The Scripps Molecular Screening Center's chemical probes lead to the validation of a new target in sepsis.
- Niessen, F., F. Schaffner, C. Furlan-Freguia, R. Pawlinski, G. Bhattacharjee, J. Chun, C.K. Derian, P. Andrade-Gordon, H. Rosen, and W.Ruf. 2008. Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation.
Probes
| ADAMTS | MMP-8 | MMP-13 | RAR | ROCK | S1P1 | S1P2 | S1P3 | SF1 | SHP | TLR4 |TSRI has submitted a total of 12 Probe Reports to the NIH. These Probe Reports covered a wide range of molecular targets, as listed below.
Target: ADAMTS
ADAMTS-4 inhibition was assayed against a subset (n=960) of the Library of
Pharmacologically Active Compounds1280 (LOPAC1280) (catalog no. LO1280, Sigma),
using a collagen model FRET substrate (fSSPa). Five compounds that inhibited
ADAMTS-4 activity greater than the hit-cuttoff of 59.5% (calculated as the average %
inhibition plus 3 times the standard deviation) at a concentration of 1 μM were identified
(piceatannol; (R, R)-cis-diethyltetrahydro-2, 8-chrysenediol; (S)-(+)-camptothecin; IIK7;
and 8-(p-sulfophenyl)theophylline. Secondary dose response studies and reversed-phase
high-performance liquid chromatography (RP-HPLC) assays were performed to eliminate
compounds that inhibit nonspecifically (e.g., interact with the substrate) or interfere with
substrate fluorescence. Only piceatannol (CID: 667639/ SID: 24278620), a red wine
polyphenolic compound and nonreceptor tyrosine kinase inhibitor, was confirmed as a
novel inhibitor of ADAMTS-4, with an IC50 value of 1 μM. Because collagen model
FRET substrates such as fSSPa have distinct conformational features that may interact
with exosites, nonactive site-binding inhibitors can be identified via this approach.
Target: MMP-8
The MMP-8 probe was originally identified from ultra high throughput screens for MMP-13 inhibitors. We used a series of assays employing secondary substrate binding sites (exosites) as collagen model FRET substrates to determine the activities of compounds against the collagenolytic enzyme MMP-13. Primary ultra high throughput screening tested the ability of 64,925 compounds to inhibit MMP-13-mediated cleavage of the fluorogenic triple-helical peptide (fTHP), modeled after the consensus binding and cleavage site in type I-III collagens. A total of 46 compounds were active in this screen and selected for further testing. Additional assays including RP-HPLC selectivity screening, ABPP selectivity profiling, and substrate assay IC50 screens against other collagenases such as MMP-1 and -8, as well as other proteases such as MMP-9, revealed a single compound as a selective, potent MMP-8 inhibitor. This compound demonstrated an improvement over prior art. The properties of this compound and screening details are provided in this MMP-8 probe report.
Target: MMP-13
We used a series of assays employing secondary substrate binding sites (exosites) as collagen model FRET substrates to determine the activities of compounds against the collagenolytic enzyme MMP-13. Two compounds with a core pyrimidinedione structure were identified as selective MMP-13 inhibitors. Primary ultra high throughput screening tested the ability of 64,925 compounds to inhibit MMP-13-mediated cleavage of the fluorogenic triple-helical peptide (fTHP), modeled after the consensus binding and cleavage site in type I-III collagens. A total of 46 compounds were active in this screen and selected for further testing. Additional assays including RP-HPLC selectivity screening, ABPP selectivity profiling, and substrate assay IC50 screens against other collagenases such as MMP-1 and -8, as well as other proteases such as MMP-9, revealed two compounds as selective MMP-13 inhibitors. These compounds demonstrated improvements over prior art. The properties of these compounds and additional screening details are provided in this MMP-13 probe report.
Target: RAR
We describe here the syntheses and initial structure activity relationship (SAR) studies of RAR isoform inverse agonists based on isoquinolinone scaffolds. One of the RAR selective probes characterized here (SID = 46499846) is a novel analog of compounds originally identified as active against SF-1 in PubChem BioAssays AID 525, 599, and 600. However, not all assays for the RAR campaign are found in PubChem. These assays, including analog synthesis, nuclear receptor (NR) library profiling assay, and cytotoxicity (CellTiter-Glo) assays are detailed here. Please refer to the original SF-1 and SF-1 optimization probe reports for details .
The inverse agonisht Probe Report describing TSRI probe development efforts and compounds of interest resulting from the RAR inverse agonist campaign has been submitted to the NIH and manuscripts submitted for publication. Due to confidentiality agreements, this RAR Probe Report will be embargoed until these documents are officially accepted. Please contact , TSRI, for more information.
