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Browsing by Author "Cassera, Maria B."

Now showing 1 - 11 of 11
  • 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)
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    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)
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    Biological and biochemical characterization of the extracellular signal-regulated kinase 8  homolog (TbERK8) in Trypanosoma brucei
    Valenciano Murillo, Ana Lisa (Virginia Tech, 2016-05-02)
    Trypanosoma brucei species are vector-borne protozoan parasites that cause Human African typanosomiasis (HAT) and nagana in cattle. In humans, the diseases caused by these parasites are fatal if left untreated. Treatments for these diseases are complicated because the approved drugs for treatment are ineffective against the parasites and have many toxic side effects associated with their use. There is a clear need to identify new therapeutics that are less toxic and more effective against T. brucei. Our approach for identifying new therapies is to identify novel targets in the parasite that can be modulated by small molecules. The mitogen-activated protein kinases (MAPK) pathway is a three-tiered signaling cascade that regulates cell responses to stimuli and are involved in essential processes. MAPKs can regulate differentiation, virulence, apoptosis, cell cycle and gene expression, which makes MAPKs interesting drug targets in T. brucei. The extracellular-signal regulated kinase 8 homolog in T. brucei (TbERK8) is essential for survival in bloodstream form T. brucei. The work in this dissertation involves characterizing this T. brucei MAPK to better understand its biological function and identify small molecules that can inhibit its activity to kill the parasite. Here, we report that TbERK8 is an atypical MAPK kinase that is able to autophosphorylate and no upstream kinases that activate TbERK8 have been identified. We have demonstrated that TbERK8 is able to phosphorylate the proliferating cell nuclear antigen homolog in T. brucei (TbPCNA). This is in contrast to the reported function the human ERK8 and PCNA homologs that form a stable complex in normal breast cells which does not result in PCNA phosphorylation. We also report here that TbPCNA is phosphorylated on three residues localized to a unique insertion loop by TbERK8. TbPCNA is tightly regulated in the parasites such that either upregulating or downregulating its expression arrests T. brucei proliferation. Although, this mechanism of phosphorylation is unique to TbPCNA, the role that such phosphorylation has in regulating TbPCNA is not known. Finally, we have identified small molecules that can selectively inhibit either TbERK8 or HsERK8, demonstrating that TbERK8 can be selectively inhibited to kill the parasite. The unique properties of TbERK8 can be exploited by small molecules that can be developed into new parasite-specific therapies that kill T. brucei with fewer side effects to the patients.
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    Connections Between Inositol Phosphate Signaling and Energy Responses in Plants
    Williams, Sarah Phoebe (Virginia Tech, 2015-11-19)
    The ability for an organism to sense and respond appropriately to its environment is often critical for survival. One mechanism for this is the inositol phosphate (InsP) signaling pathway. This work focuses on the role of InsP signaling in maintaining energy homeostasis in the plant. InsP signaling is connected to energy sensing in plants via a protein complex containing both the inositol polyphosphate 5-phosphatases (5PTase13) and the Sucrose non-Fermenting Related Kinase 1 (SnRK1). SnRK1 is considered a fuel gauge for the plant cell that senses energy status and reprograms growth appropriately. While the SnRK1.1 gene has been well studied, the role other SnRK1 isoforms play in energy or stress signaling is less well understood. This work examined the role of 3 SnRK1 isoforms in energy signaling, finding that SnRK1.1 and SnRK1.2 are regulated and function differently in Arabidopsis. The second part of this work focuses on the inositol pyrophosphates, which are a novel group of InsP signaling molecules containing diphosphate or triphosphate chains (i.e. PPx) attached to the inositol ring. These PPx-InsPs are emerging as critical players in the integration of cellular metabolism and stress signaling in non-plant eukaryotes. Most eukaryotes synthesize the precursor molecule, myo-inositol (1,2,3,4,5,6)-hexakisphosphate (InsP6), which can serve as a signaling molecule or as storage compound of inositol, phosphorus, and minerals. Even though plants produce huge amounts of InsP6 in seeds, almost no attention has been paid to whether PPx-InsPs exist in plants, and if so, what roles these molecules play. This work details the presence of PPx-InsPs in plants and delineates two Arabidopsis gene products (AtVip1 and AtVip2) capable of PP-InsP5 synthesis. We further examined the subcellular location of enzymes connected to PPx-InsP synthesis as well as the developmental and tissue specific patterns of expression of the genes that encode these enzymes. We localized the enzymes involved in InsP6 and PPx-InsP production to the nucleus and endoplasmic reticulum (ER). The subcellular compartmentalization of PPx-InsP signaling may be unique to plants. An increased understanding in the pathways involved in energy sensing and metabolic response may reveal novel strategies to improve crops for yield and viability in the future.
