Ultrahigh-throughput Screening Research Articles

Abstract 4763: Targeting p300 for inhibiting triple negative breast cancer

Abstract Breast cancer (BC) can be classified into four main subtypes: normal-like, HER2 overexpressing, luminal A and B, and basal-like BC. Normal-like BC cells exhibit gene expression pattern resembling basal epithelial cells. Erb-B-2/HER2-positive BC cells display high levels of Her2 expression. Luminal tumors show heightened expression of luminal cytokeratins and genes specific to the luminal epithelial cells, and are generally ER positive. Basal-like BC is characterized by the lack of expression of ER, progesterone receptor (PgR), and HER2. Triple-negative BC (TNBC) is defined by the lack of expression of ER, PgR and HER2 with distinct molecular, histological and clinical characteristics. Among the four BC subtypes, TNBC shares many of the molecular features of basal-like BC. Patients with TNBC have worse prognosis than those with other types of BC due to residual diseases that are resistant to chemotherapy and display heightened tendency to disseminate to distant organs. Available targeted therapies are ineffective for TNBC. Interestingly, cells derived from TNBC contain an elevated proportion of tumor cells with stem cell traits. These cells, known as cancer stem cells (CSCs), can self renew, differentiate into other types of cells in the tumor bulks, and efficiently promote tumor regrowth. CSCs are highly resistant to chemotherapy due to the expression of antiapoptotic genes. Several highly active signaling pathways, including cytokine-JAK-STAT3, Wnt-β-catenin, and Hippo-TAZ cascades, support the survival and sustained self-renewal of CSCs. One remarkable feature of these diverse signaling pathways is the reliance on the functions of the coactivators and acetyltransferases p300 and its paralog CBP for the expression of the effector genes that underlie the CSC phenotypes. We therefore hypothesize that inhibition of p300/CBP is an effective strategy for treating TNBC. Using an unbiased ultra high-throughput screening, we have identified small chemical compounds that were highly toxic to TNBC cell line MDA-MB-231 and potently inhibited the enzymatic activity of p300. Validation experiments revealed that a specific compound, L002, with the IC50 of 1.98 µM against p300 in vitro suppressed histone acetylation at several known sites of p300 acetylation in cell-based assay. L002 was highly toxic to MDA-MB-231 cells but much less so to MCF7 derived from a luminal BC. Experiments are underway to examine the effects of L002 on the survival of breast CSCs. (This work was supported by Bankhead-Coley Cancer Research Program of the Florida Department of Health.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4763. doi:1538-7445.AM2012-4763

A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution

In vitro screening systems based on the coupled transcription and translation of genes using cell-free systems have a number of attractive features for protein engineering and directed evolution. We present a completely in vitro ultrahigh-throughput screening platform using droplet-based microfluidics. Single genes are compartmentalized in aqueous droplets, dispersed in inert carrier oil, and amplified using the polymerase chain reaction (PCR). After amplification, the droplets, now containing 30,000 copies of each gene, are fused one-to-one with droplets containing a cell-free coupled transcription-translation (IVTT) system and the reagents for a fluorogenic assay. Fluorescence-activated electrocoalescence with an aqueous stream is then used to selectively recover genes from droplets containing the desired activity. We demonstrate, by selecting mixtures of lacZ genes encoding the enzyme β-galactosidase and lacZmut genes encoding an inactive variant, that this system can sort at 2000 droplets s(-1): lacZ genes were enriched 502-fold from a 1 : 100 molar ratio of lacZ : lacZmut genes. Indeed, the false positive and false negative error rates were both <0.004 and the results indicate that enrichment is not limited by the sorting efficiency, but by the co-encapsulation of multiple genes in droplets, which is described by the Poisson distribution. Compared to screening using microtiter plate-based systems, the volume and cost of PCR and IVTT reagents are reduced by almost 10(5)-fold, allowing the screening of 10(6) genes using only 150 μL of reagents.

Abstract B22: Next-generation markers and targets in breast cancer: an integrative omics approach.