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Target: ROCK
Rho-Kinase is a serine/threonine kinase involved in the regulation of smooth muscle contraction and cytoskeletal reorganization of nonmuscle cells. Its inhibition is known to promote the smooth muscle relaxation. Thus, small-molecule inhibitors of Rho-Kinase may be effective probes for treatment of cerebral vasospasm and potentially effective for treatment of angina, hypertension, arteriosclerosis, and erectile dysfunction.
This experiment’s specific aim was to find potent inhibitors for Rho-Kinase. “Kinase-Glo᾿, an ATP depletion assay was used to find inhibitors that are specific to the ATP binding site.
Three primary structural classes have been used as ROCK inhibitors: The isoquinoline scaffold, the 4-aminopyridine scaffold, and the indazole scaffold. Among these three, the indazoles contains perhaps the most potent ROCK inhibitors based on data from published papers and patent applications, but this scaffold is the least well characterized both in vitro and in vivo. Thus, several small focused libraries around 5-aminoindazole were prepared in Scripps Florida in order to further explore this scaffold as potential novel ROCK inhibitors. After in-house biological assays, SR-715 was identified as a good candidate from the 1-(4-(1H-indazol-5-yl amino)piperidin-1-yl)-2-hydroxy(or 2-amino) sub-library.
Target: S1P1
MLSCN Probe Report of 
Agonist, Antagonist
Obtain commercially available probes for S1P1 from:
Avanti Polar Lipids
Cayman Chemical
508 structures were identified as active in the primary assay and EC50
values were determined in the agonist (AG), potentiator (PTR), and
parental cell line of S1P1.
The 508 structures were clustered using a 0.6 similarity threshold and
Leadscope fingerprints to identify 109 clusters and 169 singletons. The top
10 clusters and singletons were identified based on their best activity in
the agonist assay and all clusters that show activity in the parental assay were
removed.
Target: S1P2
The Probe Report describing TSRI probe development efforts and compounds of interest resulting from the S1P2 agonist campaign has been submitted to the NIH and manuscripts submitted for publication. Due to confidentiality agreements, this S1P2 Probe Report will be embargoed until these documents are officially accepted. Please contact , TSRI, for more information.
Target: S1P3
65 structures were identified as active in the primary assay and EC50
values were determined in the agonist assay for S1P3.
The 65 structures were clustered using a 0.7 similarity threshold and
Leadscope fingerprints to identify 13 clusters and 19 singletons. The top 10
clusters and singletons were identified based on their best activity.
Structural classes that show activity against S1P1 were removed.
Target: SF1
MLSCN initial inhibitors, and modified inhibitors Probe Reports
The Steroidogenic factor 1 (SF-1, also known as NR5A1) is a transcription factor belonging to the superfamily of nuclear receptors (NRs). Evidence from deletion studies performed in vivo suggests that SF-1 is implicated in cancer and obesity. Whereas most of the NRs has been extensively characterized, SF-1 still remains poorly investigated at a pharmacological level. In this report, we describe the identification of selective chemical probes of SF-1 by ultra high-throughput screening. A set of 64,908 compounds from the MLSCN library has been screened in a miniaturized 1536-well plate format transactivation cell-based assay, allowing the selection of 359 primary hits. We used a closely related cell-based assay targeting another nuclear receptor, the Retinoic Acid Receptor-related orphan receptor A (RORA), as a counterscreen to remove both promiscuous and non-selective compounds. Two analog compounds showing a submicromolar selective inhibition of SF-1 have been identified. These compounds represent valuable investigational tools that may help characterize SF-1 therapeutic potential. | Top |
Target: SHP
Probe development efforts have identified activators of the orphan nuclear receptor SHP (small heterodimer partner). A probe report that summarizes the assays performed for this campaign and that details the structures and activities of the agonist probes has been submitted to the NIH. Due to confidentiality agreements, the SHP Probe Report will be embargoed until further notice.
Please contact , TSRI, for more information.
Target: TLR4
We screened the Maybridge HitFinder library consisting of 16,000 compounds and reported the results of
the screen, counterscreening and followup characterization of the confirmed hits. Screening the
16,000 compound Maybridge Hitfinder collection resulted in a plate average Z’ of 0.74. We selected 45
primary hits of which 10 confirmed in the primary assay. Dry powder aliquots of these 10
compounds were purchased from Maybridge and used for the remaining characterizations. A fluorescent
microscope counterscreen using Hela cells containing full-length beta-lactamase was run with the 10
confirmed hits. The counterscreen assay eliminated 5 false positive compounds that directly inhibit betalactamase.
Compounds were tested at 10 uM for inhibition of MyD88 co-immunoprecipitation with TLR4CD. In the
time course study, 4 of the 5 compounds were active after 120 minutes. The expression levels
of TLR4CD and MyD88 (input) were not changed by the addition of test compounds; indicating that the
compounds directly affect the binding between TLR4 and MyD88.
Compounds were also tested for their inhibitory effects on TLR4 signaling. LPS stimulation was
blocked by 50% in the presence of 0.37 uM of SID 26543390.
