-PCR with Platinum® Taq we screened two libraries: 1) LOPAC Library from Sigma; 2) a selective library from System. (B) RT-PCR with PDE4B specific primers. (C) RT-PCR with PDE4D specific primers. 1) 1kb DNA ladder; 2) Chembridge. Afterwards, two novel PDE10A specific inhibitors have been discovered Negative control; 3) ACTOne-PDE10A; 4) PDE10A-4B/4D knock down. and confirmed. Fig. 5 Assay Conditions for Compound Screening on Introduction Fig. 2 Kinetic Curves of ACTOne PDE4 Cells in PDE10A Cell Line with PDE4B/4D Knock Down PDEs hydrolyze 3', 5'-cyclic nucleotides including cAMP and cGMP, to their corresponding A B 5'-nucleotide monophosphates AMP and GMP. Both cAMP and cGMP are important sec- Response to Ro20-1724 (cAMP Change) ond messengers that mediate the responses of a variety of and neurotransmit- P F c o m p o u n d a t 4 7 0 n M , 6 0 -m in tr e a tm e n t P F c o m p o u n d a t 9 4 0 n M , 6 0 -m in tr e a tm e n t ters. PDEs are responsible for terminating cellular responses to hormones and neuro- 3 .5 3 .5 3 .0 3 .0 transmitters, which is critical for maintaining proper intracellular signaling events. There

2 .5 2 .5 o are 11 families of PDEs from 21 different [1,2]. Each PDE family is distinguished o 2 .0

2 .0 i

i

t

t

a

a R functionally by unique enzymatic characteristics and pharmacological profile as well as R 1 .5 1 .5 distinct tissue distribution and cellular expression patterns [3,4]. Because PDEs regulate a 1 .0 1 .0 variety of cellular functions, they have become important drug targets for the treatment of 0 .5 0 .5 several diseases including sexual dysfunction, asthma, chronic obstructive pulmonary dis- 0 .0 0 .0 s s s s s s s s s s s s ll ll ll ll ll ll ll ll ll ll ll ll e e e e e e e e e e e e ease, neurodegenerative diseases (Parkinson’s disease and Alzheimer’s), diabetes, vas- c c c c c c c c c c c c 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 5 0 5 0 1 2 5 0 5 0 cular diseases, osteoporosis cancer and rheumatoid arthritis [2,5,6]. 1 1 2 1 1 2 PDE10A has been compellingly validated for . Remarkably, high levels of PDE10A mRNA and protein are expressed specifically in the medium spiny projection C D neurons of the mammalian striatum [7]. The striatum is the largest component of the basal P F c o m p o u n d a t 4 7 0 n M 1 0 0 0 0 c e ll/w e ll P F c o m p o u n d a t 9 4 0 n M 1 0 0 0 0 c e ll/w e ll ganglia circuit that regulates motor and cognitive functions. Dysfunctions in the basal gan- 3 .5 4 glia circuits have been linked to various CNS disorders, including schizophrenia [8]. Thus, 3 .0

the specific expression of PDE10A in these neurons raises hopes that PDE10A-targeted 3

o

o

i i drugs could potentially treat schizophrenia with improved efficacy and better tolerability. t

2 .5 t a

Kinetic response of ACTOne cells optimized for PDE4 assay to Ro20-1724 using ACTOne Membrane a R Administration of potent and selective inhibitors to rats increase both cGMP and cAMP R Potential Dye Kit. ACTOne cells optimized for PDE4 assay were plated overnight in 100 μl culture me- 2 levels in the striatum [9]. As a result, phosphorylation of the downstream effectors CREB dium on a 96 well Biocoat poly-D lysine coated plate. The next day, cells were dye-loaded with 100 μl/ 2 .0 and ERK, two markers for enhanced neuronal activation, are increased, suggesting well of ACTOne Membrane Potential Dye. After 2 hour of incubation at room temperature, dilutions of 1 .5 1 PDE10A inhibitors could treat diseases characterized by a reduced activation of neurons, 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 Ro20-1724 were added to the plate by a FlexStation (Molecular Devices), and the data was recorded si- 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 tim e (m in ) such as schizophrenia. Deletion of the PDE10A gene blunts locomotor response when multaneously. tim e (m in ) challenged with N-methyl-D-aspartate (NMDA) receptor antagonist phencyclidine (PCP) and significantly delays the acquisition of conditioned avoidance, a behavioural response (A) and (B): Different numbers of the PDE10A cells with PDE4B/4D knocked down in 20 l of culture medium were Fig. 3 Cell Based Assay Vs Biochemical plated into each well of a 384-well plate. On the 2nd day, an equal volume of 1X ACTOne membrane potential dye predictive of clinical activity [10]. Consistent with these genetic studies, was added into each well. The cell plate (with dye) was incubated at room temperature in the dark for 2 hours. The cell PDE10A inhibitors are found to attenuate conditioned avoidance, and to inhibit PCP- and Assay for PDE10A (cAMP) plate was placed on a Hamamatsu FDSS 6000 and the baselines (F0) were recorded. Two different concentrations of amphetamine (-releasing agent)-stimulated locomotor activity in rats and mice. PF-2545920 were added into the wells (without Ro 20-1724). The plate was recorded on a Hamamatsu FDSS 6000 Furthermore, they antagonize apomorphine-induced climbing in mice, block NMDA an- again 60 min later. (C) and (D): PDE10A cells (PDE4B/4D KD) were plated into each well of a 384-well plate at a tagonist-induced deficits in prepulse inhibition of acoustic startle response in rats, while density of 10,000 cells/well in 20 l culture medium. On the 2nd day, an equal volume (20 ml) of 1X ACTOne mem- 6 brane potential dye was added into each well. The cell plate (with dye) was incubated at room temperature in the dark improving baseline sensory gating in mice. Particularly encouraging results have been ob- A:Cell Based Assay for 2 hours. Two different concentrations of PF-2545920 were added into the wells (without Ro 20-1724). The plate tained in animal models of the negative and cognitive symptoms of the psychosis, where was recorded on a Hamamatsu FDSS 6000 every 10 min. these compounds increase sociality in BALB/cJ mice, enhance social odor recognition in 5 mice, and improve novel object recognition in rats, suggesting targeting PDE10A could potentially treat all positive, negative, and cognitive [11]. 4 Fig. 6 Confirmation of PDE10A Specific Hits Codex BioSolutions developed a cell-based HTS assay that measures PDE10A activity PDE10A using proprietary ACTOne™ technology (Fig. 1). The technology utilizes a permanently- Parental

