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Open Access Publications from the University of California

Scholarly Works (26 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2018)

Zika Virus (ZIKV), which is associated with microcephaly and Guillain-Barré syndrome, brought a serious public health impact to most of Latin America countries in 2015 and 2016 during its endemic outbreak. Still, there is no effective antiviral drug or vaccine for ZIKV infection, addressing urgency to develop therapeutic solutions for ZIKV infection. Previously, using high throughput screening (HTS) technology, several potential hit molecules against ZIKV have been discovered, and Methotrexate (MTX) was identified as one of them. While MTX has been clinically used as a chemotherapy and anti-rheumatoid reagent, in this study, I investigated the mechanism of action of MTX against Zika virus (ZIKV) in vitro using Vero cells and human neural stem cells (hNSCs). In plaque assays, the antiviral effects of MTX against ZIKV were studied, showing a ten-fold decrease of the virus titer. To understand the antiviral mechanism of action of MTX, we focused on MTX’ target enzyme, dihydrofolate reductase (DHFR), and its pathways. To confirm that the antiviral effect of MTX is caused from antagonism of DHFR, a downstream metabolite of DHFR pathway, Leucovorin, was used as together with MTX as co-treatment for ZIKV-infected host cells. Surprisingly, addition of Leucovorin rescued the ZIKV replication during MTX treatment, supporting the idea that the antiviral effect of MTX might be facilitated through inhibition of DHFR. The antagonism of DHFR leads to a decrease of the de novo synthesis of purines and pyrimidines. Additional antiviral mechanism of MTX were studied using GAT (Glycine, Adenosine, and Thymidine) medium in which adenosine alone showed the rescue effect on ZIKV replication from MTX treatment.

    Cover page: Mechanism of Action of Methotrexate Against Zika Virus
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2019)

    Chagas disease caused by a protozoan called Trypanosoma cruzi is a neglected tropical disease and a leading cause of heart failure in Latin America where it is endemic. Due to migration of asymptomatic infected population, it is now also present in North America, Europe, Japan and Australia. Drugs currently available to treat Chagas disease are limited to benznidazole and nifurtimox, both with severe side effects, and are more efficient when administered early in the course of the infection. Thus, there is a need for the discovery of alternative and more efficient drugs. In the identification process of potential drug targets, metabolic genes are good candidates, since they are critical for cellular growth and survival [1]. In addition, genome-scale metabolic models (GEMs) [31] have been developed to accurately predict metabolic capabilities from genome sequences. This work developed an extended GEM, hereafter referred to as iIS312, of the published and validated T. cruzi CL Brener core metabolism model iSR215 [35]. The life cycle of the parasite is divided in three distinct stages: epimastigotes (replicative and insect-specific), trypomastigotes or metacyclic epimastigote (infective and non-replicative) and amastigotes (replicative and intracellular in humans). From iIS312, we built three stagespecific models using transcriptomics data [3] integration. Such models for other organisms (e.g. Plasmodium falciparum) have provided valuable insight of stage-specific changes in metabolism [1]. The stage-specific models were able to predict metabolic differences among the three T. cruzi stages, including essential genes and reactions, and flux distribution. The stage-specific models showed that epimastigotes present the most active metabolism among the stages, with least number of deactivated reactions. The trypomastigote model predicted the non-essentiality of pathways responsible for nucleic acid synthesis as this stage is non-replicative, and presented full metabolic activity in TCA for energy yield to move around. The amastigote model presented low activity in Glycolysis, as observed in previous studies, suggesting that this pathway is not essential in amastigotes. It also indicated that amastigotes take a shortcut in TCA, increasing the metabolic flux in the malic enzyme, as described in previous studies. Moreover, the gene essentiality predictions suggests potential drug targets, among which some have been proven lethal previously, including glutamate dehydrogenase [12], glucokinase and hexokinase [38]. Our results represents potentially a step forward towards the improvement of Chagas disease treatment. To our knowledge, these stage-specific models are the first GEM built for the stages amastigote and trypomastigote. This work is also the first to present an in silico GEM comparison among different stages in T. cruzi life-cycle.

