Fluorescence-based cell-based primary high throughput screening assay to identify inhibitors of TLR9-MyD88 binding.
Name: Fluorescence-based cell-based primary high throughput screening assay to identify inhibitors of TLR9-MyD88 binding. ..more
BioActive Compounds: 734
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Peter S. Tobias, The Scripps Research Institute (TSRI)
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: RO1 AI078288
Grant Proposal PI: Peter S. Tobias, The Scripps Research Institute (TSRI)
External Assay ID: TLR9_INH_BLA_1536_1X%INH PRUN
Name: Fluorescence-based cell-based primary high throughput screening assay to identify inhibitors of TLR9-MyD88 binding.
Toll-like receptors (TLRs) are involved in initiating inflammatory responses to a number of components of pathogens as well as markers of sterile injury. As such they initiate homeostatic responses but also pathologic responses. Our focus on TLR9 is based on its role in autoimmune disease. Auto-antibodies to DNA, RNA, or both are characteristic of systemic lupus erythematosus (SLE) (1). Auto-antibody production depends on the endosomal TLRs such as TLR9 and TLR7 that bind DNA (TLR9) or RNA (TLR7). Identifying specific inhibitors of TLR9 is important for furthering the understanding of SLE. To date, with one exception, all the non-protein molecules that alter TLR responses are ligand analogs that react with the extra-cytoplasmic ligand binding portions of the TLRs. The sole exception, TAK-242, is a TLR4 inhibitor that was withdrawn from further development as a drug (http://www.takeda.com, Feb. 20, 2009) for unspecified reasons. The assays we are developing are designed to identify small molecules that interfere with the first interaction of the receptor with the proximal intracellular signaling adapter, which in the case of TLR9 is MyD88. Such inhibitors will not be competitive with receptor ligands. They have the potential to be highly specific in their action among members of the TLR family because the interactions of MyD88 with each TLR member are unique, despite the structural similarities in the family (2). The rationale for this approach is that it will permit the identification of a new class of inhibitors enabling a variety of innovative approaches to research and therapeutics, not only for SLE, but also for other immunological processes in which the TLRs are involved.
1. Kono,D.H., Haraldsson,M.K., Lawson,B.R., Pollard,K.M., Koh,Y.T., Du,X., Arnold,C.N., Baccala,R., Silverman,G.J., Beutler,B.A. et al 2009. Endosomal TLR signaling is required for anti-nucleic acid and rheumatoid factor autoantibodies in lupus. Proceedings of the National Academy of Sciences 106:12061-12066.
2. Jiang,Z., Georgel,P., Li,C., Choe,J., Crozat,K., Rutschmann,S., Du,X., Bigby,T., Mudd,S., Sovath,S. et al 2006. Details of Toll-like receptor:adapter interaction revealed by germ-line mutagenesis. Proceedings of the National Academy of Sciences 103:10961-10966.
Toll-like receptor 9, TLR9, MyD88, enzyme complementation, receptor, reporter assay, beta-lactamase, BLA, Bla, FRET, LiveBLAzer, CHO cells, fluorescence, FL, CCF2/AM, CCF4/AM, inhibitor, inhibition, inhibit, protein-protein interaction, protein-DNA interaction, systemic lupus erythrematosus, SLE, lupus, primary, singlicate, HTS, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to identify compounds that inhibit TLR9-MyD88 binding. This assay uses a CHO cell line that was generated to stably express two beta-lactamase (BLA) fragment fusion proteins: a chimera of MyD88 fused to BLA fragment "a" and a chimera of TLR9 fused to BLA fragment "b". These cells have constitutive BLA activity due to the interaction of the TLR9 and MyD88 components of the chimeras, which brings together BLA fragments "a" and "b", to reconstitute full length BLA. As designed, test compounds from the MLSCN library which interrupt TLR9 and MyD88 interaction will disrupt reconstitution of the BLA fragments and reduce BLA activity. BLA activity is detected by measuring fluorescence of the cleavable BLA substrate CCF4-AM, which yields either blue fluorescence at 450 nm (no FRET) or green fluorescence at 535 nm (FRET). Test compounds from the MLSCN library were screened for inhibition of constitutive TLR9-MyD88 binding as measured by changes in the ratio of fluorescence emissions at 450 nm and 535 nm. Clavulanic acid, an inhibitor of BLA activity, was implemented as a non specific control. Compounds are tested in singlicate at a final nominal concentration of 4.98 uM.
The stably transfected TLR9 MyD88 CHO cells were cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) containing 10% v/v fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 10 mM HEPES, 5 mM L-Glutamine, 200 ug/mL geneticin (G418) and 1X Penicillin/Streptomycin.
Prior to assay, cells were suspended to a concentration of 200,000 cells/ml in DMEM (Phenol Red Free), containing 10% v/v fetal bovine serum, 0.1 mM NEAA, 1mM Sodium Pyruvate, 10 mM Hepes, 5mM L-Glutamine and 1X Penicillin/Streptomycin. To start the assay, 5 ul of assay media was dispensed into the first two columns of a 1536 well plate and 5 ul of cell suspension to the remaining wells (1,000 cells per well). Plates were centrifuged and then incubated at 37 C, 5% CO2 and 95% RH for 19 hours. Next, 25 nL of test compound in DMSO, Clavulanate (210 uM final concentration) or DMSO alone (0.5% final concentration) were added to the appropriate wells and incubated for 30 minutes at 37 C, 5% CO2 and 95% RH.
Then, 1 ul of the fluorogenic CCF4-AM substrate, prepared according to manufacturer's protocol (LiveBLAzer, Invitrogen), was added to each well. After 2 hours of incubation at 25 C, well fluorescence was measured on the EnVision plate reader (PerkinElmer Life Sciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 535 nm and 450 nm. Fluorescence values measured for each channel were corrected by subtracting "background" fluorescence, i.e. fluorescence measured in wells containing media only. Background corrected fluorescence emission values were then used to calculate a ratio for each well, according to the following mathematical expression:
Ratio = I450_nm / I535_nm
I represents the measured fluorescence emission intensity at the enumerated wavelength in nm.
Percent inhibition was calculated from the median ratio as follows:
%_Inhibition = 100 * ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) )
Test_Compound is defined as wells containing test compound
Low_Control is defined as wells containing 0.5% DMSO (0% inhibition)
High_Control is defined as wells containing 210 uM clavulanate in 0.5% DMSO (100% inhibition).
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-22, and for inactive compounds 22-0.
List of Reagents:
Stably-transfected CHO cell line coexpressing TLR9-Bla(b) and MyD88-Bla(a) (supplied by Assay Provider)
LiveBLAzer FRET-B/G Loading Kit (Invitrogen, part K1030)
Potassium Clavulanate (Sigma, part 33454)
DMEM, High Glucose (Invitrogen, part 11965-092)
DMEM, High Glucose, Phenol Red Free (Invitrogen, part 21063-029)
Fetal Bovine Serum (Invitrogen, part 26140-079)
NEAA (Invitrogen, part 11140-050)
Hepes (Invitrogen, part 15630-080)
Pen/Strep (Invitrogen, part 15640-055)
L-Glutamine (Invitrogen, part 25030-081)
Sodium Pyruvate (Invitrogen, part 11360-070)
Geneticin (Invitrogen, part 10131027)
T-175 Flasks (Nunc, part 159910)
1536-well plates (Greiner, part 789072)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)