Browsing by Subject "Peptide"

Now showing 1 - 4 of 4
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    Design and Synthesis of Caged Thiols for Development of Photo-Activatable Peptides, Inhibitors and Biomaterials
    (2017-06) Mahmoodi, Mohammad Mohsen
    The ability of light to traverse various chemical and biological barriers and be modulated by time and amplitude makes light- regulated molecules unique tools for a plethora of applications in the areas of chemistry and biology and biomaterials. Photo-removable protecting groups, also known as caging groups, are one of the most important light-regulated tools, which can be utilized to mask specific functional groups in molecules such that they can be cleaved on demand upon irradiation. In biological applications, this typically involves masking a biomolecule with a caging group to produce a compound whose biological activity is either increased or decreased upon uncaging. The recent development of two-photon-sensitive protecting groups, which allow uncaging using near-infrared (near-IR) irradiation, has resulted in significant improvements in the spatiotemporal resolution of uncaging as well as increased penetration with lower photo-toxicity; the latter attribute is of particular importance for the use of caged molecules in tissue samples or intact organisms that are essentially opaque to UV light. Additionally, two-photon un- caging approaches have proved to be extremely useful for creating novel biomaterials; in that strategy, laser irradiation is used to unmask a specific caged functionality pre-incorporated into a hydrogel or matrix, such that it can be used to immobilize peptides, proteins or cells in a three dimensionally controlled fashion. In this work, we analyzed the photolysis of several Bhc-protected thiol-containing peptides and small molecules. Those experiments revealed that Bhc-caged thiols exhibit variable uncaging yields and that their photolysis frequently leads to the formation of an unwanted rearrangement product. To circumvent this problem, we explored and designed two alternative highly efficient thiol caging groups that can be uncaged upon one- and two-photon irradiation. we initially explored using nitrodibenzofuran (NDBF) as a thiol caging group. Cysteine-containing peptides were prepared where the thiol was protected with an NDBF group. To probe the utility of this protecting group for biological experiments, thiol group uncaging was carried out using a K-Ras-derived peptide containing an NDBF-protected cysteine. Irradiation of that molecule in the presence of protein farnesyltransferase (PFTase) and farnesyl diphosphate (FPP) resulted in the formation of the free thiol form and subsequent enzymatic conversion to a prenylated species. In order to illustrate the utility of this strategy for the development of caged peptides that can be activated via irradiation inside live cells, the thiol of a cell-penetrating peptide known to be a substrate for palmitoyl acyltransferase was protected as a NDBF thioether. Irradiation of human ovarian carcinoma (SKOV3) cells, preincubated with the probe, resulted in migration of the peptide from the cytosol/Golgi to the plasma membrane (visualized via confocal microscopy) due to enzymatic palmitoylation. These data suggest that the NDBF group should be useful for caging thiols in peptides and potentially larger proteins assembled via native chemical ligation for biological applications. As another approach, guided by mechanistic studies of the photo-triggered isomerization of Bhc-thiols, we developed 6-bromo-7- hydroxy-3-methylcoumarin-4-ylmethyl (mBhc) as an alternative coumarin-based caging group that can afford efficient thiol release upon one- and two-photon irradiation. To test the efficiency of mBhc for thiol-protection in peptides, we have synthesized a K-Ras-derived peptide where the thiol was protected by mBhc. One- and two-photon photolysis of the caged peptide resulted in clean conversion to the free compound with no photo-isomerization. Irradiation of the caged peptide using a near-IR laser in the presence of an enzyme (protein farnesyltransferase, PFTase) resulted in the generation of a free thiol-containing peptide which was then enzymatically farnesylated. To further evaluate the utility of this novel caging group for biomaterial applications, an mBhc-protected thiol was covalently incorporated into a hydrogel. Using a 740 nm two- photon laser from a confocal microscope, patterns of free thiols were generated inside the matrix and visualized by reaction with maleimide functionalized fluorophores. Such 3D patterns could be useful for a variety of applications in tissue engineering. Such highly tuned matrices allow artificial extracellular environments to be created that can be used to study cell migration, differentiation and cell–cell interactions. Lastly, we strived to develop a novel NDBF-based caging group with red-shifted absorption maxima and improved two-photon uncaging efficiency. Inspired by previous studies, we elected to modify the structure of NDBF by adding an amine as a donor group, to generate a donor-acceptor system. Hence, 2-bromo-2-(7-(dimethylamino)-3-nitrodibenzofuran-2-yl)acetate was synthesized in 9-steps. Initial analysis of spectral properties of the designed molecule showed the absorption maxima (λmax) to be 440 nm. This is110 nm red-shifted relative to λmax of NDBF. The uncaging efficiency of this novel protecting group remains to be tested.
