Scholarly Works, Virginia Tech Center for Drug Discovery

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Method for the conversion of cephalomannine to taxol and for the preparation of n-acyl analogs of taxol
(United States Patent and Trademark Office, 1995-11-28)
The natural product cephalomannine can be converted to the important anticancer natural product taxol by a simple process involving the steps of dihydroxylation to give cephalomannine-diols, diol cleavage, benzoylation at the 2'-position and reaction with a 1,2-diamine. The same process when applied to mixtures of taxol and cephalomannine makes it possible to separate taxol from cephalomannine-diols by simple flash-chromatography after the dihydroxylation step. If the benzoylation step is avoided in the above sequence of conversions, the process leads to the free amine (N-debenzoyltaxol). In addition, the selection of an acylating reagent other than that with the benzoyl group for acylation of the free amine (N-debenzoyltaxol), allows the preparation of taxol analogs with other N-acyl substituents.
Crystal structure of the MACPF domain of human complement protein C8 alpha in complex with the C8 gamma subunit
Slade, Daniel J.; Lovelace, Leslie L.; Chruszcz, Maksymilian; Minor, Wladek; Lebioda, Lukasz; Sodetz, James M. (Academic Press – Elsevier, 2008-05-29)
Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that assemble on bacterial membranes to form a pore-like structure referred to as the "membrane attack complex" (MAC). C8 contains three genetically distinct subunits (C8α, C8β, Cγ.) arranged as a disulfide-linked C8α-γ dimer that is noncovalently associated with C8β. C6, C7 C8α, C8β and C9 are homologous. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. The C8γ subunit is unrelated and belongs to the lipocalin family of proteins that display a β-barrel fold and generally bind small, hydrophobic ligands. Several hundred proteins with MACPF domains have been identified based on sequence similarity; however, the structure and function of most are unknown. Crystal structures of the secreted bacterial protein Plu-MACPF and the human C8α MACPF domain were recently reported and both display a fold similar to the bacterial pore-forming cholesterol-dependent cytolysins (CDC). In the present study, we determined the crystal structure of the human C8α MACPF domain disulfide-linked to C8γ (αMACPF-γ) at 2.15 Å resolution. The αMACPF portion has the predicted CDC-like fold and shows two regions of interaction with C8γ. One is in a previously characterized 19-residue insertion (indel) in C8α and fills the entrance to the putative C8γ ligand binding site. The second is a hydrophobic pocket that makes contact with residues on the side of the C8γ β-barrel. The latter interaction induces conformational changes in αMACPF that are likely important for C8 function. Also observed is structural conservation of the MACPF signature motif Y/W-G-T/S-H-F/Y-X6-G-G in αMACPF and Plu-MACPF, and conservation of several key glycine residues known to be important for refolding and pore formation by CDCs.
Fluorescence Polarization Binding Assay for Aspergillus fumigatus Virulence Factor UDP-Galactopyranose Mutase
Qi, Jun; Oppenheimer, Michelle; Sobrado, Pablo (Hindawi, 2011-08-21)
Aspergillus fumigatus is an opportunistic human pathogenic fungus responsible for deadly lung infections in immunocompromised individuals. Galactofuranose (Galf) residues are essential components of the cell wall and play an important role in A. fumigatus virulence. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose to UDP-galactofuranose, the biosynthetic precursor of Galf. Thus, inhibitors of UGM that block the biosynthesis of Galf can lead to novel chemotherapeutics for treating A. fumigatus-related diseases. Here, we describe the synthesis of fluorescently labeled UDP analogs and the development of a fluorescence polarization (FP) binding assay for A. fumigatus UGM (AfUGM). High-affinity binding to AfUGM was only obtained with the chromophore TAMRA, linked to UDP by either 2 or 6 carbons with Kd values of 2.6 ± 0.2 μM and 3.0 ± 0.7 μM, respectively. These values were ~6 times lower than when UDP was linked to fluorescein. The FP assay was validated against several known ligands and displayed an excellent Z′ factor (0.79 ± 0.02) and good tolerance to dimethyl sulfoxide.
Activation of PAD4 in NET formation.
