HTS identification 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 ..
BioActive Compounds: 1288
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.
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 HTS protocol:
1) 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
2) 2 ul of Substrate working solution without mannose-6-p was added to columns 1 and 2 (positive control) 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
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) Plates were incubated at room temperature for 20 min.
6) After 20 minutes the plates were read on a ViewLux plate reader (Perkin Elmer), Ex544, Em590.
7) The screening was performed using a HighRes biosolution fully integrated HTS POD-based system
8) Data analysis was performed using CBIS software (ChemInnovations, Inc).
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).
Compounds with greater than 50% inhibition at 20 uM concentration are defined as actives of the primary screening. The primary screening actives proceed to the dose-response confirmation stage. Compounds have an IC50 < 50 are defined as actives in the dose-response confirmation stage.
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 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. The score is correlated with % displacement in the assay demonstrated by a compound at 20 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound#s 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 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 = 44 + 6*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 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.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable to this assay
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)