At A Glance



Intellectual Property & Code of Ethics

The Yale Center for Molecular Discovery has contributed to the following publications.  

Please click on the photos to read the papers. 

Scholl UI, Abriola L, Zhang C, Reimer EN, Plummer M, Kazmierczak BI, Zhang J, Hoyer D, Merkel JS, Wang W, Lifton RP: Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosterone-producing adenoma. J Clin Invest. 2017 Jun 30; 2017 Jun 12. PMID: 28604387

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.

Nishimura S, Mishra-Gorur K, Park J, Surovtseva YV, Sebti SM, Levchenko A, Louvi A, Gunel M. Combined HMG-COA reductase and prenylation inhibition in treatment of CCM. Proc Natl Acad Sci U S A. 2017 May 23;114(21):5503-5508. doi: 10.1073/pnas.1702942114. Epub 2017 May 12. PMID: 28500274

Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase and the N-bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in Drosophila glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.

Langdon CG, Platt JT, Means RE, Iyidogan P, Mamillapalli R, Klein M, Held MA, Lee JW, Koo JS, Hatzis C, Hochster HS, Stern DF. Combinatorial screening of pancreatic adenocarcinoma reveals sensitivity to drug combinations including bromodomain inhibitor plus neddylation inhibitor. Mol Cancer Ther. 2017 Mar 14. pii: molcanther.0794.2016. doi: 10.1158/1535-7163.MCT-16-0794. PMID: 28292938

Pancreatic adenocarcinoma (PDAC) is the fourth most common cause of cancer-related death in the United States. PDAC is difficult to manage effectively, with a five-year survival rate of only 5%. PDAC is largely driven by activating KRAS mutations, and as such, cannot be directly targeted with therapeutic agents that affect the activated protein. Instead, inhibition of downstream signaling and other targets will be necessary to effectively manage PDAC. Here, we describe a tiered single-agent and combination compound screen to identify targeted agents that impair growth of a panel of PDAC cell lines. Several of the combinations identified from the screen were further validated for efficacy and mechanism. Combination of the bromodomain inhibitor JQ1 and the neddylation inhibitor MLN4294 altered the production of reactive oxygen species in PDAC cells, ultimately leading to defects in the DNA damage response. Dual bromodomain/neddylation blockade inhibited in vivo growth of PDAC cell line xenografts. Overall, this work revealed novel combinatorial regimens, including JQ1 plus MLN4294, which show promise for the treatment of RAS-driven PDAC. Mol Cancer Ther; 16(6); 1041-53. ©2017 AACR.

Sulkowski PL, Corso CD, Robinson ND, Scanlon SE, Purshouse KR, Bai H, Liu Y, Sundaram RK, Hegan DC, Fons NR, Breuer GA, Song Y, Mishra-Gorur K, De Feyter HM, de Graaf RA, Surovtseva YV, Kachman M, Halene S, Günel M, Glazer PM, Bindra RS. 2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity. Sci Transl Med. 2017 Feb 1;9(375). pii: eaal2463. doi: 10.1126/scitranslmed.aal2463. PMID: 28148839

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5’-diphosphate-ribose) polymerase (PARP) inhibitors. This “BRCAness” phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

Nemec AA, Abriola L, Merkel JS, deStanchina E, DeVeaux M, Zelterman D, Glazer PM, Sweasy JB. DNA polymerase beta germline variant confers cellular response to cisplatin therapy. Mol Cancer Res. 2017 Jan 10. pii: molcanres.0227.2016. doi: 10.1158/1541-7786.MCR-16-0227-T. PMID: 28074003

Resistance to cancer chemotherapies leads to deadly consequences, yet current research focuses only on the roles of somatically acquired mutations in this resistance. The mutational status of the germline is also likely to play a role in the way cells respond to chemotherapy. The carrier status for the POLB rs3136797 germline mutation encoding P242R DNA polymerase beta (Pol β) is associated with poor prognosis for lung cancer, specifically in response to treatment with cisplatin. Here, it is revealed that the P242R mutation is sufficient to promote resistance to cisplatin in human cells and in mouse xenografts. Mechanistically, P242R Pol β acts as a translesion polymerase and prefers to insert the correct nucleotide opposite cisplatin intrastrand cross-links, leading to the activation of the nucleotide excision repair (NER) pathway, removal of crosslinks, and resistance to cisplatin. In contrast, wild-type (WT) Pol β preferentially inserts the incorrect nucleotide initiating mismatch repair and cell death. Importantly, in a mouse xenograft model, tumors derived from lung cancer cells expressing WT Pol β displayed a slower rate of growth when treated with cisplatin, whereas tumors expressing P242R Pol β had no response to cisplatin. Pol β is critical for mediating crosstalk in response to cisplatin. The current data strongly suggest that the status of Pol β influences cellular responses to crosslinking agents and that Pol β is a promising biomarker to predict responses to specific chemotherapies. Finally, these results highlight that the genetic status of the germline is a critical factor in the response to cancer treatment.Implications: Pol β has prognostic biomarker potential in the treatment of cancer with cisplatin and perhaps other intrastrand crosslinking agents. Mol Cancer Res; 15(3); 269-80. ©2017 AACR.