Abstract Introduction: Although the complete human genome sequence and ultra high throughput screening (HTP) techniques have provided us a large number of Omics based potential molecular markers and drug targets, till date, most of these markers and targets fail to pass the initial phases of clinical trials (CTs) for any given cancer creating a big obstacle in developing personalized anticancer therapeutics. Therefore, a new strategy is required to analyze these Omics data and subsequent development of superior markers and targets. This study was aimed to explore molecular insights and to identify next-generation targets and targeted therapy in breast cancer (BC) based on an integrative omics approach. Methods and Results: Using various Omics data for BC and an integrative bioinformatics strategy, we found that [1] there are more than one cross talking signalling pathways so the targets present in most BC cases. Therefore Herceptin like single targeting (HER2) drug alone cannot be 100% effective. [2] Four to five key transcription factors (TFs) directly regulate key tumorigenic pathways. [3] These TFs also regulate their targets through a number of key miRNAs. [4] Using feedback loop regulation, these miRNAs also regulate their regulatory TFs. [5] Such TFs and miRNAs can also regulate cancer stem cell micro-environment via onco-fetal oncogenes and pluripotent stem cell factors using similar feedback loop mechanism. [6] Identified TFs and miRNAs are differentially expressed in BC and may be used as early diagnostic and prognostic markers. [7] Upregulated TFs and miRNAs may be used as superior drug targets and [8] downregulated miRNAs may be considered as candidate therapeutics. Discussion: Our integrative analysis demonstrates that heterogeneous signalling cross talks are prevalent in BC aetiology. Therefore, patient specific drug targets should be identified and multi-targeting approach must be recommended. Similarly, identified new markers and targets are required to be validated for their efficacy in CTs and in developing next generation targeted therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B22.

Identification of Small-Molecule Inhibitors of the Colorectal Cancer Oncogene Krüppel-like Factor 5 Expression by Ultrahigh-Throughput Screening

The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium, where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the public domain of NIH, the Molecular Libraries Probe Production Centers Network library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was done in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC(50) < 10 μmol/L for KLF5 inhibition and EC(50) > 10 μmol/L for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized on the basis of computational, Western blot, and cell viability analyses. Both of these compounds, and two newly synthesized structural analogs of CID 5951923, significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results show the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5.

Molecular diversity and natural products

Natural products have long represented the mayor source ofdrugs,andeventodaytheyremainattheforefrontofpharma-ceutical research. Between 1982 and 2002, 28% of the NewChemicalEntities(NCE)approvedbytheFDAwerenaturalproducts or derivatives of natural products [1], a very highshare, since only less than 1% of the characterized organiccompounds are of natural origin. Natural products are stillplaying a major role in the realm of anti-cancer and anti-infectivedrugdiscovery,buttheirrelevanceinotherfieldshasconstantly declined in the wake of the introduction of “hun-gry” methodologies like combinatorial chemistry and ultra-high throughput screening. More recently, a resurgence isbeingwitnessed,owingtothegrowingawarenessthatnaturalproductsrepresentnotonlypotentialNewChemicalEntities(NCE), but also privileged structures to explore the chemi-cal space around a target. Plants, fungi, bacteria, and marineorganisms make medicinally interesting compound for rea-sonsoftheirown,thatnotnecessarilyoverlapwiththeneeds.Nevertheless, natural products are made by enzymes, andtheir shape must be complementary to that of a biologicalsurface. This makes them privileged structures in terms ofchemicalspaceofbiologicalrelevance.Naturalproductsare“sticky”towardbiologicalsurfacesnotonlyforthepresenceof functional groups capable to bind biomolecules throughthewholearsenalofnon-covalentinteraction(hydrogen-and

Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells

A compartmentalized tyramide labeling system (CoaTi) employing flow cytometry for sorting of yeast cells was developed as ultrahigh-throughput screening for Glucose oxidase (GOx) from Aspergillus niger. CoaTi combines in vitro compartmentalization technology with the CARD reporter system which uses fluorescein tyramide labels for detection of peroxidase activity. Physical connection between cells and fluorescein tyramide radicals was achieved by compartmentalization of yeast cells inside microdroplets of single water-in-oil emulsions. After reaction cells were recovered from single emulsions and sorted by flow cytometry, an error prone PCR mutant library of Glucose oxidase (GOx) containing 10(7) cells and ~10(5) of different GOx variants was screened. Mutagenic conditions of GOx mutant library were selected to generate <1 % of active GOx population in order to explore influence of high mutation frequency on GOx activity. GOx variant Mut12 that contains 5 mutations (N2Y, K13E, T30V, I94V, K152R) showed a 1.2 times decreased K(m) (22.0 vs 18.1 mM) and a 2.7 fold increased k(cat) (150 s(-1) vs 54.8 s(-1)) compared to wt GOx. Compared to the employed parent B11 GOx (16 mM, 80 s(-1)) it has a slightly increased K(m) and 1.8 times increased k(cat).