Rohrbach, Amanda S.; Slade, Daniel J.; Thompson, Paul R.; Mowen, Kerri A. (2012)
Peptidylarginine deiminases, or PADs, convert arginine residues to the non-ribosomally encoded amino acid citrulline in a variety of protein substrates. PAD4 is expressed in granulocytes and is essential for the formation of neutrophil extracellular traps (NETs) via PAD4-mediated histone citrullination. Citrullination of histones is thought to promote NET formation by inducing chromatin decondensation and facilitating the expulsion of chromosomal DNA that is coated with antimicrobial molecules. Numerous stimuli have been reported to lead to PAD4 activation and NET formation. However, how this signaling process proceeds and how PAD4 becomes activated in cells is largely unknown. Herein, we describe the various stimuli and signaling pathways that have been implicated in PAD4 activation and NET formation, including the role of reactive oxygen species generation. To provide a foundation for the above discussion, we first describe PAD4 structure and function, and how these studies led to the development of PAD-specific inhibitors. A comprehensive survey of the receptors and signaling pathways that regulate PAD4 activation will be important for our understanding of innate immunity, and the identification of signaling intermediates in PAD4 activation may also lead to the generation of pharmaceuticals to target NET-related pathogenesis.
Genetic Reporter System for Positioning of Proteins at the Bacterial Pole
Fixen, Kathryn R.; Janakiraman, Anuradha; Garrity, Sean; Slade, Daniel J.; Gray, Andrew N.; Karahan, Nilay; Hochschild, Ann; Goldberg, Marcia B. (2012)
Spatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells.
Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26 citrullination facilitates estrogen receptor alpha target gene activation
Zhang, Xuesen; Bolt, Michael; Guertin, Michael J.; Chen, Wei; Zhang, Sheng; Cherrington, Brian D.; Slade, Daniel J.; Dreyton, Christina J.; Subramanian, Venkataraman; Bicker, Kevin L.; Thompson, Paul R.; Mancini, Michael A.; Lis, John T.; Coonrod, Scott A. (2012-08-14)
Cofactors for estrogen receptor α (ERα) can modulate gene activity by posttranslationally modifying histone tails at target promoters. Here, we found that stimulation of ERα-positive cells with 17β-estradiol (E2) promotes global citrullination of histone H3 arginine 26 (H3R26) on chromatin. Additionally, we found that the H3 citrulline 26 (H3Cit26) modification colocalizes with ERα at decondensed chromatin loci surrounding the estrogen-response elements of target promoters. Surprisingly, we also found that citrullination of H3R26 is catalyzed by peptidylarginine deiminase (PAD) 2 and not by PAD4 (which citrullinates H4R3). Further, we showed that PAD2 interacts with ERα after E2 stimulation and that inhibition of either PAD2 or ERα strongly suppresses E2-induced H3R26 citrullination and ERα recruitment at target gene promoters. Collectively, our data suggest that E2 stimulation induces the recruitment of PAD2 to target promoters by ERα, whereby PAD2 then citrullinates H3R26, which leads to local chromatin decondensation and transcriptional activation.
Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane
Kenwood, Brandon M.; Weaver, Janelle L.; Bajwa, Amandeep; Poon, Ivan K.; Byrne, Frances L.; Murrow, Beverley A.; Calderone, Joseph A.; Huang, Liping; Divakaruni, Ajit S.; Tomsig, Jose L.; Okabe, Kohki; Lo, Ryan H.; Coleman, G. Cameron; Columbus, Linda; Yan, Zhen; Saucerman, Jeffrey J.; Smith, Jeffrey S.; Holmes, Jeffrey W.; Lynch, Kevin R.; Ravichandran, Kodi S.; Uchiyama, Seiichi; Santos, Webster L.; Rogers, George W.; Okusa, Mark D.; Bayliss, Douglas A.; Hoehn, Kyle L. (Elsevier, 2013)
Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.
Chemical and Biological Methods to Detect Post-Translational Modifications of Arginine
Slade, Daniel J.; Subramanian, Venkataraman; Fuhrmann, Jakob; Thompson, Paul R. (2014-02)
Posttranslational modifications (PTMs) of protein embedded arginines are increasingly being recognized as playing an important role in both prokaryotic and eukaryotic biology, and it is now clear that these PTMs modulate a number of cellular processes including DNA binding, gene transcription, protein-protein interactions, immune system activation, and proteolysis. There are currently four known enzymatic PTMs of arginine ( i.e., citrullination, methylation, phosphorylation, ADP-ribosylation), and two non-enzymatic PTMs (i.e., carbonylation, advanced glycation end-products (AGEs)). Enzymatic modification of arginine is tightly controlled during normal cellular function, and can be drastically altered in response to various second messengers and in different disease states. Non-enzymatic arginine modifications are associated with a loss of metabolite regulation during normal human aging. This abnormally large number of modifications to a single amino acid creates a diverse set of structural perturbations that can lead to altered biological responses. While the biological role of methylation has been the most extensively characterized of the arginine PTMs, recent advances have shown that the once obscure modification known as citrullination is involved in the onset and progression of inflammatory diseases and cancer. This review will highlight the reported arginine PTMs and their methods of detection, with a focus on new chemical methods to detect protein citrullination.