Farley-Barnes KI, McCann KL, Ogawa LM, Merkel J, Surovtseva YV, Baserga SJ. Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number. Cell Rep. 2018 Feb 13;22(7):1923-1934. doi: 10.1016/j.celrep.2018.01.056.

Ribosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2–3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90%) to be essential for the nucleolar functions of rDNA transcription (7), pre-ribosomal RNA (pre-rRNA) processing (16), and/or global protein synthesis (14). This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease.

Levin, M, Samuel, S, Merkel, J, Bickler, P. Varespladib (LY315920) appears to be a potent, broad-spectrum, inhibitor of snake venom phospholipase A2 and a possible pre-referral treatment for envenomation. Toxins 2016, 8(9), 248; doi:10.3390/toxins8090248. 

Working with external groups, YCMD optimized an enzymatic assay for soluble phospholipase A2 activity across 28 medically important snake venoms from six continents. Two related inhibitors were demonstrated to powerfully inhibit venoms displaying nanomolar and subnanomolar activity. Inhibitor testing preceded animal studies showing a survival benefit when the small molecule inhibitors were added before or after venom exposure.

Gale M, Sayegh J, Cao J, Norcia M, Gareiss P, Hoyer D, Merkel JS, Yan Q. Screen-identified selective inhibitor of lysine demethylase 5A blocks cancer cell growth and drug resistance. Oncotarget. Vol. 5. 2016; doi: 10.18632/oncotarget.9539. PMID: 27224921.

Lysine demethylase 5A (KDM5A/RBP2/JARID1A) is a histone lysine demethylase that is overexpressed in several human cancers including lung, gastric, breast and liver cancers. It plays key roles in important cancer processes including tumorigenesis, metastasis, and drug tolerance, making it a potential cancer therapeutic target. Chemical tools to analyze KDM5A demethylase activity are extremely limited as available inhibitors are not specific for KDM5A.

The Yan lab and YCMD characterized KDM5A using a homogeneous luminescence-based assay and conducted a high throughput screen of ~ 9,000 small molecules for inhibitors. Several compounds were identified that inhibited KDM5A with low μM in vitro IC50 values. Importantly, these compounds showed specificity and did not inhibit its close homologue KDM5B or the related H3K27 demethylases KDM6A and KDM6B.  One compound, was able to increase H3K4me3 levels in human cells and selectively inhibit the proliferation of cancer cells whose growth depends on KDM5A. This compound also hindered the emergence of drug-tolerence in EGFR-mutant lung cancer cells treated with gefitinib and HER2+ breast cancer cells treated with trastuzumab, highlighting the critical role of KDM5A demethylase activity in drug resistance. These small molecules identified from screening efforts in collaboration with YCMD are excellent tool compounds for further study of KDM5A’s demethylase activity and its contributions to cancer.

Kraehling JR, Chidlow JH, Rajagopal C, Sugiyama MG, Fowler JW, Lee MY, Zhang X, Ramírez CM, Park EJ, Tao B, Chen K, Kuruvilla L, Larriveé B, Folta-Stogniew E, Ola R, Rotllan N, Zhou W, Nagle MW, Herz J, Williams KJ, Eichmann A, Lee WL, Fernández-Hernando C, Sessa WC. Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells. Nat Commun. 2016 Nov 21;7:13516. doi: 10.1038/ncomms13516. PMID: 27869117

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

Surovtseva, Y., Jairam, V., Salem, AF., Bindra, RS., Herzon SB., Characterization of Cardiac Glycoside Natural Products as Potent Inhibitors of DNA Double-Strand Break Repair by a Whole-Cell Double Immunofluorescence Assay. J AM Chem Soc. 2016 Mar 23;138(11):3844-55. doi: 10.1021/jacs.6b00162. Epub 2016 Mar 9.

