@article{govan_young_lively_deiters_2015, title={Optically triggered immune response through photocaged oligonucleotides}, volume={56}, ISSN={["0040-4039"]}, DOI={10.1016/j.tetlet.2015.01.165}, abstractNote={Bacterial and viral CpG oligonculeotides are unmethylated cytosine-phosphate-guanosine dinucleotide sequences and trigger an innate immune response through activation of the toll-like receptor 9 (TLR9). We have developed synthetic photocaged CpGs via site-specific incorporation of nitropiperonyloxymethyl (NPOM)-caged thymidine residues. These oligonucleotides enable the optical control of TLR9 function and thereby provide light-activation of an immune response. We provide a proof-of-concept model by applying a reporter assay in live cells and by quantification of endogenous production of interleukin 6.}, number={23}, journal={TETRAHEDRON LETTERS}, author={Govan, Jeane M. and Young, Douglas D. and Lively, Mark O. and Deiters, Alexander}, year={2015}, month={Jun}, pages={3639–3642} }
 @article{dush_mciver_parr_young_fisher_newman_sannes_hauck_deiters_nascone-yoder_2011, title={Heterotaxin: A TGF-beta Signaling Inhibitor Identified in a Multi-Phenotype Profiling Screen in Xenopus Embryos}, volume={18}, ISSN={["1879-1301"]}, DOI={10.1016/j.chembiol.2010.12.008}, abstractNote={Disruptions of anatomical left-right asymmetry result in life-threatening heterotaxic birth defects in vital organs. We performed a small molecule screen for left-right asymmetry phenotypes in Xenopus embryos and discovered a pyridine analog, heterotaxin, which disrupts both cardiovascular and digestive organ laterality and inhibits TGF-β-dependent left-right asymmetric gene expression. Heterotaxin analogs also perturb vascular development, melanogenesis, cell migration, and adhesion, and indirectly inhibit the phosphorylation of an intracellular mediator of TGF-β signaling. This combined phenotypic profile identifies these compounds as a class of TGF-β signaling inhibitors. Notably, heterotaxin analogs also possess highly desirable antitumor properties, inhibiting epithelial-mesenchymal transition, angiogenesis, and tumor cell proliferation in mammalian systems. Our results suggest that assessing multiple organ, tissue, cellular, and molecular parameters in a whole organism context is a valuable strategy for identifying the mechanism of action of bioactive compounds.}, number={2}, journal={CHEMISTRY & BIOLOGY}, author={Dush, Michael K. and McIver, Andrew L. and Parr, Meredith A. and Young, Douglas D. and Fisher, Julie and Newman, Donna R. and Sannes, Philip L. and Hauck, Marlene L. and Deiters, Alexander and Nascone-Yoder, Nanette}, year={2011}, month={Feb}, pages={252–263} }
 @article{karginov_zou_shirvanyants_kota_dokholyan_young_hahn_deiters_2011, title={Light Regulation of Protein Dimerization and Kinase Activity in Living Cells Using Photocaged Rapamycin and Engineered FKBP}, volume={133}, ISSN={["0002-7863"]}, DOI={10.1021/ja109630v}, abstractNote={We developed a new system for light-induced protein dimerization in living cells using a photocaged analogue of rapamycin together with an engineered rapamycin binding domain. Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.}, number={3}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Karginov, Andrei V. and Zou, Yan and Shirvanyants, David and Kota, Pradeep and Dokholyan, Nikolay V. and Young, Douglas D. and Hahn, Klaus M. and Deiters, Alexander}, year={2011}, month={Jan}, pages={420–423} }
 @article{young_lively_deiters_2010, title={Activation and Deactivation of DNAzyme and Antisense Function with Light for the Photochemical Regulation of Gene Expression in Mammalian Cells}, volume={132}, ISSN={["1520-5126"]}, DOI={10.1021/ja100710j}, abstractNote={The photochemical regulation of biological systems represents a very precise means of achieving high-resolution control over gene expression in both a spatial and a temporal fashion. DNAzymes are enzymatically active deoxyoligonucleotides that enable the site-specific cleavage of RNA and have been used in a variety of in vitro applications. We have previously reported the photochemical activation of DNAzymes and antisense agents through the preparation of a caged DNA phosphoramidite and its site-specific incorporation into oligonucleotides. The presence of the caging group disrupts either DNA:RNA hybridization or catalytic activity until removed via a brief irradiation with UV light. Here, we are expanding this concept by investigating the photochemical deactivation of DNAzymes and antisense agents. Moreover, we report the application of light-activated and light-deactivated antisense agents to the regulation of gene function in mammalian cells. This represents the first example of gene silencing antisense agents that can be turned on and turned off in mammalian tissue culture.}, number={17}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Young, Douglas D. and Lively, Mark O. and Deiters, Alexander}, year={2010}, month={May}, pages={6183–6193} }
