Toxicity Screening of PMI Inhibitors in Hela cells
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: 51
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 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.
Description of assay :
3H-Mannose + HK -> 3H-Man-6-P + PMI <-> 3H2O + Fru-6-P
3H-Mannose + HK -> 3H-Man-6-P + PMM2 -> 3H-Man-1-P -> 3H-Glycoproteins
When cells are labeled with 3H-Mannose, the label is either found in medium as 3H2O or in labeled glycoproteins. If a compound inhibits PMI then there will be more 3H-Mannose influx through glycosylation pathway and more 3H counts would appear in glycoproteins as compare to control. A cellular assay involving labeling Hela cells in the presence of different concentrations of potential candidate compounds and determining 3H counts in isolated protein was developed in 24 well format.
PMI assay materials:
1) Hela cells
3) 35S-trans label
4) Trichloroacetic Acid
Protocol for cellular assays: Hela cells were incubated with the compounds or DMSO for 2 hours at 37oC. 3H-Mannose and 35S-methionine/cysteine was added to the cells and incubated for another hour at 37oC. Media was removed. The cells were washed once with PBS and harvested. The cell pellet was solubilized in 100 ul 10 mM Tris-Cl (pH 7.4)/1% NP-40. 20 ul was precipitated using Trichloroacetic acid (TCA) and counted. 5 ul was used in duplicate for protein estimation. 3H- and 35S incorporation was determined as cpm/ug.
Toxicity was estimated by 35S incorporation in proteins as compare to DMSO treated control. Lower incorporation as compare to control implies toxic effect.
Compounds that have "Low", Moderate or "High" toxicity are considered as "active" in this assay. All other compounds are marked as inactive.
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 and is not relevant in 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
Compounds that are inactive in this assay are assigned a score of 81
Compounds with Low Toxicity in this assay are assigned a score of 85
Compounds with Moderate Toxicity in this assay are assigned a score of 90
Compounds with High Toxicity in this assay are assigned a score of 95
Categorized Comment - additional comments and annotations
Data Table (Concise)