DNA repair pathways are being intensively investigated as targets for chemotherapeutic interventions. This paper describes development of an automated, whole-cell, high-throughput assay for the unbiased discovery of inhibitors of the non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways, the two primary pathways that ameliorate DNA double-strand breaks. The assay enables indirect measurement of DNA repair activity by monitoring the kinetics of γH2AX and 53BP1 foci resolution after treatment with ionizing radiation (IR) and candidate DNA damage response (DDR) inhibitor.  The paper also describes a unique platform of secondary assays to rank-order the potency of lead compounds and gain insights into their mechanism of action. The HTS assay was validated using known DDR and DNA damage checkpoint inhibitors, and applied to screen 2,366 structurally-diverse small molecules with known bioactivity. YCMD contributed to the publication by optimizing a high-throughput assay, conducting a screen, and running follow up assays which identified cardiac glycoside natural products as potent Inhibitors of DNA Double-Strand Break Repair.

Zhao, C., Brown, R., Tang, C., Hu, C., Schlieker C. (2016), Site-specific Proteolysis Mobilizes TorsinA from the Membrane of the Endoplasmic Reticulum in Response to ER Stress and B cell Stimulation. J. Biol. Chem. jbc.M115.709337. First Published on March 7, 2016,  doi:10.1074/jbc.M115.709337.

Torsin ATPases are the only representatives of the AAA+ ATPase family that reside in the lumen of the endoplasmic reticulum (ER) and nuclear envelope. Two of these, TorsinA and TorsinB, are anchored to the ER membrane by virtue of an N-terminal hydrophobic domain. Here we demonstrate that the imposition of ER stress leads to a proteolytic cleavage event that selectively removes the hydrophobic domain from the AAA+ domain of TorsinA, which retains catalytic activity. Both the pharmacological inhibition profile and the identified cleavage site between two juxtaposed cysteine residues are distinct from those of presently known proteases, suggesting that a hitherto uncharacterized, membrane-associated protease accounts for TorsinA processing. This processing occurs not only in stress-exposed cell lines but also in primary cells from distinct organisms including stimulated B cells, indicating that Torsin conversion in response to physiologically relevant stimuli is an evolutionarily conserved process. By establishing 5-nitroisatin as cell-permeable inhibitor for Torsin processing, we provide the methodological framework for interfering with Torsin processing in a wide range of primary cells without the need for genetic manipulation. 

Xiong, H., Reynolds, N. M., Fan, C., Englert, M., Hoyer, D., Miller, S. J. and Söll, D. (2016), Dual Genetic Encoding of Acetyl-lysine and Non-deacetylatable Thioacetyl-lysine Mediated by Flexizyme. Angew. Chem. Int. Ed.. doi:10.1002/anie.201511750.

Acetylation of lysine residues is an important post-translational protein modification. Lysine acetylation in histones and its crosstalk with other post-translational modifications in histone and non-histone proteins are crucial to DNA replication, DNA repair, and transcriptional regulation. We incorporated acetyl-lysine (AcK) and the non-hydrolyzable thioacetyl-lysine (ThioAcK) into full-length proteins in vitro, mediated by flexizyme. ThioAcK and AcK were site-specifically incorporated at different lysine positions into human histone H3, either individually or in pairs. We demonstrate that the thioacetyl group in histone H3 could not be removed by the histone deacetylase sirtuin type 1. This method provides a powerful tool to study protein acetylation and its role in crosstalk between post-translational modifications.

Carland F, Defries A, Cutler S, Nelson T: Novel Vein Patterns in Arabidopsis Induced by Small Molecules. Plant Physiol. 2016 Jan; 170(1):338-53. doi: 10.0004/pp.15.01540. PMCID: PMC4704596.

Leaf veins play a critical role in transporting water, nutrients, and signals. To identify regulators of vein patterning, Carland et al. screened more than 5000 structurally diverse small molecules for compounds that alter Arabidopsis thaliana leaf vein patterns. High throughput methods developed at YCMD included robotic dispensing of agarose into 96 well-plates and optimization of seed loading for a consistent one seed per well.   

Felsenstein KM, Saunders LB, Simmons JK, Leon E, Calabrese DR, Zhang S, Michalowski A, Gareiss P, Mock BA, Schneekloth JS Jr.: Small Molecule Microarrays Enable the Identification of a Selective, Quadruplex-Binding Inhibitor of MYC Expression. ACS Chem Biol. 2015 Nov 6. [Epub ahead of print] PMCID: PMC4719142.

The transcription factor MYC plays a pivotal role in cancer initiation, progression, and maintenance. However, it has proven difficult to develop small molecule inhibitors of MYC.  YCMD employed small molecule microarray technology to screen for compounds that bind to oncogenic MYC G-Quadruplex and potentially target gene expression.   Compounds were identified that bind to this structured DNA target and affect gene expression.  As a measure of selectivity, gene expression analysis and qPCR experiments demonstrated that MYC and several MYC target genes were downregulated upon treatment with this compound, while the expression of several other G-quadruplex driven genes was not affected.  Follow-up studies with scientists at the NCI (former YCMD scientist Jay Schneekloth and colleagues) showed that this compound is able to inhibit MYC induced cellular toxicity.