Discovery of PDK1 Kinase Inhibitors with a Novel Mechanism of Action by Ultrahigh Throughput Screening

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Correction for Agresti et al., Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

Correction for Agresti et al., Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify approximately 100 variants comparable in activity to the parent from an initial population of approximately 10(7). After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen approximately 10(8) individual enzyme reactions in only 10 h, using < 150 microL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

Microfluidic ultra–high throughput screening platform for discovery of new proteins or nucleic acids

Microfluidic ultra–high throughput screening platform for discovery of new proteins or nucleic acids

Effects of 2,4-diaminoquinazoline derivatives on SMN expression and phenotype in a mouse model for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA), one of the most common genetic causes of infant death, results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of survival motor neuron (SMN) protein. In humans, the SMN gene is duplicated; SMA results from the loss of SMN1 but SMN2 remains intact. SMA severity is related to the copy number of SMN2. Compounds which increase the expression of SMN2 could, therefore, be potential therapeutics for SMA. Ultrahigh-throughput screening recently identified substituted quinazolines as potent SMN2 inducers. A series of C5-quinazoline derivatives were tested for their ability to increase SMN expression in vivo. Oral administration of three compounds (D152344, D153249 and D156844) to neonatal mice resulted in a dose-dependent increase in Smn promoter activity in the central nervous system. We then examined the effect of these compounds on the progression of disease in SMN lacking exon 7 (SMNDelta7) SMA mice. Oral administration of D156844 significantly increased the mean lifespan of SMNDelta7 SMA mice by approximately 21-30% when given prior to motor neuron loss. In summary, the C5-quinazoline derivative D156844 increases SMN expression in neonatal mouse neural tissues, delays motor neuron loss at PND11 and ameliorates the motor phenotype of SMNDelta7 SMA mice.

Ligand-Binding Pocket Shape Differences between Sphingosine 1-Phosphate (S1P) Receptors S1P1 and S1P3 Determine Efficiency of Chemical Probe Identification by Ultrahigh-Throughput Screening

We have studied the sphingosine 1-phosphate (S1P) receptor system to better understand why certain molecular targets within a closely related family are much more tractable when identifying compelling chemical leads. Five medically important G-protein-coupled receptors for S1P regulate heart rate, coronary artery caliber, endothelial barrier integrity, and lymphocyte trafficking. Selective S1P receptor agonist probes would be of great utility to study receptor subtype-specific function. Through systematic screening of the same libraries, we identified novel selective agonist chemotypes for each of the S1P1 and S1P3 receptors. Ultrahigh-throughput screening (uHTS) for S1P1 was more effective than that for S1P3, with many selective, low nanomolar hits of proven mechanism emerging. Receptor structure modeling and ligand docking reveal differences between the receptor binding pockets, which are the basis for subtype selectivity. Novel selective agonists interact primarily in the hydrophobic pocket of the receptor in the absence of headgroup interactions. Chemistry-space and shape-based analysis of the screening libraries in combination with the binding models explain the observed differential hit rates and enhanced efficiency for lead discovery for S1P1 versus S1P3 in this closely related receptor family.

KD5170, a novel mercaptoketone-based histone deacetylase inhibitor, exerts antimyeloma effects by DNA damage and mitochondrial signaling

Histone deacetylase inhibitors have emerged as promising anticancer drugs. Using an unbiased ultrahigh throughput screening system, a novel mercaptoketone-based histone deacetylase inhibitor series was identified that was optimized to the lead compound, KD5170. KD5170 inhibited the proliferation of myeloma cell lines and the viability of CD138(+) primary myeloma cells by induction of apoptosis, accompanied by an increase of acetylation of histones and activation of caspase-3, caspase-8, and caspase-9. Treatment with KD5170 caused a loss of mitochondrial membrane potential resulting in release of apoptogenic factors such as cytochrome c, Smac, and apoptosis-inducing factor. Furthermore, KD5170 induced oxidative stress and oxidative DNA damage in myeloma cells as evidenced by the up-regulation of heme oxygenase-1 and H2A.X phosphorylation. Combination of KD5170 with proteasome inhibitor bortezomib or tumor necrosis factor-related apoptosis-inducing ligand synergistically enhanced the antimyeloma activity. We further found that resistance of myeloma cells to KD5170 was associated with activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway under treatment with KD5170. Pretreatment with the mitogen-activated protein kinase inhibitor U0126 restored sensitivity to KD5170, suggesting that the combination of KD5170 with U0126 could overcome drug resistance. Growth of myeloma tumor xenografts in KD5170-treated nude mice was significantly inhibited and survival was prolonged. Histone acetylation was increased in spleen and tumor tissues of animals treated with KD5170. Our data indicate that KD5170 has potent antimyeloma activity in vitro and in vivo, which is mediated by DNA damage and mitochondrial signaling and subsequent induction of apoptosis.

