IU Indianapolis ScholarWorks :: Browsing by Author "Georgiadis, Millie M."

Browsing by Author "Georgiadis, Millie M."

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A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1
(MDPI, 2023-11-23) Das, Debanu; Duncton, Matthew A. J.; Georgiadis, Taxiarchis M.; Pellicena, Patricia; Clark, Jennifer; Sobol, Robert W.; Georgiadis, Millie M.; King-Underwood, John; Jobes, David V.; Chang, Caleb; Gao, Yang; Deacon, Ashley M.; Wilson, David M., III; Biochemistry and Molecular Biology, School of Medicine
The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows for (i) hit generation; (ii) the mapping of protein–ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.
Activation of Gcn2 by Pharmacological Agents Designed to be Inhibitors
(2023-01) Carlson, Kenneth Reed; Wek, Ronald C.; Georgiadis, Millie M.; Liu, Yunlong; Staschke, Kirk A.; Turchi, John J.
The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stressadaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. This thesis reports that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, probes the mechanism by which this activation occurs, and compares the mechanism of Gcn2 activation by Gcn2iB to that of uncharged tRNA. In this study, Gcn2 activation was measured in cultured human cells by immunoblot and luciferase reporter assays making use of a genetic complementation assay to assess the contribution of various Gcn2 residues to its activation. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived fromGcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. However, compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.
Activation of Gcn2 by small molecules designed to be inhibitors
(Elsevier, 2023) Carlson, Kenneth R.; Georgiadis, Millie M.; Tameire, Feven; Staschke, Kirk A.; Wek, Ronald C.; Biochemistry and Molecular Biology, School of Medicine
The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stress-adaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. In this study, we report that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, and we probe the mechanism by which this activation occurs. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived from Gcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. Compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.
Advancing the Applicability of Fast Photochemical Oxidation of Proteins to Complex Systems
(2016-08) Rinas, Aimee Lynn; Jones, Lisa; Georgiadis, Millie M.; Long, Eric C.; Manicke, Nicholas
Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced increases the complexity of identifying and quantifying oxidation products. The current methods available for quantifying the extent of oxidation either involve manual analysis steps, or limit the number of searchable modifications or the size of sequence database. This creates a bottleneck which can result in a long and arduous analysis process, which is further compounded in a complex sample. In addition to the data complexity, the peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography. The recently published in cell FPOP method exemplifies where this field is headed - larger and more complex systems. This dissertation describes two new methodologies and one new technology for hydroxyl radical-mediated protein footprinting, expanding the applicability of the method. First is development of a new footprinting analysis method for both peptide and residue level analysis, allowing for faster quantification of results. This method utilizes a customized multilevel search workflow developed for an on-market search platform in conjunction with a quantitation platform developed using a free Excel add-in, expediting the analysis process. Second is the application of multidimensional protein identification technology (MudPIT) in combination with hydroxyl radical footprinting as a method to increase the identification of quantifiable peptides in these experiments. Last is the design and implementation of a flow system for in cell FPOP, which hydrodynamically focuses the cells, and when used yielded a 13-fold increase in oxidized proteins and 2 orders of magnitude increase in the dynamic range of the method.
Antibiotic Discovery Targeting Bacterial GroEL/GroES Chaperonin Systems
(2018-07-29) Kunkle, Trent A.; Johnson, Steven M.; Georgiadis, Millie M.; Hoang, Quyen Q.
The Centers for Disease Control (CDC) and World Health Organizations (WHO) have highlighted six species of highly drug-resistant bacteria, commonly termed the ESKAPE pathogens, that new antibacterials are urgently needed to treat). The ESKAPE pathogens account for over two-million infections and have healthcare costs upwards of $20 billion dollars annually. Over the past several decades, pharmaceutical companies have drastically reduced their research programs for developing new antibacterial agents. As well, bacteria are predisposed to rapidly generate resistance against these “me too” drugs, making this strategy a temporary stop-gap in our ability to fight these pathogens. This has left the burden to identify new antibiotics that function through fundamentally unique mechanisms of action to academia. Towards this goal, we are developing a unique antibacterial strategy that functions through targeting the bacterial GroEL chaperonin systems. GroEL is a molecular chaperone that helps fold proteins into their functional states. Being an essential protein, inhibiting GroEL activity leads to global aggregation and bacterial cell death. We previously reported a high-throughput screening effort that identified 235 GroEL inhibitors. A subsequent study with a subset of these inhibitors identified several that kill bacteria. To follow-up, we have synthesized 43 analogs of a hit-to-lead molecule, compound 1, containing systematic deletions of substituents and substructures to determine the essential parts of the scaffold for inhibiting GroEL and killing bacteria. Along with inhibiting GroEL, several compound 1 analogs exhibit >50-fold therapeutic windows between antibacterial efficacy and cytotoxicity to human liver and kidney cells in cell culture. Evaluation of two lead candidates (1 and 11) in a gain-of-resistance assay indicated that MRSA bacteria were not able to easily generate resistance to this compound class. Compound 1 also exhibited the ability to permeate through already established S. aureus biofilms and maintain its bactericidal effects, whereas vancomycin could not. Having established initial structure-activity relationships for the compound 1 substituents and substructures in this study, future efforts will focus on optimizing the antibacterial effects of lead candidates and reducing their off-target toxicity to human cells.