Citrulination unravels stem cells
Slade, Daniel J.; Subramanian, Venkataraman; Thompson, Paul R. (2014-05)
Maintenance of the pluripotent stem cell state is regulated by the post-translational modification of histones. The discovery that citrullination of the linker histone H1 is critical to this
Nitrogen-Containing Dimeric nor-Multiflorane Triterpene from a Turraea sp.
Rasamison, Vincent E.; Rakotondraibe, L. Harinantenaina; Slebodnick, Carla; Brodie, Peggy J.; Ratsimbason, Michael; TenDyke, Karen; Shen, Yongchun; Randrianjanaka, Lucien M.; Kingston, David G. I. (American Chemical Society, 2014-05-16)
The new triterpene turranoic acid (1) and thenew N-containing nor-triterpene turraenine (2), along with triptocallic acid B (3) and esculentoic acid (4) were isolated from leaves of a Turraea sp. Compounds 1-3 showed weak to moderate in vitro antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum strain FCM29. Compound 1 also displayed weak cytotoxic activity against the non-small lung cancer cell line H522-T1 with an IC50 value of 16.4 μM.
A novel role for protein arginine deiminase 4 in pluripotency: The emerging role of citrullinated histone H1 in cellular programming
Slade, Daniel J.; Horibata, Sachi; Coonrod, Scott A.; Thompson, Paul R. (2014-08)
Histone post-translational modifications (PTM) alter the chromatin architecture, generating ‘open’ and ‘closed’ states, and these structural changes can modulate gene expression under specific cellular conditions. While methylation and acetylation are the best-characterized histone PTMs, citrullination by the protein arginine deiminases (PADs) represents another important player in this process. In addition to “fine tuning” chromatin structure at specific loci, histone citrullination can also promote rapid global chromatin decondensation during the formation of extracellular traps (ETs) in immune cells. Recent studies now show that PAD4-mediated citrullination of histone H1 at promoter elements can also promote localized chromatin decondensation in stem cells, thus regulating the pluripotent state. These observations suggest that PAD-mediated histone deimination profoundly affects chromatin structure, possibly above and beyond that of other PTMs. Additionally, these recent findings further enhance our understanding of PAD biology and the important contributions that these enzymes play in development, health, and disease.
The Quest for a Simple Bioactive Analog of Paclitaxel as a Potential Anticancer Agent
Kingston, David G. I.; Snyder, J. P. (American Chemical Society, 2014-08-01)
Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation.
Lewis, Huw D.; Liddle, John; Coote, Jim E.; Atkinson, Stephen J.; Barker, Michael D.; Bax, Benjamin D.; Bicker, Kevin L.; Bingham, Ryan P.; Campbell, Matthew; Chen, Yu Hua; Chung, Chun-wa; Craggs, Peter D.; Davis, Rob P.; Eberhard, Dirk; Joberty, Gerard; Lind, Kenneth E.; Locke, Kelly; Maller, Claire; Martinod, Kimberly; Patten, Chris; Polyakova, Oxana; Rise, Cecil E.; Rüdiger, Martin; Sheppard, Robert J.; Slade, Daniel J.; Thomas, Pamela; Thorpe, Jim; Yao, Gang; Drewes, Gerard; Wagner, Denisa D.; Thompson, Paul R.; Prinjha, Rab K.; Wilson, David M. (2015-03)
PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications
Guo, Sujuan; Liang, Yanping; Murphy, Susan F.; Huang, Angela; Shen, Haihong; Kelly, Deborah F.; Sobrado, Pablo; Sheng, Zhi (Taylor & Francis, 2015-03-01)
The lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagy-modulating drugs.