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    Cripowellins Pause Plasmodium falciparum Intraerythrocytic Development at the Ring Stage
    Butler, Joshua H.; Painter, Heather J.; Bremers, Emily K.; Krai, Priscilla; Llinás, Manuel; Cassera, Maria B. (MDPI, 2023-03-13)
    Cripowellins from Crinum erubescens are known pesticidal and have potent antiplasmodial activity. To gain mechanistic insights to this class of natural products, studies to determine the timing of action of cripowellins within the asexual intraerythrocytic cycle of Plasmodium falciparum were performed and led to the observation that this class of natural products induced reversible cytostasis in the ring stage within the first 24 h of treatment. The transcriptional program necessary for P. falciparum to progress through the asexual intraerythrocytic life cycle is well characterized. Whole transcriptome abundance analysis showed that cripowellin B “pauses” the transcriptional program necessary to progress through the intraerythrocytic life cycle coinciding with the lack of morphological progression of drug treated parasites. In addition, cripowellin B-treated parasites re-enter transcriptional progression after treatment was removed. This study highlights the use of cripowellins as chemical probes to reveal new aspects of cell cycle progression of the asexual ring stage of P. falciparum which could be leveraged for the generation of future antimalarial therapeutics.
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    Identification and Regulatory Role of E3 Ligases in the Time-Dependent Degradation of the Circadian Factor Period 2
    Liu, Jingjing (Virginia Tech, 2016-06-20)
    Circadian rhythms are self-sustained, 24h, biological oscillatory processes that are present in organisms ranging from bacteria to human. Circadian rhythms, which can be synchronized by external cues, are important for organisms to adjust their behavior, physiological activity, and metabolic reactions to changes in environmental conditions. Another well-established oscillatory mechanism that shares common organizational and regulatory features with the circadian system, is the cell division cycle. Recent findings reveal that some essential regulators are common to both the cell cycle and the circadian clock. The first half of my thesis (Chapter 2-3) focuses on the function of Period 2 (Per2), a key regulatory component of the negative feedback arm of the clock and tumor suppressor protein, as a modulator of cell cycle response. We found that Per2 binds the C-terminus end of the tumor suppressor p53 thus forming a trimeric complex with p53's negative regulator Mdm2 and preventing Mdm2-mediated p53's ubiquitination and degradation. Thus, Per2 stabilizes p53 under unstressed conditions allowing for basal levels of the protein to exist and be available for a rapid response to take place in case of any stressed signals. Our experiments prove that Per2 plays an indispensible role in p53 signaling pathway. The second half of my thesis (Chapter 4-5) focuses on how Mdm2 and Per2 interplay regulate Per2 availability and its impact on circadian clock function. My research found that Mdm2 targets Per2 for ubiquitination as Mdm2 depletion stabilizes Per2 and, conversely, Mdm2 ectopic expression shorten Per2's half-life. Accordingly, association of Per2 to Mdm2 maps C-terminus of the p53 binding region in Mdm2 and thus, the RING domain remains accessible. Next, we tested the hypothesis that Mdm2-dependent ubiquitination of Per2 directly impacts circadian clock period length. Accordingly, addition of sempervirine nitrate (SN), a specific molecular inhibitor of Mdm2, to MEF cells abrogated Per2 ubiquitination leading to the accumulation of a stable pool of Per2. By recording the oscillatory behavior of the Per2:Luc reporter system in MEF cells treated with SN at different circadian times, we found that inhibition of Mdm2 E3 ligase activity promoted phase advance only when treatment took place during the degradation period. This is in agreement with our findings that radiation, but not light pulses, causes the same phase behavior. Considering the established role of both Mdm2 and p53 in the response of cells to genotoxic stress and Per2 in modulating the clock, the existence of the Mdm2-Per2-p53 complex opens the possibility of various stimuli triggering regulatory mechanisms converging in a critical node. Overall, our work provides a holistic view of how signals are integrated at multiple levels to ensure that environmental signals are sense and responses triggered timely.
