Browsing by Subject "Click chemistry"

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    Applications of substrate analogues for studies of prenyltransferase enzymes.
    (2010-04) DeGraw, Amanda Jane
    Prenyltransferase enzymes serve a variety of important biological functions from the creation of natural rubber to the modification of signal transduction proteins. The focus of this thesis is twofold; to understand better the isoprenoid chain elongation prenyltransferase enzymes, which catalyze the extension of isoprene units, and the protein prenyltransferases, which catalyze the transfer of an isoprenyl diphosphate to a protein or peptide. Substrate analogues have proven to be versatile tools for probing the identity, structure, mechanism, and function of various prenyltransferase enzymes. Described here are a variety of analogues of prenyltransferase substrates towards the study of prenyltransferase enzymes. A photoactive phosphonophosphate-containing analogue of farnesyl diphosphate (FPP) that can covalently modify proteins it is bound to upon irradiation with light was characterized and applied towards the identification of cis-prenyltransferase protein(s) involved in rubber biosynthesis. Kinetic and structural studies with this analogue and protein farnesyltransferase (PFTase) or protein geranylgeranyltransferase type-I (PGGTase-I) demonstrate that this probe is a good mimic of isoprenoid diphosphates. The phosphonophosphate linkage resulted in enhanced stability of the analogues, allowing it to be used as a label and identify a specific protein in rubber biosynthesis, rubber elongation factor (REF). The cross-linking of REF with a photoactivatible analogue of FPP suggests that REF can interact with isoprenoid diphosphates during rubber biosynthesis and this interaction may be key for the process. However, results indicated it is unlikely that REF is the sole protein responsible for rubber synthesis, thus prompting further work in the area. A caged compound is a biologically relevant molecule rendered inactive by a link to a chemical group (the "cage") through a photolabile bond. A series of photoactivatable protein prenyltransferase substrate analogues were created to achieve temporal control of prenyltransferase activity. Detailed characterization of these probes was performed to explore their applicability in protein prenylation studies. The first generation of caged PFTase analogues contain a nitrobenzyl-based photolabile group incorporated at the distal phosphate of the isoprenoid diphosphate substrate or the sulfhydryl side-chain of the cysteine residue in a CAAX peptide substrate. Kinetic studies of caged isoprenoid diphosphates demonstrated that they are poor substrates for PFTase but, upon irradiation, can efficiently release FPP upon irradiation which can be utilized for catalysis. The caged CAAX peptide photo releases the parent peptide with similar kinetics to the caged isoprenoid diphosphates. When caged the CAAX peptide does not function as a substrate, but is able to bind with efficient capacity and nearly identical conformation as compared to the photo-released peptide and does not interfere with the binding of the isoprenoid diphosphate substrate. These results lead to a wide variety of experiments where temporal control over protein prenylation is necessary. A second set of isoprenoid diphosphate analogues was created, bearing an azide or alkyne moiety. These analogues were applied as chemical proteomic probes for studying the mammalian protein prenylome. Cells were treated with either the alcohol, which is converted into the diphosphate by cellular kinases or the diphosphate isoprenoid analogue. These analogues were then appended onto prenylated proteins and through Cu(I)-catalyzed cycloaddition with a corresponding azide- or alkyne-modified fluorophore, direct visualization of prenylated proteins was accomplished. Application of this same reaction with a biotinylated capture reagent allowed for enrichment of the modified proteins and subsequent identification by liquid chromatography-tandem mass spectrometry (LC-MS). This work is still in progress.
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    Exploitation of prenylation in biomolecules: cell-penetrating prenylated peptides and prenyalted proteins.
    (2009-05) Wollack, James W.
    Protein prenylation is a common post-translational modification present in eukaryotic cells. Many key proteins involved in signal transduction pathways are prenylated and inhibition of prenylation can be useful as a therapeutic intervention. While significant progress has been made in understanding protein prenylation in vitro, we have been interested in studying this process in living cells, including the question of where prenylated molecules localize. Here, we describe the synthesis and in vivo analysis of a series of fluorescently labeled multifunctional peptides, based on the C-terminus of the naturally prenylated protein CDC42. These peptides were shown to have intrinsic cell-penetrating abilities and enter cells through a passive transport mechanism and localize to the endomembrane surrounding the nucleus. Their cell-penetrating properties were shown to be mostly due to their prenylation state and not their peptide sequence. Once discovered other derivatives of these peptides were used to study peptide prenylation and enzymatic processing in living cells. Also in this work other peptides and proteins were modified with non-natural prenyl diphosphates. This work aimed at honing in on the smallest alkyne or azide labeled prenyl diphosphate that is a substrate for PFTase. An alkyne containing PFTase substrate was identified that contained only 9 non-hydrogen atoms. The substrate was used to modify "Caax Box" containing proteins and peptides. Further proteolysis of the "Caax Box" allowed for alkyne modification of biomolecules with the addition of only a single modified cysteine. This result allows for the addition of alkyne functionality without the addition of a long hydrophobic chain that can hinder a biomolecules solubility or reactivity.
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    Synthesis and evaluation of parthenolide analogues: chemical probes and therapeutic agents
    (2013-03) Wang, Dan
    Cancer stem cells (CSCs), also known as tumor-propagating cells or tumor-initiating cells, are subpopulations of undifferentiated, highly tumorigenic cells found within bulk tumors. The rapid advances of cancer research and development of relative technologies have provided more and more evidence for the existence of CSCs, as well as the important roles they play in drug resistance and disease relapse of cancer. However, because of their quiescent nature and the similarities to normal stem cells, eradicating CSCs presents a challenging task. Chapter one provides an overview of cell surface markers of CSCs. Those markers are potential diagnostic macromolecules and targets for drug delivery.Parthenolide (PTL) is a sesquiterpene lactone natural product isolated from Mexican Indian medicinal herb Tanacetum parthenum (feverfew plant), a known medical herb utilized for centuries. PTL has been extensively studied as an anticancer agent, showing significant efficacy towards a wide spectrum of human cancer cells. In 2005, the identification of PTL as the first stand-alone and selective cytotoxic agent against the acute myeloid leukemia CSCs further heightened its therapeutic potential. However, the mechanism of action of PTL's CSC inhibitory activity is still an area of debate. Our efforts to elucidate the molecular targets of PTL is described in chapter two. The design and synthesis of two PTL affinity probes with diverse biological activity as well as their utilization in comparative and competitive protein pull-down experiments to enrich the cellular protein targets of PTL is presented. Although exhibiting promising anticancer and anti-CSC activities, the modest biological potency and poor water solubility prevent further development of PTL. Chapter three describes our efforts to synthesize PTL analogues, as well as our strategy to prepare water-soluble PTL prodrugs.