 @article{chou_young_deiters_2010, title={Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro- and Eukaryotic Cells}, volume={11}, ISSN={["1439-7633"]}, DOI={10.1002/cbic.201000041}, abstractNote={A light-activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site-specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradiation with nonphototoxic UV light of 365 nm removes the ortho-nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro- and eukaryotic systems allowed for the photochemical activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradiation of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradiation with UV light led to the activation of T7RNAP, thereby inducing RNA polymerization and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.}, number={7}, journal={CHEMBIOCHEM}, author={Chou, Chungjung and Young, Douglas D. and Deiters, Alexander}, year={2010}, month={May}, pages={972–977} }
 @article{wilkins_marionni_young_liu_wang_di salvo_deiters_cropp_2010, title={Site-Specific Incorporation of Fluorotyrosines into Proteins in Escherichia coli by Photochemical Disguise}, volume={49}, ISSN={["0006-2960"]}, DOI={10.1021/bi100013s}, abstractNote={Fluorinated analogues of tyrosine can be used to manipulate the electronic environments of protein active sites. The ability to selectively mutate tyrosine residues to fluorotyrosines is limited, however, and can currently only be achieved through the total synthesis of proteins. As a general solution to this problem, we genetically encoded the unnatural amino acids o-nitrobenzyl-2-fluorotyrosine, -3-fluorotyrosine, and -2,6-difluorotyrosine in Escherichia coli. These amino acids are disguised from recognition by the endogenous protein biosynthetic machinery, effectively preventing global incorporation of fluorotyrosine into proteins.}, number={8}, journal={BIOCHEMISTRY}, author={Wilkins, Bryan J. and Marionni, Samuel and Young, Douglas D. and Liu, Jia and Wang, Yan and Di Salvo, Martino L. and Deiters, Alexander and Cropp, T. Ashton}, year={2010}, month={Mar}, pages={1557–1559} }
 @article{young_connelly_grohmann_deiters_2010, title={Small Molecule Modifiers of MicroRNA miR-122 Function for the Treatment of Hepatitis C Virus Infection and Hepatocellular Carcinoma}, volume={132}, ISSN={["1520-5126"]}, DOI={10.1021/ja910275u}, abstractNote={MicroRNAs are a recently discovered new class of important endogenous regulators of gene function. Aberrant regulation of microRNAs has been linked to various human diseases, most importantly cancer. Small molecule intervention of microRNA misregulation has the potential to provide new therapeutic approaches to such diseases. Here, we report the first small molecule inhibitors and activators of the liver-specific microRNA miR-122. This microRNA is the most abundant microRNA in the liver and is involved in hepatocellular carcinoma development and hepatitis C virus (HCV) infection. Our small molecule inhibitors reduce viral replication in liver cells and represent a new approach to the treatment of HCV infections. Moreover, small molecule activation of miR-122 in liver cancer cells selectively induced apoptosis through caspase activation, thus having implications in cancer chemotherapy. In addition to providing a new approach for the development of therapeutics, small molecule modifiers of miR-122 function are unique tools for exploring miR-122 biogenesis.}, number={23}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Young, Douglas D. and Connelly, Colleen M. and Grohmann, Christoph and Deiters, Alexander}, year={2010}, month={Jun}, pages={7976–7981} }
