Browsing by Subject "Inhibitors"

Now showing 1 - 3 of 3
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    Modeling the Effects of Small Molecule Therapeutics on Glycolysis and Lactate Flux
    (2018-09) Schroeder, Joseph S; O'Brien, Conor M; Hu, Wei-Shou
    Chinese Hamster Ovary (CHO) cells are widely used in the industrial production of commercial therapeutics. One key aspect to the productivity of these cells is their high rate of glucose consumption. The high rate of glucose consumption is paired with a high output of lactate which can lead to negative culture performance. The rate of glucose consumption and lactate production can be modulated by a number of chemicals, some of which are being explored as therapeutic drugs, which affect the activities of the enzymes involved in glucose metabolism. This research aimed to evaluate the effects caused by small molecule therapeutics on CHO cells’ metabolism using a mathematic model of glucose metabolism. To model the therapeutics, established kinetic information for these therapeutics was implemented into a metabolic model. Then different concentrations of therapeutics were explored to assess their effects on metabolism. In addition, combinations of therapeutics were examined to study the effects of more drastic changes to metabolism. These therapeutics showed a large impact to the bistability of glucose metabolism as well as the lactate flux. These outcomes were important due to the potential to increase the productivity of CHO cells for industrial use as well as decreasing cell death. Thus, these therapeutics could be used to reduce lactate production in cells allowing for higher productivity.
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    Synthesis and applications of caged thiols for studying protein prenylation.
    (2012-02) Abate Pella, Daniel
    Ras proteins are a subfamily of small GTP-binding proteins that are involved in various critical cellular processes including cell growth, survival and nuclear transport. It has been reported that roughly 30% of human cancers are derived from mutations of Ras, and prenylation is a key step that activates their oncogenicity. Commercial inhibitors of prenylation have been successful at arresting Ras activation and can be categorized into two families: farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase inhibitors (GGTIs). The focus of this thesis is to explore the use of photoremovable protecting groups (caging groups) to better understand the process of prenylation by caging the critical thiol residues of FTIs, GGTIs and peptides. The caging group bromohydroxy coumarin (bhc) was covalently bound to the thiol of the FTI L-744,832 in order to inactivate the inhibitor. This caged FTI was evaluated with respect to its one- and two-photon uncaging kinetics and ability to release FTI upon photolysis. Analysis shows that bhc photolysis occurs more rapidly compared to the most frequently used family of nitrobenzyl-based cages, and that FTI is produced with good yields upon one- and two-photon excitation. Bhc-FTI was then tested on different cell lines in order to show that upon irradiation FTI is released that inhibits Ras farnesylation (observed via Western blot analysis), Ras membrane localization (detected by confocal microscopy), and downstream signaling (fibroblast morphology). This same approach was utilized to cage FTI with bromohydroxy quinoline (BHQ). The covalent inactivation of FTI with BHQ was employed to cage the active site thiol (BHQ-FTI) and active site amine (BHQ-FTI urethane). Kinetic evaluation suggests that BHQ-FTI uncages faster than bhc-FTI but it produces little FTI upon photolysis due to the formation of unreactive photoproducts. Despite its poor yield, one photon cell experiments with BHQ-FTI resulted in the inhibition of Ras farnesylation, Ras membrane localization and downstream signaling. Quantitation and biological experiments with BHQ-FTI urethane are ongoing. Peptides that are substrates of protein farnesyltransferase (PFTase) were caged with bhc and BHQ at their crucial thiol that is targeted for farnesylation. Upon one-photon photolysis peptides caged with BHQ show poor yields of free peptide while bhc-caged ones result in good peptide production. One of these caged peptides was subjected to an in vitro farnesylation assay to show that no farnesylation occurs, but upon one- and two-photon irradiation farnesylated peptide can be detected. Application of this caged peptide to study the mechanism of farnesylation via X-ray crystallography is under way. Certain Ras proteins are alternatively geranylgeranylated and retain full function when farnesylation has been inhibited; as a result, GGTI-286 was caged with bhc to study this phenomenon. The synthesis of this GGTI and the inactivation of its thiol via covalent bonding with bhc is described here. The kinetic analysis of bhc-GGTI as well as its quantitation and biological testing are a work still in progress.