Protein Arginine Deiminase 2 Binds Calcium in an Ordered Fashion: Implications for Inhibitor Design
Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.; Zhang, Ying; Fuhrmann, Jakob; Rempel, Don; Bax, Benjamin D.; Coonrod, Scott A.; Lewis, Huw D.; Guo, Min; Gross, Michael L.; Thompson, Paul R. (American Chemical Society, 2015-04-01)
Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.
HIV-1 drug discovery: targeting folded RNA structures with branched peptides
Wynn, Jessica E.; Santos, Webster L. (The Royal Society of Chemistry, 2015-04-28)
Human immunodeficiency virus type 1 (HIV-1) is an RNA virus that is prone to high rates of mutation. While the disease is managed with current antiretroviral therapies, drugs with a new mode of action are needed. A strategy towards this goal is aimed at targeting the native three-dimensional fold of conserved RNA structures. This perspective highlights medium-sized peptides and peptidomimetics used to target two conserved RNA structures of HIV-1. In particular, branched peptides have the capacity to bind in a multivalent fashion, utilizing a large surface area to achieve the necessary affinity and selectivity toward the target RNA.
Transition metal diamine complexes with antimicrobial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA)
Karpin, George W.; Morris, David M.; Ngo, Mai T.; Merola, Joseph S.; Falkinham, Joseph O. III (Royal Society of Chemistry, 2015-06)
Pentaalkylcyclopentadienyl (Cp*R) iridium (Ir) and cobalt (Co) 1,2-diamine complexes were synthesized. Susceptibility of Staphylococcus aureus and recent patient methicillin-resistant S. aureus (MRSA) isolates to the transition metal–diamine complexes were measured by broth microdilution and reported as the MIC and MBC. Hemolytic activities of the transition metal-complexes as well as toxicity toward Vero cells were also measured. The transition metal complex of Cp*RIr with cis-1,2-diaminocyclohexane, had strong antibi- otic activity against S. aureus and MRSA (MIC = 4 μg mL−1, MBC = 8 μg mL−1) strains and killed 99% of S. aureus cells in 6 hours. Stronger antibiotic activity was associated with the presence of octyl linked to the cyclopentadienyl group and cyclohexane as the diamine backbone. Activity was greatly diminished by tri- or tetramethylation of the nitrogen of the diamine. A cyclopentadienylcobalt complex of cis-1,2-diamino- cyclohexane also showed significant anti-microbial activity against both S. aureus and MRSA strains. The absence of hemolytic activity, Vero cell cytotoxicity and the significant anti-microbial activity of several members of the family of compounds reported suggest this is an area worth further development.
Antimalarial 5,6-Dihydro-alpha-pyrones from Cryptocarya rigidifolia: Related Bicyclic Tetrahydro-alpha-Pyrones Are Artifacts
Liu, Yixi; Rakotondraibe, L. Harinantenaina; Brodie, Peggy J.; Wiley, Jessica D.; Cassera, Maria B.; Miller, James S.; Ratovoson, F.; Rakotobe, Etienne; Rasamison, Vincent E.; Kingston, David G. I. (American Chemical Society, 2015-06-01)
Antiproliferative Compounds from Cleistanthus boivinianus from the Madagascar Dry Forest
Liu, Yixi; Young, Kelly; Rakotondraibe, L. Harinantenaina; Brodie, Peggy J.; Wiley, Jessica D.; Cassera, Maria B.; Callmander, Martin W.; Rakotondrajaona, R.; Rakotobe, Etienne; Rasamison, Vincent E.; TenDyke, Karen; Shen, Yongchun; Kingston, David G. I. (American Chemical Society, 2015-07-01)
Chemical Proteomic Platform To Identify Citrullinated Proteins
Lewallen, Daniel M.; Bicker, Kevin L.; Subramanian, Venkataraman; Clancy, Kathleen W.; Slade, Daniel J.; Martell, Julianne; Dreyton, Christina J.; Sokolove, Jeremy; Weerapana, Eranthie; Thompson, Paul R. (2015-11-20)
Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and are routinely used for disease diagnosis. Protein citrullination is also increased in cancer and other autoimmune disorders, suggesting that citrullinated proteins may serve as biomarkers for diseases beyond RA. To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins. More than 20 of these are involved in RNA splicing, suggesting, for the first time, that citrullination modulates RNA biology. Overall, this chemical proteomic platform will play a key role in furthering ourunderstanding of protein citrullination in rheumatoid arthritis and potentially a wider spectrum of inflammatory diseases.

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