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BioAssay: AID 1536

Confirmation of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphate.

Congenital Disorders of Glycosylation (CDG) are autosomal recessive defects in the synthesis of N-linked oligosaccharide chains. CDG group I (CDG-I) defects are defined as those caused by mutations in genes encoding enzymes used for the synthesis and transfer of lipid linked oligosaccharide (LLO) to newly synthesized proteins in the lumen of the ER. The steps in this pathway and the genes more ..
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 Tested Compounds
 Tested Compounds
All(13)
 
 
Active(4)
 
 
Inactive(9)
 
 
 Tested Substances
 Tested Substances
All(16)
 
 
Active(4)
 
 
Inactive(12)
 
 
AID: 1536
Data Source: Burnham Center for Chemical Genomics (BCCG-A140-PMI-10x-Assay-dry powder)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2009-03-05
Modify Date: 2010-12-29

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 4
Related Experiments
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AIDNameTypeProbeComment
1209HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay.Confirmatory depositor-specified cross reference
1220HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphateConfirmatory depositor-specified cross reference
1545Summary - Compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay.Summary2 depositor-specified cross reference
1553Screening for Phosphomannose Isomerase inhibitors in cellular based assay using Hela cells.Other depositor-specified cross reference
1620Toxicity Screening of PMI Inhibitors in Hela cellsOther depositor-specified cross reference
1020Counter Screen for Glucose-6-Phosphate Dehydrogenase-based Primary AssayScreening same project related to Summary assay
1217uHTS Identification of Diaphorase Inhibitors and Chemcical Oxidizers: Counter Screen for Diaphorase-based Primary AssaysScreening same project related to Summary assay
1535Confirmation of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay.Confirmatory same project related to Summary assay
1655Counter screen SAR assay for PMM2 inhibitors via a fluorescence intensity assayConfirmatory same project related to Summary assay
1666SAR assay for compounds that inhibit PHOSPHO1Confirmatory same project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Number: R03 MH082386-01
Assay Provider: Dr. Hudson H. Freeze, Sanford-Burnham Medical Research Institute, San Diego, CA

Congenital Disorders of Glycosylation (CDG) are autosomal recessive defects in the synthesis of N-linked oligosaccharide chains. CDG group I (CDG-I) defects are defined as those caused by mutations in genes encoding enzymes used for the synthesis and transfer of lipid linked oligosaccharide (LLO) to newly synthesized proteins in the lumen of the ER. The steps in this pathway and the genes encoding them are very similar from yeast to human. It requires 30-40 single gene products, each dependent on the previous step in the linear sequence to produce and transfer the LLO to protein. Therefore, mutations in any step may cause a type of CDG. There is considerable overlap in the clinical presentations between different types of CDG and a broad diversity within each type. The most common form of CDG, called Type Ia (CDG-Ia), is caused by defects in PMM2 (Man-6-P to Man-1-P), the gene that encodes phosphomannomutase. Mortality is 20% in the first 5 yrs, but then patients stabilize. Currently, there is no treatment for the CDG-Ia.

CDG-Ib patients, who are deficient in phosphomannose isomerase (PMI) catalyzing conversion of Man-6-P to Fru-6-P, are successfully treated with free mannose. Unfortunately, mannose therapy is not effective for CDG-Ia patients, most likely due to efficient Man-6-P consumption in the PMI reaction. It is believed that patients with Congenital Disorder of Glycosylation Type Ia (CDG-Ia) will benefit from dietary mannose if there is a simultaneous reduction of phosphomannose isomerase (PMI) activity. This would allow a modest intracellular accumulation of Man-6-P and drive metabolic flux into the glycosylation pathway using the residual PMM2 activity. It is assumed that a non-competitive inhibitor would work best in this setting.

The purpose of this assay is to identify non-competititve inhibitors of human PMI. This is accomplished by using a G6PD- NADPH-coupled assay. In the assay PMI activity is detected through conversion of its product, fructose-6-phosphate, to glucose-6-phosphate catalyzed by phosphoglucose isomerase (PGI) and subsequent oxidation of glucose-6-phosphate to 6-phosphogluconolactone concomitant with NADP-to-NADPH conversion catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). The NADPH is then detected via a resazurin-diaphorase fluorogenic reaction.