 @article{chou_young_deiters_2009, title={A Light-Activated DNA Polymerase}, volume={48}, ISSN={["1521-3773"]}, DOI={10.1002/anie.200901115}, abstractNote={When the time is right: The widely applied Thermus aquaticus DNA polymerase was rendered light-activatable by incorporation of the photocaged amino acid ortho-nitrobenzyl tyrosine in place of a key tyrosine residue in the active site (see picture). As the modified enzyme was completely inactive until irradiated with UV light, temporal regulation of polymerase activity was possible.}, number={32}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Chou, Chungjung and Young, Douglas D. and Deiters, Alexander}, year={2009}, pages={5950–5953} }
 @article{bereman_young_deiters_muddiman_2009, title={Development of a Robust and High Throughput Method for Profiling N-Linked Glycans Derived from Plasma Glycoproteins by NanoLC-FTICR Mass Spectrometry}, volume={8}, ISSN={["1535-3907"]}, DOI={10.1021/pr9002323}, abstractNote={Recent investigations continue to emphasize the importance of glycosylation in various diseases including cancer. In this work, we present a step-by-step protocol describing a method for N-linked glycan profiling of plasma glycoproteins by nanoflow liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). A large experimental space was initially explored and is described herein. Three internal standards were spiked into the sample and provided normalization of plasma glycan abundance across different experimental conditions. Incubation methods and times and the effect of NP40 detergent on glycan abundance were explored. It was found that an 18-h incubation with no detergent led to the greatest ion abundance; however, data could be obtained in less than one day from raw plasma samples utilizing microwave irradiation or shorter incubation periods. The intersample precision of three different glycans was less than 5.5% (RSD) when the internal standards were added prior to the initial processing step. The high mass measurement accuracy (<3 ppm) afforded by the FTICR mass spectrometer provided confident identifications of several glycan species.}, number={7}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Bereman, Michael S. and Young, Douglas D. and Deiters, Alexander and Muddiman, David C.}, year={2009}, month={Jul}, pages={3764–3770} }
 @article{edwards_young_deiters_2009, title={Light-Activated Cre Recombinase as a Tool for the Spatial and Temporal Control of Gene Function in Mammalian Cells}, volume={4}, ISSN={["1554-8937"]}, DOI={10.1021/cb900041s}, abstractNote={Cre recombinase catalyzes DNA exchange between two conserved lox recognition sites. The enzyme has extensive biological application, from basic cloning to engineering knock-out and knock-in organisms. Widespread use of Cre is due to its simplicity and effectiveness, but the enzyme and the recombination event remain difficult to control with high precision. To obtain such control we report the installation of a light-responsive o-nitrobenzyl caging group directly in the catalytic site of Cre, inhibiting its activity. Prior to irradiation, caged Cre is completely inactive, as demonstrated both in vitro and in mammalian cell culture. Exposure to non-damaging UVA light removes the caging group and restores recombinase activity. Tight spatio-temporal control over DNA recombination is thereby achieved.}, number={6}, journal={ACS CHEMICAL BIOLOGY}, author={Edwards, Wesleigh F. and Young, Douglas D. and Deiters, Alexander}, year={2009}, month={Jun}, pages={441–445} }
 @article{young_garner_yoder_deiters_2009, title={Light-activation of gene function in mammalian cells via ribozymes}, ISSN={1359-7345 1364-548X}, url={http://dx.doi.org/10.1039/b819375d}, DOI={10.1039/b819375d}, abstractNote={A ribozyme based gene control element enabled the spatio-temporal regulation of gene function in mammalian cell culture with light.}, number={5}, journal={Chem. Commun.}, publisher={Royal Society of Chemistry (RSC)}, author={Young, Douglas D. and Garner, R. Aaron and Yoder, Jeffrey A. and Deiters, Alexander}, year={2009}, pages={568–570} }
 @article{young_teske_deiters_2009, title={Open-Vessel Microwave-Mediated [2+2+2]-Cyclotrimerization Reactions}, ISSN={["1437-210X"]}, DOI={10.1055/s-0029-1218149}, abstractNote={[2+2+2]-Cyclotrimerization reactions often suffer from low reaction rates and low chemoselectivity. We are reporting a solution to both problems through the use of open-vessel conditions for microwave-mediated cyclotrimerization reactions.}, number={22}, journal={SYNTHESIS-STUTTGART}, author={Young, Douglas D. and Teske, Jesse A. and Deiters, Alexander}, year={2009}, month={Nov}, pages={3785–3790} }