    • 5 supplemental PDFs
    • 1 supplemental file
    Cover page: Essential metabolic pathways in Trypanosoma cruzi
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2023)
    Infections of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have triggered a global pandemic with millions of deaths worldwide. Herein, the synthesis of functionalized Re(i) tricarbonyl complexes as inhibitors of the SARS-CoV-2 main protease, also referred to as the 3-chymotrypsin-like protease (3CLpro), is presented. The metal complexes were found to inhibit the activity of the enzyme with IC50 values in the low micromolar range. Mass spectrometry revealed that the metal complexes formed a coordinate covalent bond with the enzyme. Chiral separation of the enantiomers of the lead compound showed that one enantiomer was significantly more active than the other, consistent with specific binding and much like that observed for conventional organic small molecule inhibitors and druglike compounds. Evaluation of the lead compound against SARS-CoV-2 in a cell-based infection assay confirmed enantiospecific inhibition against the virus. This study represents a significant advancement in the use of metal complexes as coordinate covalent inhibitors of enzymes, as well as a novel starting point for the development of novel SARS-CoV-2 inhibitors.
      Cover page: Enantioselective inhibition of the SARS-CoV-2 main protease with rhenium( i ) picolinic acid complexes
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2023)
      Recent antimalarial drug discovery has been a race to produce new medicines that overcome emerging drug resistance, whilst considering safety and improving dosing convenience. Discovery efforts have yielded a variety of new molecules, many with novel modes of action, and the most advanced are in late-stage clinical development. These discoveries have led to a deeper understanding of how antimalarial drugs act, the identification of a new generation of drug targets, and multiple structure-based chemistry initiatives. The limited pool of funding means it is vital to prioritize new drug candidates. They should exhibit high potency, a low propensity for resistance, a pharmacokinetic profile that favours infrequent dosing, low cost, preclinical results that demonstrate safety and tolerability in women and infants, and preferably the ability to block Plasmodium transmission to Anopheles mosquito vectors. In this Review, we describe the approaches that have been successful, progress in preclinical and clinical development, and existing challenges. We illustrate how antimalarial drug discovery can serve as a model for drug discovery in diseases of poverty.
        Cover page: Antimalarial drug discovery: progress and approaches.
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2020)
        Marine Cyanobacteria (blue-green algae) have been shown to possess an enormous potential to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Here, we report the isolation and structure determination of palstimolide A, a complex polyhydroxy macrolide with a 40-membered ring that was isolated from a tropical marine cyanobacterium collected at Palmyra Atoll. NMR-guided fractionation in combination with MS2-based molecular networking and isolation via HPLC yielded 0.7 mg of the pure compound. The small quantity isolated along with the presence of significant signal degeneracy in both the 1H and 13C-NMR spectra complicated the structure elucidation of palstimolide A. Various NMR experiments and solvent systems were employed, including the LRHSQMBC experiment that allows the detection of long-range 1H-13C correlation data across 4-, 5-, and even 6-bonds. This expanded NMR data set enabled the elucidation of the palstimolide's planar structure, which is characterized by several 1,5-disposed hydroxy groups as well as a tert-butyl group. The compound showed potent antimalarial activity with an IC50 of 223 nM as well as interesting anti-leishmanial activity with an IC50 of 4.67 µM.
          Cover page: Palstimolide A: A Complex Polyhydroxy Macrolide with Antiparasitic Activity
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2018)
          Trypanosoma cruzi parasites are the causative agents of Chagas disease. These parasites infect cardiac and gastrointestinal tissues, leading to local inflammation and tissue damage. Digestive Chagas disease is associated with perturbations in food absorption, intestinal traffic and defecation. However, the impact of T. cruzi infection on the gut microbiota and metabolome have yet to be characterized. In this study, we applied mass spectrometry-based metabolomics and 16S rRNA sequencing to profile infection-associated alterations in fecal bacterial composition and fecal metabolome through the acute-stage and into the chronic stage of infection, in a murine model of Chagas disease. We observed joint microbial and chemical perturbations associated with T. cruzi infection. These included alterations in conjugated linoleic acid (CLA) derivatives and in specific members of families Ruminococcaceae and Lachnospiraceae, as well as alterations in secondary bile acids and members of order Clostridiales. These results highlight the importance of multi-'omics' and poly-microbial studies in understanding parasitic diseases in general, and Chagas disease in particular.