KD7150, a Novel Mercaptoketone-Based HDAC Inhibitor, Exerts Potent Anti-Multiple Myeloma Effects In Vitro and In Vivo by Induction of DNA Damage and Mitochondrial Signaling.

Histone deacetylase (HDAC) inhibitors have emerged as a class of novel and promising anti-cancer agents and their activities are currently being investigated in both the pre-clinical and clinical settings. Using an unbiased, ultrahigh throughput screening system a novel mercaptoketone-based HDAC inhibitor KD5170 was identified. This novel non-hydroxamic acid non-benzamide compound was profiled for its anti-myeloma activity in vitro and in vivo. KD5170 inhibited the proliferation of multiple myeloma (MM) cell lines and the viability of CD138+ primary MM cells by induction of apoptosis. This was accompanied by an increase of acetylation of histones and activation of caspase-3, -8 and -9. Treatment with KD5170 caused a loss of mitochondrial membrane potential measured by JC-1 staining and resulted in release of apoptogenic factors such as cytochrome c, smac, and AIF from mitochondria. Furthermore, KD5170 induced oxidative stress and oxidative DNA damage in myeloma cells, as evidenced by the upregulation of heme oxygenase-1 and H2A.X phosphorylation, respectively. Combination of KD5170 with the proteasome inhibitor bortezomib or TNF-related apoptosis-inducing ligand synergistically enhanced the anti-myeloma activity. Resistance of MM cells to KD5170 was associated with activation of the ERK/MAPK pathway under treatment with KD5170. Pretreatment with the MAPK-inhibitor restored the sensitivity to KD5170, suggesting that the combination of KD5170 with a MAPK inhibitor (U0126) could overcome drug resistance. Growth of myeloma tumor xenografts in KD5170-treated nude mice was significantly inhibited (1401 mm3 ± 596 versus 442 mm3 ± 221; P = .009) and survival was prolonged (9 days and 17.5 days; P= .0095) compared to vehicle-treated mice. Accordingly, our in vivo data showed that histone acetylation was markedly upregulated in tela in spleen or tumor tissues of the animals treated with KD5170 as early as 2 hours. In conclusion, our data show that KD5170 has anti-myeloma activity in vitro and in vivo, mediated by the induction of apoptosis due to DNA damage and mitochondrial signaling.

Unsupervised Fluorescence Lifetime Imaging Microscopy for High Content and High Throughput Screening

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.

Ultrahigh‐Throughput Screening to Identify E. coli Cells Expressing Functionally Active Enzymes on their Surface

We show here that E. coli bacteria that display esterases or lipases on their cell surface together with horseradish peroxidase (HRP) are capable of hydrolysing carboxylic acid esters of biotin tyramide. The tyramide radicals generated by the coupled lipase-peroxidase reaction were short-lived and therefore became covalently attached to reactive tyrosine residues that were located in close vicinity on the surface of a bacterial cell that displayed lipase activity. Up to 120 000 biotinylated tyramide derivatives could be covalently coupled through HRP activation to the surface of a single living E. coli cell. Differences in cellular esterase activity were found to correlate with the amount of biotin tyramide deposited on the cell surface. Selective biotin tyramide labelling of cells that had lipase activity allowed their isolation by magnetic cell sorting from a 1:10(6) mixture of control cells. This strategy of covalently attaching a biotin label to esterase-proficient bacteria might open new avenues to ultrahigh-throughput screening of enzyme libraries for hydrolytic enzymes with enhanced activities or enantioselectivities.

Droplets as Microreactors for High‐Throughput Biology

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Droplets as Microreactors for High‐Throughput Biology

Inspired by the principles of biological evolution, biologists--and others--have in recent decades harnessed the power of "natural" selection to sift through huge libraries of genes and find those with desirable properties. At the same time, the demand for high-throughput biochemical and genetic assays and screens has driven the development of increasingly miniaturised assay systems. An exciting synergy is now emerging between these two fields, whereby the tools of ultrahigh-throughput screening promise to open up new directions in molecular engineering.

Functional Cell‐Surface Display of a Lipase‐Specific Chaperone

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

α-Methyltryptamine sulfonamide derivatives as novel glucocorticoid receptor ligands

α-Methyltryptamine sulfonamides were identified as human glucocorticoid receptor (hGR) ligands in an ultra high throughput screening (UHTS) campaign. Described will be the hit-to-lead activities, including parallel and single point analog synthesis to map the scaffold. Ligands were identified that exhibited 30 nM binding to hGR. The SAR and selectivity of these compounds will be discussed.