Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (HTRF) Assay
Chronic pain (neuropathic pain, inflammatory pain, cancer pain) is a major health problem. Opiate-based drugs, such as morphine and morphine derivatives, are the primary standard of care for the treatment of chronic pain. Unfortunately, patients develop tolerance to opiates due to desensitization of the opiate receptor. Thus, alternative anti-nociceptive ("pain killing") pathways need to be explored for treatment of chronic pain. ..more
BioActive Compounds: 567
Data Source: Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1 R03 MH085675-01
Assay Provider: Dr Ilya Bezprozvanny, UT Southwestern Medical Center , Dallas, TX
Chronic pain (neuropathic pain, inflammatory pain, cancer pain) is a major health problem. Opiate-based drugs, such as morphine and morphine derivatives, are the primary standard of care for the treatment of chronic pain. Unfortunately, patients develop tolerance to opiates due to desensitization of the opiate receptor. Thus, alternative anti-nociceptive ("pain killing") pathways need to be explored for treatment of chronic pain.
The N-type voltage-gated Ca2+ channels (CaV2.2s) in dorsal root ganglia neurons is a well validated target for chronic pain (1, 2). We previously demonstrated the interaction between CaV2.2 and the first PDZ domain of molecular adaptor protein Mint1 (3). In additional experiments we demonstrated that carboxyl-terminal association of CaV2.2 protein with the Mint1-PDZ domain plays an important role in synaptic targeting and function of N-type Ca2+ channels (4).
The following describes a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) HTS assay to identify small molecule inhibitors of association between Mint1-PDZ domains and N-type Ca2+ channel carboxyl-terminal peptide (NC peptide). Compounds that disrupt the interaction would result in decrease of TR-FRET ratio and are detected in the assay. Small molecules identified in the screening would serve as useful probes for studies of synaptic function of N-type Ca2+ channels, its validation as a therapeutic target and/or as starting point for development of anti-nociceptive therapeutic agents with novel mechanism of action.
1. Malmberg AB, Yaksh TL: Effect of continuous intrathecal infusion of omega-conopeptides, N-type calcium-channel blockers, on behavior and antinociception in the formalin and hot-plate tests in rats. Pain 1995; 60:83-90.
2. Malmberg AB, Yaksh TL: Voltage-sensitive calcium channels in spinal nociceptive processing: blockade of N- and P-type channels inhibits formalin-induced nociception. J Neurosci 1994; 14:4882-4890.15
3. Maximov A, Sudhof TC, Bezprozvanny I: Association of neuronal calcium channels with modular adaptor proteins. J Biol Chem 1999; 274:24453-24456.
4. Maximov A, Bezprozvanny I: Synaptic targeting of N-type calcium channels in hippocampal neurons. J Neurosci 2002; 22:6939-6952.
1) Biotin, glutathione S-transferase (GST)-fusion protein (GST-M15) and biotinylated (biotin-) N-type Ca2+ channel carboxyl-terminal peptide (biotin-NC peptide) corresponding to the carboxyl-terminal tail of CaV2.2 protein were supplied by assay provider.
2) Anti-GST monoclonal antibody labeled with europium cryptate (EuK-anti-GST-mAb (CisBio cat# 61GSTKLB) and streptavidin-XL665 (CisBio cat# 610SAXLB ).
3) Potassium Fluoride (KF) (Aqua Solutions cat# P3901)
4) Tween-20 (Sigma cat# 9416)
5) Bovine Serum Albumin (Sigma cat# A7888)
1. Assay buffer: 25 mM HEPES, pH 7.2, 100mM NaCl,0.1% BSA, 0.005% Tween-20
2. Mint1-PDZ working solution (1.75X final concentration) contains 3.5 nM GST-MINT-PDZ and 0.63 nM EuK-anti-GST-mAb
3. Biotin-NC peptide-XL665 solution (3.5X final concentration) contained 167 nM biotin-NC peptide and 46.6 nM streptavidin XL665.
4. Biotin solution (3.5X final concentration): 167 nM Biotin 3.5X final concentration) and 46.6 nM streptavidin XL665.
5. TR-FRET enhancing solution (7X final concentration): 3.2 M KF.
Primary Screen and Single-concentration confirmation
1) Dispense 4 uL Mint1-PDZ working solution to entire 1536-well white plate (Corning# 3725)
2) Using a HighRes biosolution pintool equipped with V&P Scientific pins add 70 nL of DMSO (col 1-4) or 2-mM compound solutions in DMSO (col 5-48)
3) Incubate for 30 mins
4) Dispense 2 uL Biotin working solution to columns 1 & 2
5) Dispense 2 uL of Biotin-NC peptide working solutions to columns 3-48
6) Incubate for 30 mins
7) Dispense 1 uL 3.2 M KF to entire plate
8) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge.
9) Read plate on a BMG Labtech PheraStar in a Homogeneous Time-Resolved Fluorescence mode (Ex: 337 nm; Em: 620/665 nm) within 10 min
The rat Mint1 and human a1B constructs were used because they were available in the assay provider's lab.
Compounds that demonstrated an inhibition of >= 50% and 0.5 <= F_ratio <= 1.5 at 20 uM concentration are defined as actives in primary and confirmation HTS assays. Compounds with Inhibition >= 50% that demonstrate F_ratios outside these boundaries are optically interfering with the assay (quenching or fluorescent) and the results are marked as "inconclusive".
The compounds identified as primary screening actives proceed to single-concentration confirmation stage. These compounds were retested in quadruplicate at a single 20 uM concentration in a confirmation screen.
Compounds that were actives of the confirmation assay were confirmed in the dose-response confirmation assay. Compounds that demonstrate IC50 values in the range of analyzed concentrations remain "active" in the outcome column.
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 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 assay is as follows:
1) First tier (0-40 range) is reserved for primary screening and single concentration confirmation data-the score is correlated with % inhibition in the assay demonstrated by a compound at 20 uM concentration:
a. If % inhibition is less than 0%, then the assigned score is 0
b. If % inhibition is greater than 100%, then the assigned score is 40/(1+(F_ratio - 1)^2)
c. If % inhibition is between 0% and 100%, then the calculated score is (%inhibition)*0.4/(1+(F_ratio-1)^2)
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- or pathway-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 this 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 all 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 in this assay
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)