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BioAssay: AID 1216

HTS identification of compounds activating phosphomannose isomerase (PMI) via a fluorescence intensity assay using a near- saturating concentration of mannose 6-phosphat

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(194156)
 
 
Active(656)
 
 
Inactive(193500)
 
 
 Tested Substances
 Tested Substances
All(194226)
 
 
Active(656)
 
 
Inactive(193570)
 
 
 Related BioAssays
 Related BioAssays
AID: 1216
Data Source: Burnham Center for Chemical Genomics (SDCCG-A0610-PMI-Activator-SaturatedSubstrate-FluoAssay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-03-07
Modify Date: 2010-10-28

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 656
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.

One of the common forms of CDG, called Type Ib (CDG-Ib), is caused by a deficiency in phosphomannose isomerase (PMI) which catalyzes the reversible conversion of Man-6-P to Fru-6-P. These cases are successfully treated with free mannose. Identification of specific activators of PMI could provide new tools in further characterization of diverse CDG phenotypes and potentially creating new clinical approaches to treating CDG-Ib and other related disorders.

The purpose of this assay is to identify activators of human PMI; it was specifically formatted to enable their identification. The PMI activity is measured using a PGI-G6PD-diaphorase 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. This condition is expected to make the assay specific for the activators that would work best in the presence of elevated substrate concentration.
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, 0.0046 mg/ml phosphoglucose isomerase, 0.00003 mg/ml PMI, 0.0018 mg/ml G6PDH.
4) Negative control (NC) solution - 50 mM HEPES, 1.6 U/ml Diaphorase, 0.2 mM Resazurin

PMI HTS protocol:

1) 2 ul of negative control solution was added to columns 1 and 2 of a Costar 1536-well black plate (cat #3724) using a Thermo Multidrop Combi dispenser
2) 2 uL of Substrate working solution was added to columns 3-48 of a Costar 1536-well black plate (cat #3724) using a Thermo Multidrop Combi dispenser
3) 40 nL of 100% DMSO was added to columns 1-4 using a HighRes biosolutions pintool and V&P Scientific pins. Columns 3-4 are positive controls (PC).
3) 40 nL of 2 mM compounds in 100% DMSO were dispensed in columns 5-48 using a HighRes biosolutions pintool and V&P Scientific pins
4) 2 uL of Enzyme working solution was added to the whole plate using a Thermo Multidrop Combi dispenser.
5) Final reagent concentrations are:
a)50 mM HEPES, pH 7.4
b)1.0 mM Mannose-6-phosphate (except columns 1-2)
c)4.5 mM MgCl2
d)0.22 mM NADP+
e)0.005% Tween 20
f)0.1 mM Resazurin
g)coupling enzymes: 2.3 ug/ml phosphoglucose isomerase, 0.9 ug/ml G6PDH, 0.8 U/ml Diaphorase
h)15 ng/ml human PMI
i)1% DMSO (all columns)
j)20 uM compounds (columns 3-48)
6) Plates were incubated at room temperature for 20 min.
7) After 20 minutes the plates were read on a ViewLux plate reader (Perkin Elmer), Ex544, Em590.
8) The screening was performed using a HighRes biosolution fully integrated HTS POD-based system
9) Data analysis was performed using CBIS software (ChemInnovations, Inc).
Comment
PMI activation was calculated using the following formula:
Activation Factor (AF) = (Signal_Well - Mean_NC)/(Mean_PC - Mean_NC),
where Signal_Well corresponds to luminescence signal in the well with a compound, Mean_NC and Mean_PC correspond to mean values of corresponding controls in the plate.

Compounds with greater than or equal to 2-fold activation (AF >= 2) of PMI at 20 uM concentration are defined as actives of the primary screening.

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 and the score is correlated with PMI activation factor demonstrated by a compound at 20 uM concentration:
a. If AF<1, then the assigned score is 0
b. For all other AF values, Score = 40 - 40/AF
This formula results in a score that is equal 20 for AF=2 and asymptotically approaches 40 with increasing AF values.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_QualifierThis qualifier is to be used with the next TID, EC50. If qualifier is "=", EC50 result equals to the value in that column; if qualifier is ">", EC50 result is greater than that value.String
2EC50EC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(EC50)Standard Error of EC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat
5Max_ValueThe AF value asymptotically approached by the dose response curve at saturating compound concentrationsFloat
6AF_20uM (20μM**)The AF value of PMI in primary screening at 20 uM.Float
7Mean_NCMean fluorescent signal of negative controls in the corresponding plateFloatRFU
8StdDev_NCStandard deviation (n=64) of negative controls in the corresponding plateFloatRFU
9Mean_PCMean fluorescent signal of positive controls in the corresponding plateFloatRFU
10StdDev_PC Standard deviation (n=64) of positive controls in the corresponding plateFloatRFU

** Test Concentration.
Additional Information
Grant Number: R03 MH082386-0

Data Table (Concise)
Classification
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