Assay provider results from the probe development effort to identify inhibitors of prolyl oligopeptidase-like enzyme (PREPL): fluorescence-based cell-based gel-based competitive click chemistry Activity-Based Protein Profiling (CC-ABPP) inhibition of PREPL in situ
Name: Late stage assay provider results from the probe development effort to identify inhibitors of prolyl oligopeptidase-like enzyme (PREPL): fluorescence-based cell-based gel-based competitive click chemistry Activity-Based Protein Profiling (CC-ABPP) inhibition of PREPL in situ. ..more
BioActive Compounds: 5
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_FLINT_1X%INH_INSITU
Name: Late stage assay provider results from the probe development effort to identify inhibitors of prolyl oligopeptidase-like enzyme (PREPL): fluorescence-based cell-based gel-based competitive click chemistry Activity-Based Protein Profiling (CC-ABPP) inhibition of PREPL in situ.
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, activity-based protein profiling, ABPP, click chemistry, CC-ABPP, fluorescence, gel-based, fluorophosphonate-alkyne, FP-alkyne, in situ, Neuro-2A, 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 powder samples of test compounds can inhibit Prepl in situ using gel-based click chemistry activity-based protein profiling (CC-ABPP). In this assay, cultured cells are incubated with test compound followed by the cell-permeable alkyne-functionalized fluorophosphonate (FP-alkyne) activity-based probe. Cells are harvested, homogenized, reacted with an azide-functionalized rhodamine (Rh-azide) fluorescent reporter tag under click chemistry reaction conditions, resolved by SDS-PAGE, and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel.
Neuro-2A cells were plated at 1.5 x 10^6 cells per well in six-well plates. After 24 hours at 37 C in a humidified incubator, cells were transfected with pCMV_mPrepL using Lipofectamine 2000 transfection reagent. Two days post-transfection, inhibition and labeling were carried out in the intact cells before harvesting as follows: First, the medium covering the cells was removed and replaced with medium containing 50 uM inhibitor or DMSO only and incubated for 1.5 hours. The cell-permeable FP-alkyne probe was then added to the medium to a final concentration of 10 uM and incubated for 1 hour. Cells were washed three times with PBS and harvested with a cell scraper. The cell pellet was washed three times (by suspending in 1 mL of PBS, centrifuging at 1400g for 3 minutes, and then aspirating PBS). After the final wash, the cell pellet was resuspended in 80 uL of PBS. Cells were lysed by seven freeze/thaw cycles and centrifuged at 4 C for 30 minutes (20000 x g). The supernatant was transferred to a new tube and protein concentration adjusted to 4 mg/mL with PBS. To 43 uL of 4 mg/mL protein sample were added 2 uL of 1.25 mM rhodamine-azide, TCEP (1 uL, 50 mM), TBTA ligand (3 uL, 1.67 mM), and CuSO4 (1 uL, 50 mM). This reaction was allowed to proceed for 1 hour at room temperature and then quenched with SDS-PAGE loading buffer, resolved by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the target band relative to a DMSO-only (no compound) control.
The % inhibition was then calculated as follows:
%_Inhibition = ( 1- ( IOD_Test_Compound - Median_IOD_Low_Control ) / ( Median_IOD_High_Control - MedianIOD_Low_Control ) ) * 100
Test_Compound is defined as target treated with test compound.
High_Control is defined as target treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
PubChem Activity Outcome and Score:
Compounds with greater than 50% inhibition were considered active. Compounds with less than or equal to 50% 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 100-63, and for inactive compounds 42-0.
List of Reagents:
Neuro-2A cells (provided by Assay Provider)
pCMV_mPrepL (Open Biosystems Clone ID 3585402)
Lipofectamine 2000 (Invitrogen 11668-019)
PBS (Cellgro 20-031-CV)
FP-alkyne (provided by Assay Provider)
Rh-azide (Invitrogen T10182)
TBTA ligand (SigmaAldrich 678937)
TCEP (SigmaAldrich 75259)
CuSO4 (Fisher C493-500)
This assay was performed by the assay provider with powder samples of test compounds.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)