Jane-Wit D, Surovtseva YV, Qin L, Li G, Liu R, Clark P, Manes TD, Kashgarian M, Kirkiles-Smith NC, Tellides G, Pober J: Complement membrane attack complexes activate noncanonical NF–kB by forming an Akt+NIK+ signalosome on Rab5+ endosomes. Proc Natl Acade Sci USA, 2015 Aug 4;112(31):9686-91. doi:10.1073/pnas. 1503535112. Epub 2015 Jul 20. PMCID: PMC4534258.

The paper investigates the mechanism of non-canonical NF-kB pathway activation by complement membrane attack complexes (MAC).  The study elucidates important steps of pathway activation, describes the role of clathrin-mediated endocytosis and Rab5+ endosomes in MAC signaling, and has important translational implications by suggesting new therapeutic targets for controlling complement-mediated inflammation.  YCMD contributed to the publication by optimizing a high-throughput assay and conducting a genome-wide siRNA screen, which identified endocytosis and vesicular trafficking genes as important players in the MAC-induced non-canonical NF-kB pathway activation. 

Stachelek GC, Peterson-Roth E, Liu Y, Fernandez RJ 3rd, Pike LR, Qian JM, Abriola L, Hoyer D, Hungerford W, Merkel J, Glazer PM: YU238259 is a Novel Inhibitor of Homology-dependent DNA Repair that Exhibits Synthetic Lethality and Radiosensitization in Repair-deficient Tumors. Mol Cancer Res. 2015 Jun 26; 2015 Jun 26. PMCID: PMC4618103.

DNA repair pathways are potential targets for cancer therapy strategies. This paper describes a high throughput cell-based screen for small molecules that were preferentially toxic to cells lacking the FANCD2 and BRCA2 repair proteins, but had low toxicity in isogenic, repair-proficient cells. Work performed at YCMD included development of the synthetic lethal assay, high throughput screening, hit follow-up by the synthetic lethal assay, chemistry-based analysis of the hits and assessment of structure activity relationships (SAR), hit expansion by catalog and new molecule design and synthesis to improve the potency and solubility of the lead molecule. A novel series of compounds that are synthetically lethal to HDR-deficient cells was identified.

Langdon, C. G., Held, M. A., Platt, J. T., Meeth, K., Iyidogan, P., Mamillapalli, R., Koo, A. B., Klein, M., Liu, Z., Bosenberg, M. W. and Stern, D. F. (2015), The broad-spectrum receptor tyrosine kinase inhibitor dovitinib suppresses growth of BRAF-mutant melanoma cells in combination with other signaling pathway inhibitors. Pigment Cell & Melanoma Research. doi: 10.1111/pcmr.12376. PMID: 25854919.

While BRAF inhibitors have revolutionized treatment of mutant BRAF metastatic melanomas, resistance develops rapidly. Treatment with dovitinib in combination with several agents inhibits cell growth more effectively than either agent alone. Inhibition of BRAF-mutant melanoma and colorectal carcinoma cell growth was measured after incubation with combinations of dovitinib with other small molecules in a screen at YCMD. This work highlights the potential effectiveness of dovitinib with second agents to improve BRAF-mutant melanoma treatment, regardless of their sensitivity to BRAF inhibitors.

Scortegagna M, Lau E, Zhang T, Feng Y, Sereduk C, Yin H, De SK, Meeth K, Platt JT, Langdon CG, Halaban R, Pellecchia M, Davies MA, Brown K, Stern DF, Bosenberg M, Ronai ZA. PDK1 and SGK3 Contribute to the Growth of BRAF-Mutant Melanomas and Are Potential Therapeutic Targets. Cancer Res. 2015 Apr 1;75(7):1399-412. doi: 10.1158/0008-5472.CAN-14-2785. PMCID: PMC4383687.

Recent Braf::Pten mutant studies have shown PDK1 to be involved in melanoma development. Interactions between PDKi (a PDK1 inhibitor) and 45 other agents were determined in a “one versus many” screen at YCMD. Specifically, melanoma cell growth was measured after incubation with the PKDi combinations. This work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment.

Nelson JW, Plummer MS, Blount KF, Ames TD, Breaker RR. Small molecule fluoride toxicity agonists. Chem Biol. 2015 Apr 23;22(4):527-34. doi: 10.1016/j.chembiol.2015.03.016. PMCID: PMC4383687.

Fluoride is a ubiquitous anion that inhibits a wide variety of metabolic processes. Here, we report the identification of a series of compounds that enhance fluoride toxicity in Escherichia coli and Streptococcus mutans. These molecules were isolated by using a high-throughput screen (HTS) at YCMD for compounds that increase intracellular fluoride levels as determined via a fluoride riboswitch reporter fusion construct. A series of derivatives were synthesized at YCMD to examine structure-activity relationships, leading to the identification of compounds with improved activity.

