No description available.

Doctor of Philosophy / Department of Biochemistry / John M. Tomich / Peptide-based delivery systems show great potential as safer drug delivery vehicles. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. We have designed and synthesized a set of 15 and 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated spheres with 50-150 nm diameters (confirmed by TEM and DLS). Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these structures are further stabilized by β-sheet hydrogen bonding and are stable at very low concentrations and even in the presence of SDS, urea and trypsin as confirmed by circular dichroism spectroscopy. Given sufficient time, they fuse together to form larger assemblies and trap compounds of different sizes within the enclosed space. They are prepared using a protocol that is similar to preparing lipid vesicles. We have shown that different concentrations of the fluorescent dye, 5(6)-Carboxyfluorescein can be encapsulated in these assemblies and delivered into human lens epithelial cells and MCF-7 cells grown on coverslips. Besides fluorescent dyes, we have delivered the plasmid (EGFP-N3, 4.7kb) into N/N 1003A lens epithelial cells and observed expression of EGFP (in the presence and absence of a selection media). In the case of large molecules like DNA, these assemblies act as nanoparticles and offer some protection to DNA against certain nucleases. Linear peptides that lacked a branching point and other branched peptides with their sequences randomized did not show any of the lipid-like properties exhibited by the branched peptides. The peptides can be chemically decorated with target specific sequences for use as DDS for targeted delivery.

The central nervous system (CNS) is susceptible to several disorders that can affect the structure or function of the brain or spinal cord, such as stroke and spinal cord injury (SCI). CNS disorders are complex, frequently causing failure of cognitive, motor and sensory functions. Unfortunately, there are only a few care alternatives for patients with CNS disorders, due to the limited capacity of the CNS to spontaneously regenerate; what expresses the need to develop innovative solutions, such as scaffolds that also could act as drug delivery systems to promote tissue and functional repairs in the CNS. To achieve this goal, three main projects were developed in this thesis. In the first project, a novel drug releasing duraplasty that can be applied as part of decompressive craniectomy (DC) was designed and tested. While DC can significantly reduce the risk of death, this procedure does not reverse the stroke damage. Thus, biosynthesized cellulose (BC) was used to produce a new duraplasty loaded with growth factors. The in vivo animal studies revealed that our duraplasty had excellent biocompatibility when implanted onto rodents’ brains. In the second project, BC tubes were prepared and nerve growth factor was incorporated into the tubes to be used as potential nerve guides to assist with the reconstitution of nerve tissues across SCI lesion. Physical and mechanical properties of the drug delivery systems produced were evaluated and compared to the neural native tissue. In addition, cell cultures demonstrated that growth factors released from both drug delivery systems were bioactive for over 7 days. In the third project, linear and 2-branched peptides were synthesized as potential bioactive molecules to improve tissue regeneration. These peptides, containing the RGDS sequence, were synthesized through Solid Phase Peptide Synthesis and characterized by mass spectrometry, high-performance liquid chromatography, and their conformational structures were analyzed by an energy minimized 3D model. In summary, this thesis explores the use of BC as drug releasing systems, which are promising and clinically relevant strategies to enhance nerve regeneration for many patients facing physical, mental and financial strains due to stroke, SCI or other difficult-to-cure injuries to the CNS.

Binding of the Rev protein with Rev Response Element (RRE) RNA present in singly- and unspliced mRNA transcripts is necessary for the replication of HIV-1. This interaction transports the mRNA transcripts from the nucleus to the cytoplasm for translation of the necessary structural and enzymatic proteins for the newly budding virus as well as for providing its genetic material. Given the high rate of mutation in HIV-1, the highly conserved and pertinent RRE RNA is of high interest for pharmaceutical intervention. Consequently, a branched peptide library containing unnatural amino acids was developed to target RRE RNA with the goal of increasing stability, potency, selectivity, and in vivo activity for RRE RNA. An unnatural amino acid branched peptide library (46,656 sequences) was synthesized and screened against RRE IIB and several hits in the sub-micromolar regime were found. A number of hits demonstrated selectivity in the presence of other RNAs in addition to two hits, 4A5 and 4B3, significantly inhibiting HIV-1 growth in vitro. These peptides inhibited HIV-1 replication in a concentration dependent manner and were demonstrated to be non-toxic. Further analysis of 4A5 and 4B3 via footprinting and SHAPE-MaP experiments determined that these peptides blocked binding of Rev through binding at the primary and secondary binding sites of RRE RNA. Sphingosine 1-phosphate (S1P) is a signaling molecule that plays a role in various biological processes including immunity, neurogenesis, and angiogenesis. The role S1P plays is largely determined by its location, in which Spinster homolog 2 (spns2) and mfsd2b are the two known transporters. The two transporters exist in different cell types and cellular localizations, with spns2-produced S1P being responsible for trafficking of lymphocytes. As such, spns2 has become of interest for therapeutic targeting in autoimmune and inflammatory diseases. To validate spns2 as a target in pharmaceutical intervention, a series of spns2 inhibitors were developed. A screening of a library of inhibitors found that compound SLP7120922 demonstrated inhibition of spns2 transport activity. The design, synthesis, and biological evaluation of inhibitors based on SLP7120922 is described. Modifications to the lipophilic tail region were performed with one compound 4.40f discovered to be potent, minimally toxic, and active in vivo. A series of modifications to the head region were then conducted that evaluated linear head derivatives with alkyl-, amide-, and amino acid-based groups. A number of compounds are reported that demonstrate good in vitro activity and minimal toxicity with two compounds, 4.48b and 4.52c, showing favorable in vivo activity in mice. / Doctor of Philosophy / Human immunodeficiency virus (HIV-1) has a high rate of mutation, which commonly leads to the need for many types of medications throughout the lifetime of a patient. In order to design a therapeutic that the virus has a low chance of growing resistance to, a target needs to be chosen with a low mutation rate. One such target is the Rev Response Element (RRE) RNA and it is necessary for the virus to replicate. A protein named Rev binds to RRE RNA in order for RRE to carry out its pertinent function. To block this function we have chosen branched peptides to target the RNA. Peptides are made of the same building blocks of proteins, but are much shorter than proteins. The peptides described here are made up of modified building blocks, called unnatural amino acids. This work describes the generation of an unnatural amino acid branched peptide library and how it was screened in order to find branched peptides that bind RRE RNA. Many peptides were found to bind RRE RNA but two in particular, 4A5 and 4B3, were the best binders that inhibited HIV-1 growth. The remainder of the work describes how these peptides bind to RRE RNA, while demonstrating that they are non-toxic and bind HIV-1 in a concentration dependent manner. A transporter protein termed Spinster homolog 2 (spns2) transports a signaling molecule known as sphingosine 1-phosphate (S1P). For our immune system to function properly, spns2 has to transport S1P to the appropriate places to signal to immune cells. Unfortunately, this is a problem in autoimmune and inflammatory diseases, such as multiple sclerosis, due to these diseases having an overactive immune system. A potential way to treat these diseases would be by inhibiting spns2. This work describes the design, synthesis, and biological evaluation of spns2 inhibitors. Many compounds were found to inhibit spns2 to a degree, but three compounds, in particular, show potent and effective inhibition in mice.