Ft/F0 3 (A) (B) 110 110 expressed modified cyclic nucleotide-gated channel (CNG) to monitor intracellular cAMP T = 60 min Parental Cell-no Ro T = 60 min Parental Cell-no Ro Parental Cell-with Ro Parental Cell-with Ro changes in live cells. To assay PDE10A activity, a Gs-coupled GPCR is over-expressed to 90 PDE10A-no Ro 90 PDE10A-no Ro 2 PDE10A-with Ro PDE10A-with Ro constitutively activate membrane bound adenylyl cyclases, leading to cAMP synthesis. 70 70

The produced cAMP is hydrolyzed by PDE10A so the steady cAMP is kept at low level. RFU 50 RFU 50 Upon PDE activity inhibition, the cellular cAMP level rises quickly and is measured by 1 CNG biosensor. The assay will sensitively and reliably identify PDE10A inhibitors in high- 30 30 throughput campaigns. As the first commercial cell-based PDE10A assay available to the 0.1 1 10 100 1000 10000 100000 10 10 pharmaceutical and biotech industry, it will greatly accelerate the drug discovery efforts for PF2545920 (nM) -10 -10 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 schizophrenia disease, one of the leading public health challenges Compound A (nM) Compound B (nM)

100 (D) 140 (c) 120 Summary T = 60 min Control Cell-no Ro Compound A ACTOne-PDE10A B: Biochemical Assay 120 Control Cell-with Ro 100 T = 60 min PF-2545920 A first commercial cell based PDE10A assay has been developed with a modified cyclic 80 PDE10A-no Ro 100 PDE10A-with Ro nucleotide-gated ion channel as the biosensor. 80 80

60 RFU The assay sensitivity is comparable to the biochemical assay and can be used for HTS. 60 60 RFU 40 LOPAC Library from Sigma and a selective library from Chembridge were screened with 40

20 20 the newly developed PDE10A cell based assay. 40 Two novel PDE10A specific inhibitors have been discovered and confirmed. 0 0 % Inhibition % 1 10 100 1000 10000 100000 0.1 1 10 100 1000 10000 100000 20 Compound C (nM) Cpd (nM) References ACTOne PDE10A or the parental cells (without PDE10A) were plated on a 384-well assay plate at 10K/well. 1. Houslay MD, Baillie GS, Maurice DH. Circulation Research 2007; 100:950–966. 0 On the 2nd day, an equal volume of 1X ACTOne membrane potential dye with or without 20 M Ro20-1724 2. Lugnier C. Pharmacology and Therapeutics 2006; 109:366–398. -10 -9 -8 -7 -6 -5 -4 was added into each well. After two hours incubation, different concentrations (5X of the final concentrations) 3. Conti M, Beavo J. Annual review of biochemistry 2007; 76:481–511. PF-2545920, Log(M) of the compounds (A-C) were prepared in 1X DPBS with 2.5% DMSO.10 l of above solutions was added into 4. Jeon YH, Heo YS, Kim CM, Hyun YL, Lee TG, Ro S, Cho JM. Cellular and Molecular each well. The cell plate was read on a FlexStation after 60 min of compounds addition. (D) Dose response A) ACTOne PDE10A (cAMP) or the parental cells were plated overnight in 20 μl culture medium on a Life Sciences 2005; 62:1198–1220. curves of compound A and PF-2545920 on ACTOne-PDE10A cells in the presence of 10 M Ro 20-1724 384-well plate. The next day, cells were loaded with 20 μl/well of ACTOne Membrane Potential Dye 5. Bender AT, Beavo JA. Pharmacological Reviews 2006; 58:488–520.. with 50 M Ro20-1724 and incubated at room temperature for 2hr. Different concentrations of 6. Menniti FS, et al. Nature Reviews Drug Discovery 2006; 5:660–670. PF2545920 were prepared in DPBS.10 l of compound solution was added into each well and the data Codex BioSolutions, Inc. 7. Seeger TF, et al. Brain Res 2003, 985:113–126. was recorded on a Hamamatsu’s FDSS 6000. 8. Graybiel AM. Curr Biol 2000, 10:R509–R511. B) 75 ng of PDE10A (from BPS Bioscience) was pre-treated with different concentrations of 401 Professional Drive, Suite 160, Gaithersburg, MD 20879 9. Torremans A, Ahnaou A, et al. Acta Neurobiol Exp 2010, 70:13-9. PF2545920 at RT for 20 min. Then the assay buffer containing 10 mM Tris (pH7.4), 10 mM MgCl2, Tel: (240)-632-8810; Fax: (240)-632-8820; 10. Siuciak JA, et al. Neuropharmacology 2006, 51:374-85. 0.05% Tween-20, 200 mM substrate (cAMP) and 5 kU was added. The total volume for is

11. Grauer SM, et al. J Pharmacol Exp Ther 2009, 331:574-590. 50 l. The reaction was kept at 37oC for 20 min. The OD was measured at 620 nM on a plate reader. E-Mail: [email protected]