 @article{young_govan_lively_deiters_2009, title={Photochemical Regulation of Restriction Endonuclease Activity}, volume={10}, ISSN={["1439-7633"]}, DOI={10.1002/cbic.200900090}, abstractNote={Removal by the light: The photochemical regulation of restriction endonucleases, which are important enzymes in molecular biology, has been investigated. Photolabile protecting groups have been installed on DNA substrates and have been demonstrated to inhibit restriction endonuclease activity until removed by UV light irradiation. Interestingly, these groups do not appear to dramatically affect initial binding of the enzyme to the DNA substrate, but rather prevent recognition of the specific cleavage site.}, number={10}, journal={CHEMBIOCHEM}, author={Young, Douglas D. and Govan, Jeane M. and Lively, Mark O. and Deiters, Alexander}, year={2009}, month={Jul}, pages={1612–1616} }
 @article{young_lusic_lively_deiters_2009, title={Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization}, volume={37}, number={8}, journal={Nucleic Acids Research}, author={Young, D. D. and Lusic, H. and Lively, M. O. and Deiters, A.}, year={2009} }
 @article{edwards_young_deiters_2009, title={The effect of microwave irradiation on DNA hybridization}, volume={7}, ISSN={["1477-0539"]}, DOI={10.1039/b903609a}, abstractNote={The effect of microwave irradiation on DNA/DNA hybridization has been studied under controlled power and temperature conditions. It was discovered that microwave irradiation led to the melting of double-stranded deoxyoligonucleotides well below their thermal melting temperature and independent of the length of the deoxyoligonucleotides. These observations indicate a specific interaction of microwaves with DNA, and have important implications in the chemical or enzymatic processing of DNA under microwave heating.}, number={12}, journal={ORGANIC & BIOMOLECULAR CHEMISTRY}, author={Edwards, Wesleigh F. and Young, Douglas D. and Deiters, Alexander}, year={2009}, pages={2506–2508} }
 @article{mciver_young_deiters_2008, title={A general approach to triphenylenes and azatriphenylenes: total synthesis of dehydrotylophorine and tylophorine}, ISSN={["1359-7345"]}, DOI={10.1039/b811068a}, abstractNote={A convergent and flexible synthesis of substituted triphenylenes, azatriphenylenes, and the cytotoxic alkaloids dehydrotylophorine and tylophorine has been developed.}, number={39}, journal={CHEMICAL COMMUNICATIONS}, author={McIver, Andrew and Young, Douglas D. and Deiters, Alexander}, year={2008}, pages={4750–4752} }
 @article{young_lusic_lively_yoder_deiters_2008, title={Gene Silencing in Mammalian Cells with Light-Activated Antisense Agents}, volume={9}, ISSN={1439-4227 1439-7633}, url={http://dx.doi.org/10.1002/cbic.200800627}, DOI={10.1002/cbic.200800627}, abstractNote={On and off the spot: The spatio-temporal light-activation of gene silencing was achieved through the complete inactivation of phosphorothioate antisense agents by installing light-removable (caged) groups at specific sites, as illustrated here. Full antisense activity was restored through a brief irradiation with UV light at 365 nm.}, number={18}, journal={ChemBioChem}, publisher={Wiley}, author={Young, Douglas D. and Lusic, Hrvoje and Lively, Mark O. and Yoder, Jeffrey A. and Deiters, Alexander}, year={2008}, month={Dec}, pages={2937–2940} }
 @article{young_deiters_2008, title={Light-regulated RNA-small molecule interactions}, volume={9}, ISSN={["1439-4227"]}, DOI={10.1002/cbic.200800051}, abstractNote={Regulating RNA with light. An RNA aptamer capable of specifically binding one isomer of a photoisomerizable spiropyran small molecule was engineered. The reversibility of the light-switchable aptamer recognition was then characterized by SPR imaging. This RNA–small molecule pair has significant implications in the light-regulation of biological functions and the reversible control of gene expression. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2008/z800051_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.}, number={8}, journal={CHEMBIOCHEM}, author={Young, Douglas D. and Deiters, Alexander}, year={2008}, month={May}, pages={1225–1228} }
 @article{young_edwards_lusic_lively_deiters_2008, title={Light-triggered polymerase chain reaction}, ISSN={["1364-548X"]}, DOI={10.1039/b715152g}, abstractNote={Photochemical control of the polymerase chain reaction has been achieved through the incorporation of light-triggered nucleotides into DNA.}, number={4}, journal={CHEMICAL COMMUNICATIONS}, author={Young, Douglas D. and Edwards, Wesleigh F. and Lusic, Hrvoje and Lively, Mark O. and Deiters, Alexander}, year={2008}, pages={462–464} }