RNA is essential for the transfer of genetic information, as the central dogma of biology dictates. The role of RNA, however, is not limited to serving as an information shuttle between DNA and fully functional protein. Indeed, RNA has experienced a surge of interest in the field of chemical biology for its other critical roles in biology including those in control of transcription, translation, splicing, genetic replication, and catalysis. RNA has proven to be a difficult and complex target for the design of small molecular ligands because of its structural heterogeneity and conformational flexibility. Yet, the highly folded tertiary structures of these oligomers present unique scaffolds which designed ligands should be able to selectively target. To that end, two branched peptide libraries ranging in size from 4,096–46,656 unique sequences were screened for their ability to bind HIV-1 related RNA structures, the transactivation response element (TAR) and the Rev response element (RRE). In addition to discovering a mid-nanomolar branched peptide ligand for TAR, the first branched boronic acid peptide library designed to target RNA was screened for binding to RRE. Each of these efforts resulted in the identification of selective binders to their respective RNA targets, and the unnatural branching of these compounds was demonstrated to provide a multivalent binding interaction with the RNA. Furthermore, these compounds were shown to be cell permeable and displayed little to no cytotoxicity in HeLa and A2780 cells. / Ph. D.

RNAs have gained significant attention in recent years because they can fold into well-defined secondary or tertiary structures. These three dimensional architectures provide interfaces for specific RNA-RNA or RNA-protein interactions that are essential for biological processes in a living system. These discoveries greatly increased interest in RNA as a potential drug target for the treatment of diseases. Two of the most studied RNA based regulatory systems are HIV-1 trans-activating response element (TAR)/Tat replication pathway and Rev response element (RRE)/Rev export pathway. To efficiently target TAR and RRE RNA, we designed and synthesized three generations of branched peptide libraries that resulted in medium sized molecules. The first generation of BPs were discovered from screening a one-bead one-compound library (4,096 compounds) against HIV-1 TAR RNA. One peptide FL4 displayed a binding affinity of 600 nM to TAR RNA, which is tighter than its native protein counterpart, Tat. Biophysical characterization of these BP demonstrated that "branches" in BPs impart multivalency, and they are cell permeable and non-toxic. The second generation peptides were discovered from an on-bead high-throughput screening of a 3.3.4 branched peptide boronic acids (BPBAs) library that bind selectively to the tertiary structure of RRE IIB. The library comprised of 46,656 unique sequences. We demonstrate that our highest affinity BPBA (BPBA1) selectively binds RRE IIB in the presence of competitor tRNAs as well as against six RRE IIB structural variants. Further, we show that the boronic acid moieties afford a novel binding mode towards RNA that is tunable; their Lewis acidity has critical effects on binding affinity. In addition, biophysical characterizations provide evidence that "branching" in these peptides is a key structural motif for multivalent interactions with the target RNA. Finally, RNA footprinting studies revealed that the BPBA1 binding site encompasses a large surface area that spans both the upper stem as well as the internal loop regions of RRE IIB. BPBA1 is cell permeable and non-toxic. In the next generation of branched peptides, a 3.3.4 branched peptide library composed of 4,096 unique sequences that featured boronic acid and acridine moieties was designed. We chose acridine as the amino acid side chain due to its potential for π-stacking interaction that provides high binding affinity to RNA target. The library was screened against HIV-1 RRE IIB RNA. Fifteen peptides were sequenced and four contained acridine alone and/or in conjunction with boronic acid moieties displayed dissociation constants lower than 100 nM. The ribonuclease protection assays of A7, a sequence that contains both boronic acid and acridine residues, showed a similar protection pattern compared to previous peptide BPBA1, suggesting that the 3.3.4 branched peptides shared similar structural elements and contacted comparable regions of the RRE IIB RNA. The results from this research indicated that "branching" in peptides imparts multivalent interactions to the RNA, and that functional groups such as boronic acid and acridine are key structural features for efficient binding and selectivity for the folded RNA target. We demonstrated that the branched peptides are cell permeable and non-toxic. / Ph. D.