This assay is performed in the presence of 10x-Km concentrations of the PMI substrate, mannose-6-phosphate, to help to ensure the identification of non-competitive inhibitors.

This dose response assay is developed and performed to confirm hits originally identified in "HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphate" (AID 1220) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
Protocol
PMI assay materials:

1) Human PMI protein was provided by Dr. Hudson Freeze (Sanford-Burnham Medical Research Institute, San Diego, CA).
2) Substrate working solution: 50 mM HEPES, pH 7.4, 2.0 mM Mannose-6-phosphate, 1.6 U/ml Diaphorase, 0.2 mM Resazurin.
3) Enzyme working solution: 50 mM HEPES, pH 7.4, 0.44 mM NADP+, 9.048 mM MgCl2, 0.01% Tween 20, 4.6 ug/ml phosphoglucose isomerase, 30 ng/ml PMI, 1.8 ug/ml G6PDH.

PMI Dose-response confirmation protocol:

1) 9 uL of Substrate working solution was added to columns 3-24 of a Greiner 384-well black plate (cat # 784076) using a WellMate bulk dispenser (Matrix)
2) 9 ul of Substrate working solution without mannose-6-p was added to columns 1 and 2 (positive control)
3) Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration.
4) 2 uL compounds in 10% DMSO were transferred into columns 3-22. Columns 1-2 and 23-24 contained 4 uL of 10% DMSO.
5) 9 uL of Enzyme working solution was added to the whole plate using a Thermo Multidrop Combi dispenser.
6) Plates were incubated at room temperature for 30 min.
7) The plates were read on an Analyst plate reader (Molecular Devices), Ex544, Em590.
8) Data analysis was performed using CBIS software (ChemInnovations, Inc).
Comment
Compounds with an IC < 100 uM are considered to be active in this assay.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the PMI assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the PMI assay is as follows:

1) First tier (0-40 range) is reserved for primary screening data data and is not applicable to this assay.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to this assay.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues

a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound's inhibitory potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the PMI inhibition assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pEC50 - 3)*QC,
where pEC50 is a negative log(10) of the EC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
2IC50*IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat
5Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was (1) excluded and (0) means the point was not excluded, for the titration series going from low to high compound concentrations.String
6% inhibition at 0.390625 uM_first_point (0.390625μM**)% inhibition at a given concentrationFloat%
7% inhibition at 0.78125 uM_first_point (0.78125μM**)% inhibition at a given concentrationFloat%
8% inhibition at 1.5625 uM_first_point (1.5625μM**)% inhibition at a given concentrationFloat%
9% inhibition at 3.125 uM_first_point (3.125μM**)% inhibition at a given concentrationFloat%
10% inhibition at 6.25 uM_first_point (6.25μM**)% inhibition at a given concentrationFloat%
11% inhibition at 12.5 uM_first_point (12.5μM**)% inhibition at a given concentrationFloat%
12% inhibition at 25 uM_first_point (25μM**)% inhibition at a given concentrationFloat%
13% inhibition at 50 uM_first_point (50μM**)% inhibition at a given concentrationFloat%
14% inhibition at 100 uM_first_point (100μM**)% inhibition at a given concentrationFloat%
15Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was (1) excluded and (0) means the point was not excluded, for the titration series going from low to high compound concentrations.String
16% inhibition at 0.390625 uM_second_point (0.390625μM**)% inhibition at a given concentrationFloat%
17% inhibition at 0.78125 uM_second_point (0.78125μM**)% inhibition at a given concentrationFloat%
18% inhibition at 1.5625 uM_second_point (1.5625μM**)% inhibition at a given concentrationFloat%
19% inhibition at 3.125 uM_second_point (3.125μM**)% inhibition at a given concentrationFloat%
20% inhibition at 6.25 uM_second_point (6.25μM**)% inhibition at a given concentrationFloat%
21% inhibition at 12.5 uM_second_point (12.5μM**)% inhibition at a given concentrationFloat%
22% inhibition at 25 uM_second_point (25μM**)% inhibition at a given concentrationFloat%
23% inhibition at 50 uM_second_point (50μM**)% inhibition at a given concentrationFloat%
24% inhibition at 100 uM_second_point (100μM**)% inhibition at a given concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03 MH082386-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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