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    Determining Cleavage Site Sequences to Characterize the Active Site of BACE1 Aspartic Protease
    (2015-04) Heink, Nicole
    Proteases play key roles in physiology and disease development due to their active role in the regulation of other proteins. Understanding and characterizing the active site of relevant proteases provides information vital to the production of inhibitors and elucidates potential native substrates that may be affected by inhibitors. Once the preferred sequence of the active site of a target protease, such as β-secretase (BACE1), has been defined, protease inhibitors can be created to treat or manage diseases such as Alzheimer's disease (AD). BACE1 is an aspartic protease that is overexpressed in the AD brain and is believed to initiate the AD disease pathway. As such, it is a strong candidate for drug design. However, chronic administration of BACE1 inhibitors could result in undesirable side effects due to the impairment of its ability to hydrolyze native substrates; therefore, the amino acid peptide sequence preferentially cleaved by BACE1 needs to be characterized. Not only will this indicate potential substrates that may be affected by BACE1 inhibition, it will also aid in the synthesis of viable inhibitors. Recently, a novel method for determing the cleavage sequence of proteases was reported. Proteomic identification of cleavage sites (PICS) is a method designed to accurately identify cleavage sequence preferences and neighbor interactions in the cleavage site that influence protease cleavage. This method gives additional information with less bias than previous methods used to characterize protease active sites. Multiple controls were used to confirm the validity of the procedure. These controls demonstrated our ability to successfully identify the amino acid sequence preferences for well characterized proteases. We were thus able to confidently use PICS to obtain the sequence of amino acids preferentially cleaved by BACE. BACE1A has two noticeable characteristics for the amino acid sequence cleaved: aromatic amino acids are preferred in the P1 site and leucine is strongly preferred in P2'. Other preferred amino acids are observed in the sequence, but not to the extent of P1 and P2'. Neighbor interactions were also investigated. Positive cooperativity resulted with leucine or valine in P3 and with phenylalanine or tyrosine in P1. There were strong interactions between valine in P3 and phenylalanine in P1 and between tyrosine in P1 and valine in P2'. Sequence preferences were also investigated for BACE2A, which exhibited both similarities and differences between BACE1A and BACE2A. The next step in this research will be to use knowledge of the preferred cleavage sites to determine physiological substrates of BACE1A. This will reveal more information about the natural function of BACE1A and identify potential side effects of its inhibition.
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    Self-assembling Phosphoramidate Pronucleotides: Enzymatic Regulation and Application Towards Therapeutic Delivery
    (2019-08) West, Harrison
    Prior characterizations of the nucleoside phosphoramidate moiety have centered upon the ability of amine containing side-chains to mask the negative charge inherent to monophosphorylated nucleosides for the purpose of enhancing their passive movement across biological membranes. When used for the intracellular delivery of therapeutic nucleoside monophosphate analogs, these molecules are referred to as phosphoramidate “ProTides” and represent an important class of antiviral and anticancer prodrugs. The primary aim of this thesis to build upon these works and present the nucleoside phosphoramidate moiety as a multifunctional regulator of molecular self-assembly. Appendage of nucleoside phosphoramidates to molecules such as self-assembling peptides was shown to modulate the self-assembling properties of the molecules through alteration of the non-covalent interactions between individual monomers and nanostructure assemblies. Additionally, the nucleoside phosphoramidate moiety was found to impart enzyme responsive qualities. Histidine triad nucleotide binding proteins (Hints), an enzyme class that possesses phosphoramidase activity, were found to regulate the assembly of nucleoside phosphoramidate bearing nanostructures by inducing ionic interaction mediated crosslinking after enzymatic hydrolysis. Chemical modification of self-assembling peptides with nucleoside phosphoramidates bearing non-natural and therapeutic nucleosides was also achieved to effect the first ever demonstrated self-assembling phosphoramidate ProTides as one-component nanomedicines. The developed formulations are currently under investigation for localized delivery of cancer chemotherapeutic prodrugs
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    A Solution Towards Weight Management: Discovery, Design, Synthesis, and Characterization of Melanocortin Ligands
    (2021-05) Koerperich, Zoe
    The melanocortin receptors are a family of rhodopsin-like (family A) G protein-coupled receptors (GPCRs) that are known to have a variety of conserved physiological functions. The following chapters of this dissertation explore four different projects designed to examine this receptor family through: 1) synthesis of novel ligands using traditional structure activity relationships (SAR), 2) use of a novel mixture based positional scanning approach for ligand discovery, 3) synthesis of endogenous ligand mimetics with established scaffolds, and 4) use of various functional, binding affinity, and proximity assays for in vivo evaluation. Chapter 1 serves as an introduction to the melanocortin receptor (MCR) family as well as ligands (endogenous and synthetic) that are well known in the field. This chapter also introduces the concept of selectivity between the five melanocortin receptors, particularly the centrally expressed melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R). Chapter 2 describes all the materials and methods used in Chapters 3-6 for peptide synthesis, characterization, and pharmacological evaluation. Chapter 3 describes the exploration of single nucleotide polymorphisms in the antagonist signaling molecule Agouti-related Protein (AgRP) by incorporating them into a modified AgRP octapeptide macrocyclic scaffold. Chapter 4 investigates the roles of ligands in protein:protein interactions, specifically those of the MC4R and the kappa opioid receptor (KOR). Chapter 5 demonstrates the complexity involved in designing a selective ligand through focused tetrapeptide SAR and functional assessment in a cAMP accumulation assay. Chapter 6 outlines the discovery of a completely novel MC3R antagonist scaffold using a mixture based positional scanning approach. Lastly, Chapter 7 broadly summarizes the dissertation presented and reflects upon knowledge and experienced gained, advancements to the field, and potential future directions for the work presented in this thesis.