 @article{young_nichols_kelly_deiters_2008, title={Microwave activation of enzymatic catalysis}, volume={130}, ISSN={["0002-7863"]}, DOI={10.1021/ja802404g}, abstractNote={Microwave irradiation can be used to regulate biocatalysis. Herein, the utilization of hyperthermophilic enzymes in a microwave reactor is reported. While these enzymes are inactive at low temperatures, they can be activated with microwave irradiation. To the best of our knowledge, this is the first illustration of a specific microwave effect in enzymatic catalysis.}, number={31}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Young, Douglas D. and Nichols, Jason and Kelly, Robert M. and Deiters, Alexander}, year={2008}, month={Aug}, pages={10048-+} }
 @article{young_torres-kolbus_deiters_2008, title={Microwave-assisted synthesis of unnatural amino acids}, volume={18}, ISSN={["0960-894X"]}, DOI={10.1016/j.bmcl.2008.09.025}, abstractNote={Microwave irradiation has been proven to be a useful tool in the rapid assembly of racemic unnatural amino acids in only two steps. Additional benefits of this methodology are the commercial availability of the inexpensive starting materials and the high yields and high purities of the final amino acid products.}, number={20}, journal={BIOORGANIC & MEDICINAL CHEMISTRY LETTERS}, author={Young, Douglas D. and Torres-Kolbus, Jessica and Deiters, Alexander}, year={2008}, month={Oct}, pages={5478–5480} }
 @article{gumireddy_young_xiong_hogenesch_huang_deiters_2008, title={Small-molecule inhibitors of microRNA miR-21 function}, volume={47}, ISSN={["1521-3773"]}, DOI={10.1002/anie.200801555}, abstractNote={MicroRNAs (miRNAs) have recently emerged as an important class of gene regulators, and their misregulation has been linked to a variety of cancers. Small molecule inhibitors of miRNAs would be important tools to elucidate the detailed mechanisms of miRNA function and provide lead structures for the development of new therapeutics. We are reporting a cellular screen for miRNA pathway inhibitors and the first small molecule modifiers of miRNA function.

miRNAs are single-stranded noncoding RNAs of 21-23 nucleotides. They are a novel class of gene regulators that function by binding to the 3’ untranslated region of target messenger RNAs leading to either suppression of their translation or acceleration of their degradation.[1] The majority of miRNAs are initially transcribed as primary miRNAs (primiRNAs),[2] which are further processed in the nucleus by the enzyme Drosha, thus transforming pri-miRNAs into shorter stem-loop-structured, double-stranded RNAs called precursor miRNAs (pre-miRNAs).[3] Pre-miRNAs are then transported from the nucleus to the cytoplasm and are processed by Dicer into mature miRNAs.[4] Mature miRNAs enter the effector complex, called the RNA-induced silencing complex (RISC), to then target single-stranded complementary mRNAs (Supporting Figure 1).[5] It is estimated that miRNAs are involved in the regulation of about 30% of all genes and almost every genetic pathway.[6] Moreover, recent evidence suggests that they can function as oncogenes and tumor suppressors.[7, 8] Thus, small molecule regulation of misregulated miRNAs has the potential to provide a new area of therapeutics. So far, specific miRNA inhibition has been only achieved by antisense nucleic acids.[9] We developed an assay for small molecule inhibitors of miRNA function and discovered potentially specific miRNA pathway inhibitors. Although inhibitors of the siRNA pathway have been identified,[10] to our knowledge no small molecule inhibitors of the miRNA pathway have been reported. We selected miR-21 as a target miRNA due to its documented function as an anti-apoptotic factor in cancer cells and its elevated levels in various cancers such as breast, ovarian, and lung cancer as well as glioblastomas.[7, 11] Lentiviral reporter constructs for miRNA activity were assembled by introducing the complementary sequences of mature miR-21, the specificity control miR-30, or a negative control linker sequence (a site with no detectable recognition by natural miRNAs) downstream of a luciferase reporter gene (Supporting Figure 2). These plasmids serve as sensors to detect the presence of specific mature miRNAs (Scheme 1).