Narayan A, Bommakanti A, Patel AA. High-throughput RNA profiling via up-front sample parallelization. Nat Methods. 2015 Apr; 12(4):343-6. doi: 10.1038/nmeth.3311b. PMCID: PMC4451056.

Analysis of gene expression within diverse specimens underpins our understanding of cellular physiology and disease. The Patel laboratory utilized the liquid handling infrastructure at YCMD to array microRNA to allow for an efficient platform for META RNA profiling.


Farley KI, Surovtseva Y, Merkel J, Baserga SJ. Determinants of mammalian nucleolar architecture. Chromosoma. 2015 Feb 12. doi: 10.1007/s00412-015-0507-z. PMCID: PMC4534358.

The nucleolus is a highly dynamic organelle with a complex structure.  In this paper, nucleolar architecture was assayed at YCMD using a panel of mammalian cell lines.  Specifically, nucleoli were immunostained using anti-fibrillarin antibodies and imaged on InCell2200, and their number and morphology was quantified using Cell Profiler pipeline developed at YCMD.  We showed that nucleolar number varies greatly among mammalian cells, demonstrating the complexity of processes governing nucleolar development. 

Popa A, Zhang W, Harrison M, Goodner K, Goodwin EC, Kazakov T, Burd C, DiMaio, D. (2015) Direct binding of the retromer to human papillomavirus 16 minor capsid protein L2 mediates endosome exit during virus entry.  PLoS Pathogens, e1004699. doi: 10.1371/journal.ppat.1004699. PMCID: PMC4334968.

This paper presents follow up studies performed on hits identified in an siRNA screen conducted at YCMD.

Hebda JA, Magzoub M, Miranker AD. Small molecule screening in context: Lipid-catalyzed amyloid formation. Protein Science. Vol. 23. IS 10. SN: 1469-896X. pp. 1341 – 1348. 2014. doi: 10.1002/pro.2518. PMID: 25043951; PMCID: PMC4286996.               

Islet Amyloid Polypeptide (IAPP) is a 37-residue hormone cosecreted with insulin by the β-cells of the pancreas. Amyloid fiber aggregation of IAPP has been correlated with the dysfunction and death of these cells in type II diabetics. The likely mechanisms by which IAPP gains toxic function include energy independent cell membrane penetration and induction of membrane depolarization. Although the relationship between amyloid formation and toxicity is poorly understood, the fact that conditions promoting one also favor the other suggests related membrane active structural states. This paper describes a novel high throughput screening protocol that capitalizes on this correlation to identify compounds that target membrane active species. Out of 960 known bioactive compounds screened, 37 compounds were identified as active toward IAPP fiber formation. Several compounds tested in secondary cell viability assays also demonstrate cytoprotective effects. YCMD contributed to the publication by assisting in designing and performing the screen of the MicroSource Gen-Plus library described in the publication.

Colavito S, Zou MR, Yan Q, Nguyen DX, Stern DF Significance of glioma-associated oncogene homolog 1 (GLI1) expression in claudin-low breast cancer and crosstalk with the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway. Breast cancer research : BCR. 2014; 16(5):444. PubMed [journal] PMCID: PMC4303124.

The recently identified claudin-low subtype of breast cancer is enriched for cells with stem-like and mesenchymal-like characteristics, has a poor prognosis, and has few targeted treatment options. Using a high throughput inhibitor screen at YCMD, we identified high expression of glioma-associated oncogene homolog 1 (GLI1), the effector molecule of the hedgehog (Hh) pathway, as a critical determinant of cell lines that have undergone an epithelial to mesenchymal transition (EMT). Our results uncover crosstalk between NFκB and GLI1 signals and suggest that targeting these pathways may be effective against the claudin-low breast cancer subtype.

Zhang W, Kazakov T, Popa A, Goodwin EC, DiMaio D. (2014) Vesicular trafficking of incoming human papillomavirus 16 to the Golgi apparatus and endoplasmic reticulum requires g-secretase activity. mBio 5(5):e01777-14. doi:10.1128/mBio.01777-14. 2014. PMCID: PMC4172078.

This paper presents follow up studies performed on hits identified in an siRNA screen conducted at YCMD.

Pirruccello M, Nandez R, Idevall-Hagren O, Alcazar-Roman A, Abriola L, Berwick SA, Lucast L, Morel D, DeCamilli P Identification of inhibitors of inositool 5-phosphatases through multiple screening strategies. ACS Chem Biol. 2014 Jun 20;9(6):1359-68. doi: 10.1021/cb500161z. PMCID: PMC4076014.