            Cover page: Experimental Chagas disease-induced perturbations of the fecal microbiome and metabolome
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2020)
            Chagas disease is a neglected tropical disease and a leading cause of heart failure in Latin America caused by a protozoan called Trypanosoma cruzi. This parasite presents a complex multi-stage life cycle. Anti-Chagas drugs currently available are limited to benznidazole and nifurtimox, both with severe side effects. Thus, there is a need for alternative and more efficient drugs. Genome-scale metabolic models (GEMs) can accurately predict metabolic capabilities and aid in drug discovery in metabolic genes. This work developed an extended GEM, hereafter referred to as iIS312, of the published and validated T. cruzi core metabolism model. From iIS312, we then built three stage-specific models through transcriptomics data integration, and showed that epimastigotes present the most active metabolism among the stages (see S1-S4 GEMs). Stage-specific models predicted significant metabolic differences among stages, including variations in flux distribution in core metabolism. Moreover, the gene essentiality predictions suggest potential drug targets, among which some have been previously proven lethal, including glutamate dehydrogenase, glucokinase and hexokinase. To validate the models, we measured the activity of enzymes in the core metabolism of the parasite at different stages, and showed the results were consistent with model predictions. Our results represent a potential step forward towards the improvement of Chagas disease treatment. To our knowledge, these stage-specific models are the first GEMs built for the stages Amastigote and Trypomastigote. This work is also the first to present an in silico GEM comparison among different stages in the T. cruzi life cycle.
              Cover page: Genome-scale metabolic models highlight stage-specific differences in essential metabolic pathways in Trypanosoma cruzi
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2019)
              Gallinamide A, originally isolated with a modest antimalarial activity, was subsequently reisolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs. Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (10, Ki = 0.0937 ± 0.01 nM and kinact/Ki = 8 730 000). From a protein structure and substrate preference perspective, cruzain, an essential Trypanosoma cruzi cysteine protease, is highly homologous. Our investigations revealed that gallinamide and its analogs potently inhibit cruzain and are exquisitely toxic toward T. cruzi in the intracellular amastigote stage. The most active compound, 5, had an IC50 = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect stage) and the host cell, and thus represents a new candidate for the treatment of Chagas disease.
                Cover page: Design of Gallinamide A Analogs as Potent Inhibitors of the Cysteine Proteases Human Cathepsin L and Trypanosoma cruzi Cruzain
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2016)
                  Cover page: Synthesis and Evaluation of Oxyguanidine Analogues of the Cysteine Protease Inhibitor WRR-483 against Cruzain
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2018)
                  Cysteine proteases (CPs) play key roles in the pathogenesis of protozoan parasites, including cell/tissue penetration, hydrolysis of host or parasite proteins, autophagy, and evasion or modulation of the host immune response, making them attractive chemotherapeutic and vaccine targets. This review highlights current knowledge on clan CA cysteine proteases, the best-characterized group of cysteine proteases, from 7 protozoan organisms causing human diseases with significant impact: Entamoeba histolytica, Leishmania species (sp.), Trypanosoma brucei, T. cruzi, Cryptosporidium sp., Plasmodium sp., and Toxoplasma gondii. Clan CA proteases from three organisms (T. brucei, T. cruzi, and Plasmodium sp.) are well characterized as druggable targets based on in vitro and in vivo models. A number of candidate inhibitors are under development. CPs from these organisms and from other protozoan parasites should be further characterized to improve our understanding of their biological functions and identify novel targets for chemotherapy.
                    Cover page: Cysteine proteases in protozoan parasites
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