Scheme 1

Luciferase expression under control of a miRNA binding sequence in the 3’ untranslated region (3’ UTR) provides an efficient miRNA assay. Endogenous miR-21 (HeLa cells) or exogenous miR-30 downregulate luciferase activity when paired with ...



The reporter constructs were stably introduced into HeLa cells which express high levels of miR-21, but relatively low levels of miR-30.[12] In order to test the miRNA specificity of the reporter system, cells that contained both the Luc-miR-30A reporter construct and a construct expressing exogenous primary miR-30 were assayed. These cells displayed a greatly diminished luciferase signal compared to cells with a mismatched Luc-miR-30A reporter/miR-21 combination (Supporting Figure 3), demonstrating that the Luc-miR-21 and Luc-miR-30A reporters are specific and react only to miR-21 and miR-30, respectively. The ability to detect endogenous miR-21 was proven by the fact that the Luc-miR-21 reporter, when introduced into HeLa cells, led to a 90% decreased luciferase signal in comparison to the control luciferase-linker construct, visualizing the high level of endogenous miR-21 expression in HeLa cells (Supporting Figure 4). As expected, the miR-30A reporter displayed only a modest decrease since HeLa cells express relatively low levels of endogenous miR-30.

Subsequently, a primary screen of >1000 compounds from our own compound collection and the Library of Pharmacologically Active Compounds (Sigma-Aldrich) was conducted at a 10 μM compound concentration and an initial hit compound 1 was discovered. This diazobenzene led to an increase of the luciferase signal by 251% compared to untreated cells (the DMSO control had no effect on the luciferase signal; Supporting Figure 5). Through several rounds of screening and structural modification a preliminary structure-activity relationship was developed (Supporting Figure 6). Acylation and alkylation of the amino group in 1 led to diminished activities. However, the screening of a wide range of molecules structurally related to the azobenzene core delivered the highly active compound 2 (5-fold increase of the luciferase signal at 10 μM, Figure 1 and ​and2A).2A). Other molecules derived from 2 through introduction of an amino or nitro group in the 4’ position led to 12% or 64% reduced activity, respectively. Other amide substituents displayed a substantial loss of activity (24-53%), with the exception of allyl and propyl groups, which showed 11% and 16% lower activity, respectively. An exchange of the amide for a sulfonamide delivered compounds with no activity and, interestingly, the styrene analog of 2 had a 40% lower activity. Thus far, 2 is the most effective small molecule inhibitor of microRNA miR-21 function inducing a 485% increase in the luciferase reporter signal at 10 μM. The diazobenzene 2 does not display cytotoxic effects at this concentration as determined by an MTT assay (data not shown). Dose response studies from 0-10 μM revealed a concentration dependence of the luciferase signal with an EC50 of 2 μM (Figure 1).



Figure 1

Dose-response curve of 2, revealing an EC50 of 2 μM and a luciferase signal increase of ~500% at 10 μM. The error bars represent the standard deviation from three independent measurements.





Figure 2

A) Change in luciferase signal of cells treated with 2 (10 μM) relative to a DMSO control. B) Mature or primary miRNA levels in cells treated with 2 (10 μM) relative to a DMSO control, as determined by quantitative RT-PCR. The error bars ...



Several experiments were conducted in order to investigate the mode of action of the inhibitor 2. The compound does not affect the luciferase signal in HeLa cells expressing the Luc-linker control sequence (Figure 2A), thus indicating that it does not increase the luciferase signal through means other than inhibiting the miRNA pathway. Furthermore, HeLa cells expressing both the miR-30 luciferase reporter construct and miR-30 were treated with 2. In this case, no increase of the luciferase signal was detected (Figure 2A), demonstrating that 2 is potentially specific towards miR-21 and does not have a general effect on the common miRNA pathway.

The specificity of 2 for the inhibition of miR-21 function was further validated by measuring intracellular miRNA levels via quantitative RT-PCR (Figure 2B). We found that levels of the stably expressed, exogenous mature miR-30, endogenous mature miR-93 and endogenous non-miRNA genes such as E-chaderin, ID1, RAP1A, Fibronectin are not reduced by treatment with 2 (Figure 2B, Supporting Figure 7). Gratifyingly, miR-21 expression is reduced by 78% compared to the DMSO control in HeLa cells. Quantitative RT-PCR experiments with primers specific for the primary miR-21 (pri-miR-21) sequence but not mature or precursor miR-21 revealed that the pri-miR-21 levels in cells treated with 2 were reduced by 87% (Figure 2B). Similar observations were also made in three other cell lines, MCF-7, A172, and MDA-MB-231, which endogenously express miR-21 (Supporting Figure 7 & 8). These results strongly suggest that compound 2 is an inhibitor that targets the transcription of the miR-21 gene into pri-miR-21, but not downstream processes of the common miRNA pathway.