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    Targeting Two Late-Stage Enzymes of the Mycobacterium tuberculosis Biotin Biosynthetic Pathway
    (2018-09) Bockman, Matthew
    Mycobacterium tuberculosis (Mtb), responsible for both latent and symptomatic tuberculosis (TB), remains the leading cause of mortality among infectious diseases worldwide. The rise and propagation of drug-resistant TB remains a global health crisis and has prompted researchers to investigate novel mechanisms of action for the development of antitubercular agents. Chapter 1 discusses the biotin biosynthetic pathway as a target for the development of antibiotics targeting Mtb, providing both chemical and genetic validation evidence of inhibiting this pathway in Mtb infections. This chapter thoroughly examines each enzyme in the biotin biosynthetic pathway by reviewing: the reaction it catalyzes, its mechanism of action, structural and sequence analysis, and catalogue of inhibitors known for each enzyme. The late-stage biotin synthase (BioB) and biotin protein ligase (BPL) proteins are elaborated on and will be the focus of this thesis. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. Chapter 2 reports the synthesis and evaluation of a systematic series of potent nucleoside-based bisubstrate inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KDs ≤ 2 nM. Additionally, this chapter discusses the structural interactions between the inhibitors and MtBPL using the highly-resolved x-ray co-crystal structures. Despite relatively uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MICs) ranging from 0.78 to >100 uM. Cellular accumulation studies showed a nearly ten-fold enhancement in accumulation of a C-2′-a-fluoro analogue over the corresponding C-2′-b-fluoro analogue, consistent with their differential whole-cell activity. The parent compound, Bio-AMS, was also evaluated for its pharmacokinetic (PK) parameters, and although it shows stability toward plasma and liver microsomes, Bio-AMS is rapidly cleared form CD-1 mice. From chapter 2, the potent compound Bio-AMS was shown to possess selective activity against MtBPL. However, Mtb develops spontaneous resistance to Bio-AMS with a frequency of resistance (FOR) of at least 1 x 10-7 by overexpression of Rv3406, a type II sulfatase that enzymatically inactivates Bio-AMS. In an effort to circumvent this resistance mechanism, chapter 3 describes the strategic modification of the Bio-AMS at the 5’-position to prevent enzymatic inactivation. The new analogues retain subnanomolar potency to MtBPL, and the 5′R-C-methyl derivative exhibited identical antimycobacterial activity toward: Mtb H37Rv, MtBPL overexpression, and an isogenic Rv3406 overexpression strain (MIC = 1.56 uM). Moreover, this compound was not metabolized by recombinant Rv3406 and resistant mutants to this compound could not be isolated (FOR < 1.4 x 10-10) demonstrating it successfully overcame Rv3406-mediated resistance. The natural product acidomycin, discovered in 1952 and isolated from Streptomyces spp., was originally shown to have selective antibiotic activity against Mtb grown in the absence of biotin, implying it is an antimetabolite of the biotin biosynthetic pathway. Chapter 4 fully investigates the mechanism of action and selectivity of acidomycin. Acidomycin was evaluated against an array of drug susceptible and drug resistant Mtb strains, as well as a panel of gram-positive and gram-negative pathogens, and showed remarkable selectivity to Mtb with MICs ranging from 0.096 – 6.2 uM for the Mtb strains and >100 M for the other microorganisms. Acidomycin was also shown to be a reversible, competitive inhibitor of E. coli biotin synthase (EcBioB), with a Ki of 1.5 uM, and a homology model shows substantial sequence alignment in the mycobacterial enzyme (MtBioB). The selectivity of acidomycin against E. coli versus Mtb is due to differential levels of cellular accumulation, with a 30-fold increase in the amount of acidomycin accumulated in Mtb over E. coli. In vivo, acidomycin was shown to be rapidly eliminated from CD-1 mice, is a half-life of 9.6 min, but exhibited remarkable plasma and microsomal stability. A brief series of acidomycin analogues showed a very tight SAR window for modifications, with the primary amide analogue being the best analogue with an MIC less than two-fold of acidomycin.