Small molecules that inhibit or activate these enzymes have potential therapeutic applications and will be powerful research tools.  This paper describes a high throughput biochemical screen performed at YCMD and the follow up assays that were done in the hits.  Work performed at YCMD included developing three complimentary in vitro assays against multiple enzymes and substrates, high throughput screening against two enzymes, hit triaging/clustering/expansion and testing hit compounds against a panel of 5-phosphatases and various substrates.  Two chemical scaffolds were identified and one was active in cells.

Schlamadinger D, Miranker A. Fiber-Dependent and Independent Toxicity of Islet Amyloid Polypeptide. Biophysical Journal. 2014 Dec: 107(11):2559-2566. PMCID: PMC4255681.

The 37-residue peptide hormone islet amyloid polypeptide (IAPP) plays a central role in diabetes pathology. This paper describes cell-based toxicity experiments which directly assess amyloid fiber aggregation kinetics and cytotoxicity to β-cells, and shows that amyloid formation of IAPP can be strongly inhibited by the extracellular environment of live cells.  YCMD staff has helped to develop some of the cell-based assays described in the paper.

YCMD has conducted a comprehensive meta-analysis of FDA approved drugs published in a series of Drug Discovery Today manuscripts.

Click here to see the complete listing of publications.

Sztuba-Solinska J, Shenoy SR, Gareiss P, Krumpe LRH, Le, Grice SFJ, O’Keefe BR, Schneekloth Jr. JS. Identification of Biologically Active, HIV TAR RNA-Binding Small Molecules Using Small Molecule Microarrays. J Am Chem Soc. 2014 May 12; 136, 8402-8410. doi:10.1021/ja502754f. PMCID: PMC4227816.

Identification of RNA binding small molecules remains an important therapeutic challenge. YCMD employed small molecule microarray technology to screen for compounds targeting the transactivation response RNA hairpin of HIV.  A compound was identified that binds to this structured RNA target, and follow-up studies with scientists from the NCI showed that this compound is able to inhibit HIV induced cellular toxicity.

Mannam P, Shinn AS, Srivastava A, Neamu RF, Walker WE, Bohanon M, Merkel J, Kang MJ, Dela Cruz CS, Ahasic AM, Pisani MA, Trentalange M, West AP,Shadel GS, Shen J, Elias JA, Lee PJ. MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury. Am J Physiol Lung Cell Mol Physiol. 2014 Apr; 306(7):L604-19. doi: 10.1152/ajplung.00272.2013. Epub 2014 Jan 31. PMCID: PMC3962628.

Peripheral blood mononuclear cells were assayed at YCMD for activation of MKK3 (using a phospho-MKK3/6 SureFire assay), and compared between critically ill septic patients and non-septic patients. MKK3 is a kinase involved in sepsis and mitochondrial function. MKK3 deficiency is protective and reduces the generation of reactive oxygen species.

*Chosen Best Research Paper Published in AJP-Lung by a Junior Investigator

Albright RA, Ornstein DL, Cao W, Chang WC, Robert D, Tehan M, Hoyer D, Liu L, Stabach P, Yang G, De La Cruz EM, Braddock DT. Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke. J Biol Chem. 2014 Feb 7;289(6):3294-306; doi: 10.1074/jbc.M113.505867. PMCID: PMC3916532.

Neklesa TK, Noblin DJ, Kuzin A, Lew S, Seetharaman J, Acton TB, Kornhaber G, Xiao R, Montelione GT, Tong L, Crews CM. A bidirectional system for the dynamic small molecule control of intracellular fusion proteins. ACS Chem Biol. 2013 Oct 18;8(10):2293-300. doi: 10.1021/cb400569k. Epub 2013 Aug 26. PMCID: PMC4113957.

Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function.  The Crews laboratory screened the YCMD compound library and identified compounds that can both degrade or stabilize fusion proteins of interest.

Sayegh J, Cao J, Zou MR, Morales A, Blair LP, Norcia M, Hoyer D, Tackett AJ, Merkel JS, Yan Q. Identification of Small Molecule Inhibitors of Jumonji AT-Rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen. J Biol Chem. 2013; 288: 9408-9417. doi: 10.1074/jbc.M112.419861. PMID: 23408432; PMCID: PMC3611010.

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is an attractive target for cancer therapy.  Working with YCMD, the Yan lab developed and optimized a high-throughput ALPHAscreen assay for KDM5B activity. Over 15,000 small molecules were screened and several novel KDM5B inhibitors were identified with low micromolar inhibitory activity, in addition to several known demethylase inhibitors.  These new KDM5B inhibitors are being further investigated and optimized.

