The Scripps Molecular Screening Center's chemical probes lead to the validation of a new target in sepsis:

Target: ADAMTS

 MLSCN Inhibitor Probe Report

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-13

 MLSCN Inhibitor Probe Report

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: MMP-8

 MLSCN Inhibitor Probe Report

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: NPY-Y2

MLPCN Antagonist Probe Report

Due to its expression profile and biological action, NPY Y2 is an attractive GPCR target for anxiolytic research. Additionally, Y2 is predicted to be a therapeutic target in alcoholism. It has been reported, however, that the complex structure and high molecular weight of BIIE 0246 (the current NPY Y2 antagonist) limit its usefulness as an in vivo pharmacological tool (2). It is therefore necessary to produce brain penetrant, high affinity selective ligands for the Y2 receptor.

Probe Structure & Characteristics: Twelve compounds belonging to 4 different structural scaffolds that met probe criteria as being non-toxic, cell penetrant, high affinity, and selective Y2 antagonists with IC50 values less than 10 µM for NPY Y2 in CNGC cell-based assays were identified. Additionally, at least one compound from each scaffold (5 compounds in total) identified from the original uHTS assays demonstrated submicromolar Ki values, as well as higher brain-penetrance than the current NPY Y2 antagonist, BIIE 0246, therefore satisfying CPDP goals.

Target: P97

Misfolded proteins accumulate in the endoplasmic reticulum (ER) in response to environmental stress. To reduce the burden these proteins place on the secretory pathway, eukaryotic cells have evolved a process, known as ER-associated degradation (ERAD), to recognize and eliminate these proteins. The highly conserved p97 ATPase functions in ERAD by hydrolyzing ATP needed to export ubiquitinated substrates to the cytosol for degradation by the proteasome. The discovery of p97 missense mutations in a genetic form of human dementia, the localization of p97 in ubiquitylated inclusions in affected neurons of amyotrophic lateral sclerosis (ALS) and Parkinson’s disease, and the overproduction of p97 in multiple cancers, suggests that p97 has diverse and essential cellular roles. Thus, the identification of probes that selectively target p97 activity may provide insights into the biological roles of P97.

The Probe Report describing TSRI probe development efforts and compounds of interest resulting from the P97 campaign have been submitted to the NIH and manuscripts submitted for publication. Due to confidentiality agreements, this P97 Probe Report will be embargoed until these documents are officially accepted. Please contact , TSRI, for more information.

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 agonist 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.

Target: RBBP9

MLSCN Inhibitor Probe Report

The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which activates genes essential for DNA replication. Due to the critical role of RB in regulating the cell cycle, factors that bind and regulate RB activity are considered valuable targets for preventing tumorigenesis. One such protein, RB binding protein 9 (RBBP9), is widely expressed in different tissues and upregulated in certain tumors. The RBBP9 protein contains an alpha/beta hydrolase fold which belongs to the DUF1234 domain superfamily of unknown function. Although an enzymatic activity of RBBP9 has not been reported, this protein does react with activity-based probes that target serine hydrolases, suggesting that it is a functional enzyme. Also consistent with this premise, the crystal structure of RBBP9 was recently solved and revealed a well-structured active site with a properly arranged catalytic triad. A role for RBBP9 in cellular transformation came from studies showing that RBBP9 mRNA expression is increased in transformed rat liver cell lines and human liver tumor biopsies. RBBP9-overexpressing cells form tumors when implanted into immuno-deficient mice, and RBBP9 overexpression confers resistance to TGF-β1-induced growth inhibition through its interaction with Rb and displacement of E2F. RBBP9 is also suggested to play a role in gender-related differential responses to radiation-induced cell proliferation. As a result, the identification of compounds that selectively inhibit RBBP9 activity may provide valuable probes for the study of apoptosis, cell cycle, and tumorigenesis.

Target: ROCK

MLSCN Inhibitor Probe Report

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

MLSCN Agonist Probe Report

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.

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

inhibitor

In atherosclerosis, kidney transplantation and other disease, inappropriate inflammatory esponses contribute to poor patient outcomes. Toll-like receptor (TLR) signaling has been strongly mplicated in the inflammatory response. Toll-like receptors (TLRs) recognition of microbial components and partner proteins signaling are important elements of the innate immune response. TLRs are type I ransmembrane proteins consisting of an extracellular domain and an IL1R homologous intracellular domain and are comprised of at least 9 members. One of the TLRs, TLR4, recognizes lipopolysaccharides and signals through the shared adapter protein, MyD88 leading to activation of the FKB pathway. TLR4 signals enhance the immune response to bacterial infections but excessive ignaling leads to severe inflammation and septic shock.

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. [3]. Screening the 6,000 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(Table 1). 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.