Department of Ophthalmology

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    Molecular Understanding and Modern Application of Traditional Medicines: Triumphs and Trials
    (2008-08-12) Corson, Timothy W.; Crews, Craig M
    Traditional medicines provide fertile ground for modern drug development, but first they must pass along a pathway of discovery, isolation, and mechanistic studies before eventual deployment in the clinic. Here, we highlight the challenges along this route, focusing on the compounds artemisinin, triptolide, celastrol, capsaicin, and curcumin.
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    Design and applications of bifunctional small molecules: Why two heads are better than one
    (2008-11-21) Corson, Timothy W.; Aberle, Nicholas; Crews, Craig M
    Induction of protein−protein interactions is a daunting challenge, but recent studies show promise for small molecules that specifically bring two or more protein molecules together for enhanced or novel biological effect. The first such bifunctional molecules were the rapamycin- and FK506-based “chemical inducers of dimerization”, but the field has since expanded with new molecules and new applications in chemical genetics and cell biology. Examples include coumermycin-mediated gyrase B dimerization, proteolysis targeting chimeric molecules (PROTACs), drug hybrids, and strategies for exploiting multivalency in toxin binding and antibody recruitment. This Review discusses these and other advances in the design and use of bifunctional small molecules and potential strategies for future systems.
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    Triptolide Directly Inhibits dCTP Pyrophosphatase
    (2011-07) Corson, Timothy W.; Cavga, Hüseyin; Aberle, Nicholas; Crews, Craig M
    Triptolide is a potent natural product, with documented antiproliferative, immunosuppressive, anti-inflammatory, antifertility, and antipolycystic kidney disease effects. Despite a wealth of knowledge about the biology of this compound, direct intracellular target proteins have remained elusive. We synthesized a biotinylated photoaffinity derivative of triptolide, and used it to identify dCTP pyrophosphatase 1 (DCTPP1) as a triptolide-interacting protein. Free triptolide interacts directly with recombinant DCTPP1, and inhibits the enzymatic activity of this protein. Triptolide is thus the first dCTP pyrophosphatase inhibitor identified, and DCTPP1 is a biophysically validated target of triptolide.
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    The TAg-RB Murine Retinoblastoma Cell of Origin Has Immunohistochemical Features of Differentiated Müller Glia with Progenitor Properties
    (2011-09) Pajovic, Sanja; Corson, Timothy W.; Spencer, Clarellen; Dimaras, Helen; Orlic-Milacic, Marija; Marchong, Mellone N; To, Kwong-Him; Thériault, Brigitte; Auspitz, Mark; Gallie, Brenda L
    PURPOSE: Human retinoblastoma arises from an undefined developing retinal cell after inactivation of RB1. This is emulated in a murine retinoblastoma model by inactivation of pRB by retinal-specific expression of simian virus 40 large T-antigen (TAg-RB). Some mutational events after RB1 loss in humans are recapitulated at the expression level in TAg-RB, supporting preclinical evidence that this model is useful for comparative studies between mouse and human. Here, the characteristics of the TAg-RB cell of origin are defined. METHODS: TAg-RB mice were killed at ages from embryonic day (E)18 to postnatal day (P)35. Tumors were analyzed by immunostaining, DNA copy number PCR, or real-time quantitative RT-PCR for TAg protein, retinal cell type markers, and retinoblastoma-relevant genes. RESULTS: TAg expression began at P8 in a row of inner nuclear layer cells that increased in number through P21 to P28, when clusters reminiscent of small tumors emerged from cells that escaped a wave of apoptosis. Early TAg-expressing cells coexpressed the developmental marker Chx10 and glial markers CRALBP, clusterin, and carbonic anhydrase II (Car2), but not TuJ1, an early neuronal marker. Emerging tumors retained expression of only Chx10 and carbonic anhydrase II. As with human retinoblastoma, TAg-RB tumors showed decreased Cdh11 DNA copy number and gain of Kif14 and Mycn. It was confirmed that TAg-RB tumors lose expression of tumor suppressor cadherin-11 and overexpress oncogenes Kif14, Dek, and E2f3. CONCLUSIONS: TAg-RB tumors displayed molecular similarity to human retinoblastoma and origin in a cell with features of differentiated Müller glia with progenitor properties.
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    Small-Molecule Hydrophobic Tagging Induced Degradation of HaloTag Fusion Proteins
    (2012-02) Neklesa, Taavi K; Tae, Hyun Seop; Schneekloth, Ashley R; Stulberg, Michael J; Corson, Timothy W.; Sundberg, Thomas B; Raina, Kanak; Holley, Scott A; Crews, Craig M
    The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1G12V-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small-molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models.