APE1/Ref-1 Role in Redox Signaling: Translational Applications of Targeting the Redox Function of the DNA Repair/Redox Protein APE1/Ref-1
(2012-01) Kelley, Mark R.; Georgiadis, Millie M.; Fishel, Melissa L.
The heterogeneity of most cancers diminishes the treatment effectiveness of many cancer-killing regimens. Thus, treatments that hold the most promise are ones that block multiple signaling pathways essential to cancer survival. One of the most promising proteins in that regard is APE1, whose reduction-oxidation activity influences multiple cancer survival mechanisms, including growth, proliferation, metastasis, angiogenesis, and stress responses. With the continued research using APE1 redox specific inhibitors alone or coupled with developing APE1 DNA repair inhibitors it will now be possible to further delineate the role of APE1 redox, repair and protein-protein interactions. Previously, use of siRNA or over expression approaches, while valuable, do not give a clear picture of the two major functions of APE1 since both techniques severely alter the cellular milieu. Additionally, use of the redox-specific APE1 inhibitor, APX3330, now makes it possible to study how inhibition of APE1’s redox signaling can affect multiple tumor pathways and can potentiate the effectiveness of existing cancer regimens. Because APE1 is an upstream effector of VEGF, as well as other molecules that relate to angiogenesis and the tumor microenvironment, it is also being studied as a possible treatment for age-related macular degeneration and diabetic retinopathy. This paper reviews all of APE1’s functions, while heavily focusing on its redox activities. It also discusses APE1’s altered expression in many cancers and the therapeutic potential of selective inhibition of redox regulation, which is the subject of intense preclinical studies.
BASE EXCISION REPAIR APURINIC/APYRIMIDINIC ENDONUCLEASES IN APICOMPLEXAN PARASITE TOXOPLASMA GONDII
(2012-03-19) Onyango, David O.; Sullivan, William J., Jr.; Chou, Kai-Ming; Georgiadis, Millie M.; Queener, Sherry F.; Vasko, Michael R.
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Toxoplasma infection is a serious threat to immunocompromised individuals such as AIDS patients and organ transplant recipients. Side effects associated with current drug treatment calls for identification of new drug targets. DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen species produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host immune response. Most of the oxidative DNA damage is repaired through the base excision repair (BER) pathway, of which, the apurinic /apyrimidinic (AP) endonucleases are the rate limiting enzymes. Toxoplasma possesses two different AP endonucleases. The first, TgAPE, is a magnesium-dependent homologue of the human APE1 (hAPE1), but considerably divergent from hAPE1. The second, TgAPN, is a magnesium-independent homologue of yeast (Saccharomyces cerevisiae) APN1 and is not present in mammals. We have expressed and purified recombinant versions of TgAPE and TgAPN in E. coli and shown AP endonuclease activity. Our data shows that TgAPN is the more abundant AP endonuclease and confers protection against a DNA damaging agent when over-expressed in Toxoplasma tachyzoites. We also generated TgAPN knockdown Toxoplasma tachyzoites to establish that TgAPN is important for parasite protection against DNA damage. We have also identified pharmacological inhibitors of TgAPN in a high-throughput screen. The lead compound inhibits Toxoplasma replication at concentrations that do not have overt toxicity to the host cells. The importance of TgAPN in parasite physiology and the fact that humans lack APN1 makes TgAPN a promising candidate for drug development to treat toxoplasmosis.
Base excision repair apurinic/apyrimidinic endonucleases in apicomplexan parasite Toxoplasma gondii
(2011-05) Onyango, David O.; Naguleswaran, Arunasalam; Delaplane, Sarah; Reed, April; Kelley, Mark R.; Georgiadis, Millie M.; Sullivan, William J., Jr.
DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host. The spontaneous loss of DNA bases due to hydrolysis and oxidative DNA damage in intracellular parasites is great, but little is known about the type of DNA repair machineries that exist in these early-branching eukaryotes. However, it is clear, processes similar to DNA base excision repair (BER) must exist to rectify spontaneous and host-mediated damage in Toxoplasma gondii. Here we report that T. gondii, an opportunistic protozoan pathogen, possesses two apurinic/apyrimidinic (AP) endonucleases that function in DNA BER. We characterize the enzymatic activities of Toxoplasma exonuclease III (ExoIII, or Ape1) and endonuclease IV (EndoIV, or Apn1), designated TgAPE and TgAPN, respectively. Over-expression of TgAPN in Toxoplasma conferred protection from DNA damage, and viable knockouts of TgAPN were not obtainable. We generated an inducible TgAPN knockdown mutant using a ligand-controlled destabilization domain to establish that TgAPN is critical for Toxoplasma to recover from DNA damage. The importance of TgAPN and the fact that humans lack any observable APN family activity highlights TgAPN as a promising candidate for drug development to treat toxoplasmosis.
Building better enzymes: Molecular basis of improved non‐natural nucleobase incorporation by an evolved DNA polymerase
(Wiley, 2020-02) Ouaray, Zahra; Singh, Isha; Georgiadis, Millie M.; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of Medicine
Obtaining semisynthetic microorganisms that exploit the information density of “hachimoji” DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the “hachimoji” P:Z nucleobase pair with a similar efficiency to that seen for Watson–Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the “hachimoji” Z nucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet.
Building better polymerases: Engineering the replication of expanded genetic alphabets
(Elsevier, 2020-12-11) Ouaray, Zahra; Benner, Steven A.; Georgiadis, Millie M.; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of Medicine
DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

Browsing by Author "Georgiadis, Millie M."

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