In summary, we developed a method to identify inhibitors of the miRNA pathway in live cells, specifically of miR-21 an important anti-apoptotic factor in several cancers. A screening of >1000 small organic molecules followed by a structure activity relationship analysis of an initial hit delivered the azobenzene 2 as a specific and efficient inhibitor of miR-21 expression. Research on miRNAs is still in its infancy and the biogenesis of many miRNAs (including miR-21) is not fully understood, thus specific inhibitors of the miRNA pathway (like 2) will be unique tools for the investigation of miRNAs and their involvement in various types of diseases.}, number={39}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Gumireddy, Kiranmai and Young, Douglas D. and Xiong, Xin and Hogenesch, John B. and Huang, Qihong and Deiters, Alexander}, year={2008}, pages={7482–7484} }
 @article{zou_young_cruz-montanez_deiters_2008, title={Synthesis of Anthracene and Azaanthracene Fluorophores via [2+2+2] Cyclotrimerization Reactions}, volume={10}, ISSN={["1523-7052"]}, DOI={10.1021/ol8019549}, abstractNote={A highly convergent [2+2+2] cyclotrimerization approach to anthracenes and 2-azaanthracenes has been developed. It allows for the facile introduction of the anthracene moiety on alkyne and nitrile bearing molecules and the rapid construction of compound arrays. This is showcased in the assembly of a collection of fluorophores and their photochemical evaluation.}, number={20}, journal={ORGANIC LETTERS}, author={Zou, Yan and Young, Douglas D. and Cruz-Montanez, Alejandra and Deiters, Alexander}, year={2008}, month={Oct}, pages={4661–4664} }
 @article{young_deiters_2007, title={A general approach to chemo- and regioselective cyclotrimerization reactions}, volume={46}, ISSN={["1433-7851"]}, DOI={10.1002/anie.200700802}, abstractNote={Microwave-ready heterocycles: Cobalt-catalyzed [2+2+2] cyclotrimerization of nitrile derivatives with diynes anchored to a solid support under microwave irradiation provides a universal approach to pyridine, pyridone, and iminopyridine products. The reaction is completely chemo- and regioselective, and the products are obtained in excellent yield and high purity.}, number={27}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Young, Douglas D. and Deiters, Alexander}, year={2007}, pages={5187–5190} }
 @article{young_sripada_deiters_2007, title={Microwave-assisted solid-supported alkyne cyclotrimerization reactions for combinatorial chemistry}, volume={9}, ISSN={["1520-4766"]}, DOI={10.1021/cc070068b}, number={5}, journal={JOURNAL OF COMBINATORIAL CHEMISTRY}, author={Young, Douglas D. and Sripada, Lakshminath and Deiters, Alexander}, year={2007}, pages={735–738} }
 @article{lusic_young_lively_deiters_2007, title={Photochemical DNA activation}, volume={9}, ISSN={["1523-7052"]}, DOI={10.1021/ol070455u}, abstractNote={A new photocaged nucleoside was synthesized and incorporated into DNA with the use of standard synthesis conditions. This approach enabled the disruption of specific H-bonds and allowed for the analysis of their contribution to the activity of a DNAzyme. Brief irradiation with nonphotodamaging UV light led to rapid decaging and almost quantitative restoration of DNAzyme activity. The developed strategy has the potential to find widespread application in the light-induced regulation of oligonucleotide function.}, number={10}, journal={ORGANIC LETTERS}, author={Lusic, Hrvoje and Young, Douglas D. and Lively, Mark O. and Deiters, Alexander}, year={2007}, month={May}, pages={1903–1906} }
 @article{young_deiters_2007, title={Photochemical activation of protein expression in bacterial cells}, volume={46}, ISSN={["1521-3773"]}, DOI={10.1002/anie.200700057}, abstractNote={Turning genes on with light: Photochemical control of gene expression is a versatile tool for the elucidation of biological processes and the programming of new biological functions. Activation of protein expression in prokaryotic cells through light irradiation is achieved through a photocaged small molecule. Spatiotemporal regulation of the lac operon was obtained through the application of a photocaged isopropyl-β-D-thiogalactopyranoside derivative.}, number={23}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Young, Douglas D. and Deiters, Alexander}, year={2007}, pages={4290–4292} }