Grotzke JE, Lu Q, Cresswell P. 2013. Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation. Proc Natl Acad Sci USA, 110, 3393-3398. PMCID: PMC3587246. 

Endoplasmic reticulum–associated degradation (ERAD) is a constitutive process that identifies misfolded proteins in the ER and shuttles them to the cytosol, where they can be degraded by the proteasome.  This paper reports a novel system to analyze ERAD based on mutants of split or intact Venus fluorescent protein for which fluorescence depends on enzymatic deglycosylation.  Genomewide siRNA screen was performed at YCMD and successfully identified known ERAD factors, validating performance of the system and showing that it could greatly facilitate the identification of ERAD components and investigation of this critical pathway.

Held, Matthew A.; Langdon, Casey G.; Platt, James T.; Graham-Steed, Tisheeka; Liu, Zongzhi; Chakraborty, Ashok; Bachinocchi, Antonella; Koo, Andrew; Haskings, Jonathan W.; Bosenberg, Marcus W.; Stern, David F. Genotype-Selective Combination Therapies for Melanoma Identified by High-Throughput Drug Screening. Cancer Discovery January 2013 3:1 1-3; doi:10.1158/2159-8290.CD-ITI13-01. PMCID: PMC3546137.

Single agent dose response testing was performed at YCMD to identify three concentrations to be used in pairwise drug testing against a large panel of patient-derived melanoma cell lines. The combination drug plates were made at YCMD and used in the screening. Many important insights regarding molecular phenotype and drug responsiveness were observed.

Lipovsky A, Popa A, Pimienta G, Wyler M, Bhan A, Kuruvilla L, Guie MA, Poffenberger AC, Nelson CD, Atwood WJ, DiMaio D. 2013. Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus. Proc Natl Acad Sci USA, 110, 7452-7. PMCID: PMC3645514.

This paper describes a genome-wide siRNA screen which was performed to identify cellular genes required for human papillomavirus (HPV) biology.  The screen identified numerous potential antiviral targets and many retrograde transport factors, including multiple subunits of the retromer.  High-throughput image-based siRNA assay development, whole-genome screening and screen follow-up were conducted at YCMD.

Albright, RA, Chang, WC, Robert D, Ornstein DL, Cao W, Redick ME, Young JI, De La Cruz EM, Braddock DT, 2012. NPP4 is a procoagulant enzyme on the surface of vascular endothelium. Blood, 120:4432-4440. PMCID: PMC4017314.

Sayegh J.; Cao J.; Zou MR; Morales A.; Blair LP; Norcia M.; Tachkett AJ; Merkel JS; Yan Q. Identification of small molecule inhibitors of Jumonji AT-rich interactive domain 1B (JARID1B) histone demethylase by a sensitive high throughput screen. J Biol Chem. 2013 Mar 29;288(13):9408-17. doi: 10.1074/jbc.M112.419861. Epub 2013 Feb 13. PMCID: PMC3611010.

A new assay was developed at YCMD and used to screen 15,000 small molecules for targets overexpressed in cancers. The chemiluminescent assay helped identify inhibitors for this important and emerging class of epigenetic targets.

Bannwarth L, Goldberg AB, Chen C, Turk BE. 2012. Identification of exosite-targeting inhibitors of anthrax lethal factor by high-throughput screening. Chem Biol, 19, 875-882. PMCID: PMC3408624.

Development and screening of fluorescence polarization assay was conducted at YCMD. A Center chemist synthesized modified depsidones for Structure Activity Relationship studies that inhibit protease activity.

Buckley DL, Gustafson JL, Van Molle I, Roth AG, Tae HS, Gareiss PC, Jorgensen WL, Ciuli A, Crews CM. 2012. Small-molecule inhibitors of the interaction between the E3 ligase VHL and HIF1α. Angew Chem Int Ed Engl. 51, 11463-11467. PMCID: PMC3519281.

Protein-protein interactions (PPIs) are vital to most biological processes, yet despite recent advances they remain notoriously difficult to target.  The biologically relevant interaction between the E3 ubiquitin ligase VHL and HIF1alpha regulates hypoxia driven gene expression.  Utilizing a fluorescent polarization based assay at YCMD, SAR studies of small molecule modulators of this interaction were investigated.  In addition, computational chemistry methods were utilized to guide the design of new small molecule inhibitors of this interaction.

Freed EF, Prieto JL, McCann KL, McStay B, Baserga SJ.  2012. NOL11, implicated in the pathogenesis of North American Indian childhood cirrhosis, is required for pre-rRNA transcription and processing. PLoS Genet. 2012;8. PMCID: PMC3420923.