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    Investigating the role of the Apicoplast in Plasmodium falciparum Gametocyte Stages
    Wiley, Jessica Delia (Virginia Tech, 2014-05-22)
    Malaria continues to be a global health burden that affects millions of people worldwide each year. Increasing demand for malaria control and eradication has led research to focus on sexual development of the malaria parasite. Sexual development is initiated when pre-destined intraerythrocytic ring stage parasites leave asexual reproduction and develop into gametocytes. A mosquito vector will ingest mature gametocytes during a blood meal. Sexual reproduction will occur in the midgut, leading to the production of sporozoites that will migrate to the salivary gland. The sporozoites will be injected to another human host during the next blood meal consequently, transmitting malaria. Due to decreased drug susceptibility of mature gametocytes, more investigation of the biology and metabolic requirements of malaria parasites during gametocytogenesis, as well as during the mosquito stages, are urgently needed to reveal novel targets for development of transmission-blocking agents. Furthermore, increasing drug resistance of the parasites to current antimalarials, including slowed clearance rates to artemisinin, requires the discovery of innovative drugs against asexual intraerythrocytic stages with novel mechanisms of action. Here, we have investigated the role of the apicoplast during Plasmodium falciparum gametocytogenesis. In addition, we describe drug-screening studies that have elucidated a novel mode of action of one compound from the Malaria Box, as well as identified new natural product compounds that may be serve as starting molecules for antimalarial development.
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    Metabolomics profiling reveals new aspects of dolichol biosynthesis in Plasmodium falciparum
    Zimbres, Flavia M.; Lisa Valenciano, Ana; Merino, Emilio F.; Florentin, Anat; Holderman, Nicole R.; He, Guijuan; Gawarecka, Katarzyna; Skorupinska-Tudek, Karolina; Fernandez-Murga, Maria L.; Swiezewska, Ewa; Wang, Xiaofeng; Muralidharan, Vasant; Cassera, Maria B. (2020-08-06)
    The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite's development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite.
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    Molecular target identification of antimalarial drugs using proteomic and metabolomic approaches
    Laourdakis, Christian Daniel (Virginia Tech, 2014-05-15)
    Malaria is a parasitic infectious disease that results in millions of clinical cases per year and accounts for approximately 1 million deaths annually. Because the parasite has developed resistance to all current antimalarials, new therapies are urgently needed. Purine and pyrimidine biosynthesis for DNA and RNA synthesis has been recognized as a source of therapeutic targets. Targeted metabolite profiling has aided in the understanding of several biological processes in the parasite besides drug discovery. Therefore, having a robust analytical platform to quantify the purines and pyrimidines is of a great value. For this purpose an ion pair reversed phase ultra-performance liquid chromatography in tandem with mass spectrometry method was developed and validated. In addition, the apicoplast is an organelle present in the malaria parasite and other apicomplexan parasites. It was demonstrated that the apicoplast is essential for parasite's survival. The supply of isopentenyl diphosphate and dimethylallyl diphosphate for isoprenoid biosynthesis is the sole function of this organelle in the asexual intraerythrocytic stages. Isoprenoid precursors are synthesized through the methylerythritol phosphate (MEP) pathway in the malaria parasite while humans utilize the mevalonate pathway. Therefore, the MEP pathway is a source of drug targets for drug development. Our group has identified MMV008138 as anti-apicoplast inhibitor through phenotypic screening. Preliminary data suggest that the molecular target of MMV008138 may be within the MEP pathway. We used proteomic and metabolomic approaches to identify the molecular target of MMV008138 to aid future medicinal chemistry to improve the efficacy of this inhibitor.