 @article{young_deiters_2007, title={Photochemical control of biological processes}, volume={5}, ISSN={["1477-0539"]}, DOI={10.1039/b616410m}, abstractNote={Photochemical regulation of biological processes offers a high level of control to study intracellular mechanisms with unprecedented spatial and temporal resolution. This report summarizes the advances made in recent years, focusing predominantly on the in vivo regulation of gene function using irradiation with UV light. The majority of the described applications entail the utilization of photocaging groups installed either on a small molecule modulator of biomolecular function or directly on a biological macromolecule itself.}, number={7}, journal={ORGANIC & BIOMOLECULAR CHEMISTRY}, author={Young, Douglas D. and Deiters, Alexander}, year={2007}, pages={999–1005} }
 @article{senaiar_teske_young_deiters_2007, title={Synthesis of indanones via solid-supported [2+2+2] cyclotrimerization}, volume={72}, ISSN={["0022-3263"]}, DOI={10.1021/jo7013565}, abstractNote={A new facile approach toward natural and unnatural indanones has been developed, featuring a solid-supported [2+2+2] cyclotrimerization as the key step. This strategy has been applied to the chemo- and regioselective assembly of indanone arrays and to the total synthesis of a recently isolated indanone marine natural product.}, number={20}, journal={JOURNAL OF ORGANIC CHEMISTRY}, author={Senaiar, Ramesh S. and Teske, Jesse A. and Young, Douglas D. and Deiters, Alexander}, year={2007}, month={Sep}, pages={7801–7804} }
 @article{young_deiters_2006, title={Photochemical hammerhead ribozyme activation}, volume={16}, ISSN={["1464-3405"]}, DOI={10.1016/j.bmcl.2006.02.034}, abstractNote={We report the light-activation of allosteric cis and trans acting ribozymes via decaging of a small organic molecule ligand. To achieve this effectively, we introduce an optimized N-caging group based on a nitrobenzyl core structure. This approach can potentially be employed toward a light-induced control of gene function.}, number={10}, journal={BIOORGANIC & MEDICINAL CHEMISTRY LETTERS}, author={Young, DD and Deiters, A}, year={2006}, month={May}, pages={2658–2661} }
 @article{senaiar_young_deiters_2006, title={Pyridines via solid-supported [2+2+2] cyclotrimerization}, DOI={10.1039/b515901f}, abstractNote={The formation of pyridines via a crossed [2 + 2 + 2] cycloaddition has been achieved on a solid-support for the first time.}, number={12}, journal={Chemical Communications (Cambridge, England)}, author={Senaiar, R. S. and Young, D. D. and Deiters, A.}, year={2006}, pages={1313–1315} }
 @article{young_senaiar_deiters_2006, title={Solid-supported [2+2+2] cyclotrimerizations}, volume={12}, ISSN={["0947-6539"]}, DOI={10.1002/chem.200501360}, abstractNote={The transition-metal-catalyzed [2+2+2] cyclotrimerization of a diyne and an alkyne provides a convergent route to highly-substituted aromatic rings. This reaction possesses distinct drawbacks, especially low chemo- and regioselectivities, which hamper its application in combinatorial synthesis. These problems have been solved by the development of solid-supported [2+2+2]-cycloaddition reactions. If conducted on a solid-support, this reaction enables rapid combinatorial access to diverse sets of carbo- and heterocyclic small-molecule arrays. The scope of this methodology has been investigated by examining different immobilization strategies, different diyne precursors, and a variety of functionalized alkyne reaction partners. Overall, isoindoline, phthalan, and indan libraries were assembled in good to excellent yields and with high purities.}, number={21}, journal={CHEMISTRY-A EUROPEAN JOURNAL}, author={Young, Douglas D. and Senaiar, Ramesh S. and Deiters, Alexander}, year={2006}, month={Jul}, pages={5563–5568} }