Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the prolyl oligopeptidase-like enzyme (PREPL)
Name: Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the prolyl oligopeptidase-like enzyme (PREPL). ..more
BioActive Compounds: 2221
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Alan Saghatelian, Harvard University
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 DP2 OD002374-01 Fast Track
Grant Proposal PI: Alan Saghatelian, Harvard University
External Assay ID: PREPL_INH_FP_1536_1X%INH
Name: Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the prolyl oligopeptidase-like enzyme (PREPL).
Hypotonia-cystinuria syndrome (HCS) and 2p21 deletion syndrome are debilitating and poorly understood diseases, both involving deletion of the gene encoding prolyl oligopeptidase-like enzyme (PREPL), a member of the prolyl oligopeptidase (POP) family of serine peptidases . Saghatelian and colleagues have developed an advanced LC-MS-based peptide metabolite profiling platform that allows linkage of enzymes with their endogenous substrates and establishment of their participation in specific biochemical networks; however, doing so requires selective chemical tools to functionally perturb the enzymes of interest . They used this technology to annotate the uncharacterized enzyme KIAA1363  and to identify a series of peptide substrates in the kidney for another POP family member, DPP4 . Importantly, substrate identification for KIAA1363 was only accomplished through the application of a selective small molecule KIAA1363 inhibitor, as constitutive inhibition produced widespread metabolic disturbances . Initial attempts at identifying the substrate specificity of PREPL have not been successful. Physicochemical properties of PREPL suggest that endogenous protein-protein interactions with PREPL might lead to an active form of PREPL that cannot be recapitulated in vitro. Therefore, the ability to target PREPL in vivo is a necessary step in understanding the function of this important peptidase enzyme. Development of selective inhibitors of PREPL would provide crucial tools to advance understanding of the physiological and pathological roles of this critical enzyme and might reveal new treatment possibilities for HCS and 2p21 deletion syndrome .
1. Martens, K., et al., Global distribution of the most prevalent deletions causing hypotonia-cystinuria syndrome. Eur J Hum Genet, 2007. 15(10): p. 1029-33.
2. Saghatelian, A., et al., Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry, 2004. 43(45): p. 14332-9.
3. Chiang, K.P., et al., An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling. Chem Biol, 2006. 13(10): p. 1041-50.
4. Tagore, D.M., et al., Peptidase substrates via global peptide profiling. Nat Chem Biol, 2009. 5(1): p. 23-5. Jaeken, J., et al., Deletion of PREPL, a gene encoding a putative serine oligopeptidase, in patients with hypotonia-cystinuria syndrome. Am J Hum Genet, 2006. 78(1): p. 38-51.
5. Jaeken, J., et al., Deletion of PREPL, a gene encoding a putative serine oligopeptidase, in patients with hypotonia-cystinuria syndrome. Am J Hum Genet, 2006. 78(1): p. 38-51.
6. Bachovchin, D.A., et al., Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat Biotechnol, 2009. 27(4): p. 387-94.
7. Leung, D., et al., Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat Biotechnol, 2003. 21(6): p. 687-91.
8. Liu, Y., M.P. Patricelli, and B.F. Cravatt, Activity-based protein profiling: the serine hydrolases. Proc Natl Acad Sci U S A, 1999. 96(26): p. 14694-9.
Prolyl oligopeptidase-like enzyme, PREPL, FLJ16627, KIAA0436, serine protease, hypotonia-cystinuria syndrome, HCS, 2p21 deletion syndrome, fluorescence polarization, FP-PEG-Rh, inhibit, inhibitor, inhibition, primary, primary screen, HTS, high throughput screen, 1536, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this biochemical assay is to identify compounds that act as PREPL inhibitors. This is a competitive activity-based protein profiling (ABPP) assay that uses fluorescence polarization to investigate enzyme- substrate functional interactions based on active site-directed molecular probes [5-7]. In the assay, a fluorophosphonate-polyethylene glycol-rhodamine (FP-PEG-Rh) probe that broadly targets enzymes from the serine hydrolase family is used to label PREPL in the presence of test compounds . The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). The assay is performed by incubating a library of compounds with PREPL for a defined period, adding the FP-PEG-Rh probe, and then reading fluorescence polarization at a specific time point. As designed, test compounds that act as PREPL inhibitors will prevent PREPL-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a final nominal concentration of 5.96 micromolar.
Prior to the start of the assay, 4.0 microliters of Assay Buffer (0.01% Pluronic acid, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT) containing 37.5 nanomolar of PREPL protein were dispensed into 1536 microtiter plates. Next, 30 nL of test compound in DMSO or DMSO alone (0.59% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 degrees Celsius.
The assay was started by dispensing 1.0 microliter of 375 nM FP-PEG-Rh probe in Assay Buffer to all wells. Plates were centrifuged and after 15 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm). Fluorescence polarization was read for 15 seconds for each polarization plane (parallel and perpendicular).
Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):
FP= ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )
Raw1 is defined as the S channel.
Raw2 is defined as the P channel.
The percent inhibition for each compound was calculated as follows:
100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Low_Control is defined as wells containing PREPL and DMSO.
Test_Compound is defined as wells containing PREPL in the presence of test compound.
High_Control is defined as wells containing no PREPL protein.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-11, for inactive 11-0.
List of Reagents:
Recombinant PREPL protein (supplied by Assay Provider)
FP-PEG-Rh probe (supplied by Assay Provider-Benjamin Cravatt laboratory)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic acid (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)
DTT (Invitrogen, part 15508-013)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)