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    Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond
    Van Voorhis, Wesley C.; Adams, John H.; Adelfio, Roberto; Ahyong, Vida; Akabas, Myles H.; Alano, Pietro; Alday, Aintzane; Resto, Yesmalie Aleman; Alsibaee, Aishah; Alzualde, Ainhoa; Andrews, Katherine T.; Avery, Simon V.; Avery, Vicky M.; Ayong, Lawrence; Baker, Mark; Baker, Stephen; Ben Mamoun, Choukri; Bhatia, Sangeeta; Bickle, Quentin; Bounaadja, Lotfi; Bowling, Tana; Bosch, Juergen; Boucher, Lauren E.; Boyom, Fabrice F.; Brea, Jose; Brennan, Marian; Burton, Audrey; Caffrey, Conor R.; Camarda, Grazia; Carrasquilla, Manuela; Carter, Dee; Cassera, Maria B.; Cheng, Ken Chih-Chien; Chindaudomsate, Worathad; Chubb, Anthony; Colon, Beatrice L.; Colon-Lopez, Daisy D.; Corbett, Yolanda; Crowther, Gregory J.; Cowan, Noemi; D'Alessandro, Sarah; Le Dang, Na; Delves, Michael; DeRisi, Joseph L.; Du, Alan Y.; Duffy, Sandra; El-Sayed, Shimaa Abd El-Salam; Ferdig, Michael T.; Robledo, Jose A. Fernandez; Fidock, David A.; Florent, Isabelle; Fokou, Patrick V. T.; Galstian, Ani; Javier Gamo, Francisco; Gokool, Suzanne; Gold, Ben; Golub, Todd; Goldgof, Gregory M.; Guha, Rajarshi; Guiguemde, W. Armand; Gural, Nil; Guy, R. Kiplin; Hansen, Michael A. E.; Hanson, Kirsten K.; Hemphill, Andrew; van Huijsduijnen, Rob Hooft; Horii, Takaaki; Horrocks, Paul; Hughes, Tyler B.; Huston, Christopher; Igarashi, Ikuo; Ingram-Sieber, Katrin; Itoe, Maurice A.; Jadhav, Ajit; Jensen, Amornrat Naranuntarat; Jensen, Laran T.; Jiang, Rays H. Y.; Kaiser, Annette; Keiser, Jennifer; Ketas, Thomas; Kicka, Sebastien; Kim, Sunyoung; Kirk, Kiaran; Kumar, Vidya P.; Kyle, Dennis E.; Jose Lafuente, Maria; Landfear, Scott; Lee, Nathan; Lee, Sukjun; Lehane, Adele M.; Li, Fengwu; Little, David; Liu, Liqiong; Llinas, Manuel; Loza, Maria I.; Lubar, Aristea; Lucantoni, Leonardo; Lucet, Isabelle; Maes, Louis; Mancama, Dalu; Mansour, Nuha R.; March, Sandra; McGowan, Sheena; Vera, Iset Medina; Meister, Stephan; Mercer, Luke; Mestres, Jordi; Mfopa, Alvine N.; Misra, Raj N.; Moon, Seunghyun; Moore, John P.; Rodrigues da Costa, Francielly Morais; Mueller, Joachim; Muriana, Arantza; Hewitt, Stephen Nakazawa; Nare, Bakela; Nathan, Carl; Narraidoo, Nathalie; Nawaratna, Sujeevi; Ojo, Kayode K.; Ortiz, Diana; Panic, Gordana; Papadatos, George; Parapini, Silvia; Patra, Kailash; Ngoc Pham; Prats, Sarah; Plouffe, David M.; Poulsen, Sally-Ann; Pradhan, Anupam; Quevedo, Celia; Quinn, Ronald J.; Rice, Christopher A.; Rizk, Mohamed Abdo; Ruecker, Andrea; St Onge, Robert; Ferreira, Rafaela Salgado; Samra, Jasmeet; Robinett, Natalie G.; Schlecht, Ulrich; Schmitt, Marjorie; Villela, Filipe Silva; Silvestrini, Francesco; Sinden, Robert; Smith, Dennis A.; Soldati, Thierry; Spitzmueller, Andreas; Stamm, Serge Maximilian; Sullivan, David J.; Sullivan, William G.; Suresh, Sundari; Suzuki, Brian M.; Suzuki, Yo; Swamidass, S. Joshua; Taramelli, Donatella; Tchokouaha, Lauve R. Y.; Theron, Anjo; Thomas, David; Tonissen, Kathryn F.; Townson, Simon; Tripathi, Abhai K.; Trofimov, Valentin; Udenze, Kenneth O.; Ullah, Imran; Vallieres, Cindy; Vigil, Edgar; Vinetz, Joseph M.; Phat Voong Vinh; Hoan Vu; Watanabe, Nao-aki; Weatherby, Kate; White, Pamela M.; Wilks, Andrew F.; Winzeler, Elizabeth A.; Wojcik, Edward; Wree, Melanie; Wu, Wesley; Yokoyama, Naoaki; Zollo, Paul H. A.; Abla, Nada; Blasco, Benjamin; Burrows, Jeremy; Laleu, Benoit; Leroy, Didier; Spangenberg, Thomas; Wells, Timothy; Willis, Paul A. (PLOS, 2016-07-28)
    A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.
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    Plasmodium falciparum Parasites Are Killed by a Transition State Analogue of Purine Nucleoside Phosphorylase in a Primate Animal Model
    Cassera, Maria B.; Hazleton, Keith Z.; Merino, Emilio F.; Obaldia, Nicanor, III; Ho, Meng-Chiao; Murkin, Andrew S.; DePinto, Richard; Gutierrez, Jemy A.; Almo, Steven C.; Evans, Gary B.; Babu, Yarlagadda S.; Schramm, Vern L. (PLOS, 2011-11-11)
    Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent.