Inhibition of Ape1's DNA Repair Activity as a Target in Cancer: Identification of Novel Small Molecules that have Translational Potential for Molecularly Targeted Cancer Therapy

Indiana University-Purdue University Indianapolis (IUPUI)The DNA Base Excision Repair (BER) pathway repairs DNA damaged by endogenous and exogenous agents including chemotherapeutic agents. Removal of the damaged base by a DNA glycosylase creates an apurinic / apyrimidinic (AP) site. AP endonuclease1 (Ape1), a critical component in this pathway, hydrolyzes the phosphodiester backbone 5’ to the AP site to facilitate repair. Additionally, Ape1 also functions as a redox factor, known as Ref-1, to reduce and activate key transcription factors such as AP-1 (Fos/Jun), p53, HIF-1α and others. Elevated Ape1 levels in cancers are indicators of poor prognosis and chemotherapeutic resistance, and removal of Ape1 via methodology such as siRNA sensitizes cancer cell lines to chemotherapeutic agents. However, since Ape1 is a multifunctional protein, removing it from cells not only inhibits its DNA repair activity but also impairs its other functions. Our hypothesis is that a small molecule inhibitor of the DNA repair activity of Ape1 will help elucidate the importance (role) of its repair function in cancer progression as wells as tumor drug response and will also give us a pharmacological tool to enhance cancer cells’ sensitivity to chemotherapy. In order to discover an inhibitor of Ape1’s DNA repair function, a fluorescence-based high-throughput screening (HTS) assay was used to screen a library of drug-like compounds. Four distinct compounds (AR01, 02, 03 and 06) that inhibited Ape1’s DNA repair activity were identified. All four compounds inhibited the DNA repair activity of purified Ape1 protein and also inhibited Ape1’s activity in cellular extracts. Based on these and other in vitro studies, AR03 was utilized in cell culture-based assays to test our hypothesis that inhibition of the DNA repair activity of Ape1 would sensitize cancer cells to chemotherapeutic agents. The SF767 glioblastoma cell line was used in our assays as the chemotherapeutic agents used to treat gliobastomas induce lesions repaired by the BER pathway. AR03 is cytotoxic to SF767 glioblastoma cancer cells as a single agent and enhances the cytotoxicity of alkylating agents, which is consistent with Ape1’s inability to process the AP sites generated. I have identified a compound, which inhibits Ape1’s DNA repair activity and may have the potential in improving chemotherapeutic efficacy of selected chemotherapeutic agents as well as to help us understand better the role of Ape1’s repair function as opposed to its other functions in the cell

Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1

APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC1280), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals

Document-Centric Groupware for Distributed Governmental Agencies

The distribution of the German government between Bonn and Berlin calls for the technical support for the collaborative document-based tasks performed by inter- and intra-ministerial work groups. The currently available cooperation and communication tools do not provide an integrated and homogeneous cooperation environment suitable for this application domain. This paper presents the Cooperative Document Repository, an integrated document-centric collaboration environment. The integration of cooperative document management and document processing with multi-point audio/video conferencing provides a flexible collaboration environment within which a wide range of collaborative activities can be performed. Keywords: groupware; CSCW; document-based collaboration; federal government; distributed administrations; POLIWORK; video conferencing Introduction The German unification and the resulting move of governmental agencies from Bonn to the new capital, Berlin, will result in a distributio..

Hypermedia Support for the Integrated Systems Engineering Environment MUSE

The requirements for the security,environmental and social compatibility of complex technical systems are growing. Therefore, it is reasonable to validate a planned system before realisation and production to ensure a certain quality of the system and to fulfill legal regulations. The goal of the MUSE project is to provide an integrated systems engineering environment that supports concurrent system model design and validation based on the German mechanical engineering standard VDI 2221. First, MUSE supports the designer of a technical system by providing integrated tools for the specification, simulation and validation of the system's behaviour. Secondly, MUSE supports the structured archiving of all documents produced during the systems engineering process by a hypermedia database system. In this paper, we describe the issues of the hypermedia user interface and the system-wide hypermedia database support. We show how the concepts of VDI 2221 can be represented by means of a MUSE-speci..

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HyperStorM: An Extensible Object-Oriented Hypermedia Engine

It is a well-known problem that developers of hypermedia applications need assistance for modeling and maintaining application-specific hypermedia structures. In the past, various hypermedia engines have been proposed to support these tasks. Until now, hypermedia engines either provided a fixed hypermedia data model and left extensions to the hypermedia application or they left the modeling of the hypermedia data completely to the application developer and only provided storage functionality which had to be plugged into the data model by the application developer. As an alternative, we propose an extensible object-oriented hypermedia engine which supports the specification of application semantics as application classes within the hypermedia engine, thereby supporting complex operations maintaining application-specific as well as application-independent constraints. In the HyperStorM hypermedia engine, the storage layer and the application layer of a hypermedia system are implemented ..

Glutaredoxin 1 downregulation in the substantia nigra leads to dopaminergic degeneration in mice

International audienceBackground: Parkinson's disease (PD) is characterized by a severe loss of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Perturbation of protein thiol redox homeostasis has been shown to play a role in the dysregulation of cell death and cell survival signaling pathways in these neurons. Glutaredoxin 1 (Grx1) is a thiol/disulfide oxidoreductase that catalyzes the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis.Objectives: We evaluated if the downregulation of Grx1 could lead to dopaminergic degeneration and PD-relevant motor deficits in mice.Methods: Grx1 was downregulated unilaterally through viral vector-mediated transduction of short hairpin RNA against Grx1 into the SNpc. Behavioral assessment was performed through rotarod and elevated body swing test. Stereological analysis of tyrosine hydroxylase-positive and Nissl-positive neurons was carried out to evaluate neurodegeneration.Results: Downregulation of Grx1 resulted in contralateral bias of elevated body swing and reduced latency to fall off, accelerating rotarod. This was accompanied by a loss of tyrosine hydroxylase-positive neurons in the SNpc and their DA projections in the striatum. Furthermore, there was a loss Nissl-positive neurons in the SNpc, indicating cell death. This was selective to the SNpc neurons because DA neurons in the ventral tegmental area were unaffected akin to that seen in human PD. Furthermore, Grx1 mRNA expression was substantially decreased in the SNpc from PD patients.Conclusions: Our study indicates that Grx1 is critical for the survival of SNpc DA neurons and that it is downregulated in human PD. © 2020 International Parkinson and Movement Disorder Society