Assay provider results from the probe development effort to identify inhibitors of prolyl oligopeptidase-like enzyme (PREPL): fluorescence-based biochemical assay for inhibition of anti-target prolyl endopeptidase (PEP)
Name:Assay provider results from the probe development effort to identify inhibitors of prolyl oligopeptidase-like enzyme (PREPL): fluorescence-based biochemical assay for inhibition of anti-target prolyl endopeptidase (PEP). ..more
BioActive Compounds: 2
Depositor Specified Assays
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: PEP_INH_FLINT_1X%INH
Name:Assay provider results from the probe development effort to identify inhibitors of prolyl oligopeptidase-like enzyme (PREPL): fluorescence-based biochemical assay for inhibition of anti-target prolyl endopeptidase (PEP).
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 (1). 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 (2). They used this technology to annotate the uncharacterized enzyme KIAA1363 (3) and to identify a series of peptide substrates in the kidney for another POP family member, DPP4 (4). 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 (3). 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 (5).
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.
Assay provider, Prolyl oligopeptidase-like enzyme, PREPL, FLJ16627, KIAA0436, serine protease, hypotonia-cystinuria syndrome, HCS, 2p21 deletion syndrome, inhibit, inhibitor, inhibition, prolyl endopeptidase, PEP, fluorescence, anti-target, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine whether test compounds can inhibit anti-target prolyl endopeptidase (Pep) using a substrate-based in vitro assay. In this assay, Pep is incubated with test compound followed by addition of fluorogenic substrate Z-Gly-Pro-AMC and fluorescence monitoring. As designed, test compounds that act as inhibitors will prevent enzyme-substrate interactions, giving lower fluorescence intensity.
Purified, recombinant Pep (0.70 ug/mL) in Pep assay buffer (25 mM Na2HPO4, 0.5 mM EDTA, 2 mM DTT) was preincubated with 20 uM inhibitor or DMSO only for 15 minutes. Substrate Z-Gly-Pro-AMC in Pep assay buffer was then added to a final concentration of 133 uM. After 20 minutes, the cleavage of this fluorogenic substrate was monitored on a Spectramax Gemini XS fluorescence plate reader (Molecular Devices), with excitation at 360 nm and emission at 460 nm.
The % inhibition was then calculated as follows:
%_Inhibition = (1 - ( Test_Compound - Low_Control ) / ( High_Control - Low_Control ) ) * 100
Test_Compound is defined as fluorescence intensity of sample treated with test compound.
High_Control is defined as fluorescence intensity of sample treated with DMSO only (no compound).
Low_Control is defined as fluorescence intensity of buffer only.
PubChem Activity Outcome and Score:
Compounds with greater than 20% inhibition were considered active. Compounds with less than or equal to 20% inhibition were considered inactive.
The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-80, and for inactive compounds 0-0.
List of Reagents:
Recombinant Pep (provided by Assay Provider)
Na2HPO4 (Fisher S379-212)
EDTA (Fisher BP121-500)
DTT (Fisher BP172-5)
Z-Gly-Pro-AMC (Bachem I-1145)
This assay was performed by the assay provider with liquid samples of test compounds.
** Test Concentration.
Data Table (Concise)