This paper describes yeast two-hybrid screen identifying a novel nucleolar protein NOL11, and presents a variety of assays showing that NOL11 is required for the early stages of ribosome biogenesis in humans.  siRNA NOL11 knockdown followed by nucleolar staining and imaging assays were conducted at YCMD and confirmed the role of NOL11 in ribosome biogenesis and determining nucleolar morphology.

Noblin, D.J.; Page, C.M.; Tae, H.S.; Gareiss, P.C.; Schneekloth, J.S.; Crews, C.M. A HaloTag-Based Small Molecule Microarray Screening Methodology with Increased Sensitivity and Multiplex Capabilities. ACS Chemical Biology, 2012: doi: 10.1021/cb300453k. PMCID: PMC3547077.

Small molecule microarray screening provides an affinity based method to determine compound interaction with targets of interest.  Traditionally, targets of interest are detected using antibodies or direct fluorescent labels.  In collaboration with YCMD, a new method of target identification utilizing the HaloTag technology was shown to have increased sensitivity in addition to multiplex capabilities.

Raycroft MT, Harvey BP, Bruck MJ, and Mamula MJ. 2012. Inhibition of Antigen Trafficking through Scavenger Receptor A, The Journal of Biological Chemistry 287 (8), 5310–5316. PMCID: PMC3285311.

Two small molecule inhibitors that reduce T cell immunity and antigen transfer were identified from an assay developed and screened at YCMD. Molecules such as these many help halt autoimmune disease development.

Woo CM, Beizer NE, Janso JE, Herzon SB.  2012.  Isolation of Lomaiviticins C−E, Transformation of Lomaiviticin C to Lomaiviticin A, Complete Structure Elucidation of Lomaiviticin A, and Structure−Activity Analyses. J Am Chem Soc. 134, 15285-8. PMID: 22963534.

Two small molecule inhibitors that reduce T cell immunity and antigen transfer were identified from an assay developed and screened at YCMD. Molecules such as these many help halt autoimmune disease development.

Cho Y, Vermeire JJ Merkel JS, Leng L, Du X, Bucala R, Cappello M, Lolis, E. 2012. Drug repositioning and pharmacophore identification in the discovery of hookworm MIF inhibitors, Chemistry & Biology. Volume 18, Issue 9, 23 September 2011, Pages 1089–1101. PMCID: PMC3294498.

Assays were developed at YCMD using recombinant MIF from hookworm parasites were screened for enzymatic activity against bioactive libraries. Hookworm-specific inhibitors were identified. Investigators solved co-crystal structures of inhibitor and enzyme.

Braddock, D.T. Autotaxin and lipid signaling pathways as anticancer targets. Current Opinion in Investigational Drugs (2010) 11: 629-37. PMID: 20496257.

Gareiss PC, Schneekloth AR, Salcius MJ, Seo S-Y, Crews CM. Identification and Characterization of a Peptidic Ligand for Ras. ChemBioChem 2010; 11:517-22. PMID: 20082398

Ras is oncogenic GTPase that remains an elusive drug target.  Utilizing phage display, a small peptidic ligand for Ras was identified.  A fluorescence polarization assay was developed with YCMD and used to characterize the molecular interaction.  Additionally an enzymatic assay was developed to determine the effects of the peptide on the GTPase function of Ras.

Goodwin, EC, Atwood, WJ, and DiMaio, D. High-throughput, cell-based screen for chemicals that inhibit infection by SV40 and human polyomaviruses. J. Virol 2009; 83:5630-5639. PMCID: PMC2681952.

Yi F, Zhu P, Southall N, Inglese J, Austin CP, Zheng W, Regan L. An AlphaScreen-Based High-Throughput Screen to Identify Inhibitors of Hsp90-Cochaperone Interaction. J Biomol Screen. 2009; 14: 273-81. PMCID: PMC3066041.

Rodriguez-Gonzalez A, Cyrus K, Salcius M, Kim K, Crews CM, Deshaies RJ, Sakamoto KM. Targeting steroid hormone receptors for ubiquitination and degradation in breast and prostate cancer. Oncogene. 2008 Dec 4;27(57):7201-11. PMID: 18794799.

Lewis CA, Merkel J, Miller SJ. Catalytic site-selective synthesis and evaluation of a series of erythromycin analogs. Bioorg Med Chem Lett. 2008 Nov; 18(22):60007-6011. PMCID: PMC2669672.

Saunders LP, Ouellette A, Bandle R, Chang WC, Zhou H, Misra RN, De La Cruz EM, Braddock DT. Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion. Mol Cancer Ther. 2008 Oct; 7(10):3352-3362. PMID: 18852138.

Blount K, Puskarz I, Penchovsky R, Breaker R. Development and application of a high-throughput assay for glmS riboswitch activators. RNA Biol. 2006 Apr;3(2):77-81. PMID: 17114942.