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    Delivery of Intraocular Triamcinolone Acetonide in the Treatment of Macular Edema
    (MDPI, 2012-03-15) Pickrell, Aaron; Harris, Alon; Ngo, Sandra; Amireskandari, Annahita; Stewart, Erin; Siesky, Brent; Ophthalmology, School of Medicine
    Macular edema (ME) is one of the eventual outcomes of various intraocular and systemic pathologies. The pathogenesis for ME is not yet entirely understood; however, some of the common risk factors for its development have been identified. While this investigation will not discuss the numerous etiologies of ME in detail, it appraises the two most widely studied delivery modalities of intraocular corticosteroids in the treatment of ME—intravitreal injection (IVI) and sub-Tenon’s infusion (STI). A thorough review of the medical literature was conducted to identify the efficacy and safety of IVI and STI, specifically for the administration of triamcinolone acetonide (TA), in the setting of ME in an attempt to elucidate a preferred steroid delivery modality for treatment of ME.
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    The Effect of Diluted Penetration Enhancer in Nebulized Mist versus Liquid Drop Preparation Forms on Retrobulbar Blood Flow in Healthy Human Subjects
    (MDPI, 2012-08-08) Primus, Sally; Januleviciene, Ingrida; Siesky, Brent; Gerber, Austin; Egan, Patrick; Amireskandari, Annahita; Siaudvytyte, Lina; Barsauskaite, Ruta; Harris, Alon; Ophthalmology, School of Medicine
    The aim of this study was to compare the effects of nebulized mist and liquid drop applications on retrobulbar blood flow. A prospective, non-randomized clinical trial was used to collect data from 40 healthy human eyes. Color Doppler Imaging determined peak systolic (PSV) and end diastolic (EDV) blood flow velocities and resistance index (RI) in the ophthalmic artery after both applications. Measurements were taken at baseline and at 1 min post-treatment in both eyes with 5 min measurements in the treatment eye only. p values ≤ 0.05 were considered statistically significant. Mist application to treatment eye produced an increase in 1 min and 5 min PSV and EDV (0.001 < p < 0.03) and a decrease in 5 min RI (p = 0.01), with no significant changes in PSV, EDV or RI of control eye or in treatment eye 1 min RI (p > 0.05). Drop application to treatment eye produced an increase in PSV (p < 0.001) and EDV (p = 0.01) at 1 min, with an increase in control eye 1 min PSV and EDV (p = 0.03). There were no statistically significant changes in treatment eye PSV, EDV and RI after 5 min (p > 0.05). The use of nebulized mist may provide an effective alternative to liquid drop medication application.
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    Selective Bacteriophage Screening Targeting GαqQ209L Protein
    (Office of the Vice Chancellor for Research, 2013-04-05) Khan, Abdul Karim; Sishtila, Kamakshi; Corson, Timothy W.
    Uveal melanoma is the most common intraocular cancer in adults with 2,500 patients diagnosed every year in the United States The cancer is highly chemoersistant and is able to metastasize to other parts of the body, usually the liver where it proves almost universally fatal. It is often difficult to detect the growth of the tumor without a confirmatory biopsy. The goal of this project is to find a peptide sequence specifically binding to GαqQ209l which is an oncogenic mutation causing uveal melanoma in majority of all oncogenic cases. The method of bacteriophage display was used to find a peptide ligand that will bind specifically to the Gαq Q209L; subtractive panning was used against Gαq Wild Type and glutathion. The binders were amplified and then tested using a phage ELISA. This experiment is still in progress and there are no conclusions to be made yet. In the future it is hoped to successfully find a peptide sequence that is specific to cells.
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    The First Synthesis of the Antiangiogenic Homoisoflavanone, Cremastranone
    (Royal Society of Chemistry, 2014) Lee, Bit; Basavarajappa, Halesha D.; Sulaiman, Rania S.; Fei, Xiang; Seo, Seung-Yong; Corson, Timothy W.; Department of Ophthalmology, IU School of Medicine
    An antiangiogenic homoisoflavanone, cremastranone, was synthesized for the first time. This scalable synthesis, which includes selective demethylation, could be used to develop lead molecules to treat angiogenesis-induced eye diseases. Synthetic cremastranone inhibited the proliferation, migration and tube formation ability of human retinal microvascular endothelial cells, important steps in pathological angiogenesis.
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    Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD
    (Landes Bioscience, 2014) Mitter, Sayak K.; Song, Chunjuan; Qi, Xiaoping; Mao, Haoyu; Rao, Haripriya; Akin, Debra; Lewin, Alfred; Grant, Maria; Dunn, William; Ding, Jindong; Bowes Rickman, Catherine; Boulton, Michael; Department of Ophthalmology, IU School of Medicine
    Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.