Browsing by Subject "Phage display"
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Item Characterization of the interactions of zinc with known and novel allosteric modulators of glycine receptor function(2016-12) Cornelison, Garrett Lee; Mihic, S. John; Aldrich, Richard W; Harris, R. Adron; Stavchansky, Salomon AThe glycine receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is implicated as a possible therapeutic target for the treatment of diseases such as alcoholism and inflammatory pain. In humans, four glycine receptor subtypes (α1, α2, α3, and β) co-assemble to form pentameric channel proteins as either α homomers or αβ heteromers. To date, few agents have been identified that can selectively modulate the glycine receptor, especially those possessing subtype specificity. We used a cell-based method of phage display panning, coupled with two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, to identify novel heptapeptide modulators of the α1β glycine receptor. Peptides were identified that act with selectivity on α1β and α3β compared to α2β glycine receptors. In addition, peptide activity at the glycine receptor decreased when zinc was chelated by tricine, similar to previous observations of a decrease in ethanol’s enhancing actions at the receptor in the absence of zinc. Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nanomolar to low micromolar concentrations, and inhibiting its effects at higher concentrations. As zinc is present physiologically at various concentrations within this range, it is capable of influencing glycine receptor function, including modulation by other pharmacological agents; however, the magnitude of this effect and its possible relevance are not known. I therefore investigated the utility of previously-described “zinc-enhancement insensitive” α1 glycine receptor mutants D80A, D80G, and W170S to probe for interactions between zinc and other allosteric modulators at the glycine receptor. Interestingly, I found that only the W170S mutation conferred complete abolishment of zinc enhancement across a variety of agonist and zinc concentrations. Using α1 W170S receptors, I established that in addition to ethanol, zinc also interacts with inhaled drugs of abuse, but not volatile anesthetics, to synergistically enhance channel function. Additionally, I determined that this interaction is abolished at higher zinc concentrations, when receptor-enhancing bindings sites are saturated, suggesting a mechanism by which modulators such as ethanol and inhalants are capable of increasing receptor affinity for zinc in addition to enhancing channel function on their own.Item Engineering antibody and T cell receptor fragments : from specificity design to optimization of stability and affinity(2014-12) Entzminger, Kevin Clifford; Maynard, Jennifer Anne, 1974-B and T cells comprise the two major arms of the adaptive immune response tasked with clearing and preventing infection; molecular recognition in these cells occurs through antibodies and T cell receptors (TCRs), respectively. Highly successful therapeutics, clinical diagnostics and laboratory tools have been engineered from fragments of these parent molecules. The binding specificity, affinity and biophysical characteristics of these fragments determine their potential applications and resulting efficacies. Thus engineering desired properties into antibody and TCR fragments is a major concern of the multi-billion dollar biopharmaceutical industry. Toward this goal, we (1) designed antibody specificity using a novel computational method, (2) engineered thermoresistant Fabs by phage-based selection and (3) modulated binding kinetics for a single-chain TCR. In the first study, de novo modeling was used to generate libraries of FLAG peptide-binding single-chain antibodies. Phage-based screening identified a dominant design, and activity was confirmed after conversion to soluble Fab format. Bioinformatics analysis revealed potential areas for design process improvement. We present the first experimental validation of this in silico design method, which can be used to guide future antibody specificity engineering efforts. In the second study, the variable heavy chain of a moderately stable EE peptide-binding Fab was subjected to random mutagenesis, and variants were selected for resistance to heat inactivation. Thermoresistant clones where biophysically characterized, and structural analysis of selected mutations suggested general mechanisms of stabilization. Framework mutations conferring thermoresistance can be grafted to other antibodies in future Fab stabilization work. In the third study, TCR fragment binding kinetics for a clonotypic antibody were modulated by varying valence during phage-based selection. Binding affinity and kinetics for representative variants depended on the display format used during selection, and all TCR fragments retained binding to native pMHC antigen. This work demonstrates a general engineering platform for tuning protein-protein interactions. Using a combination of computational design and phage-based screening, we have identified antibodies and TCR fragments with improved binding properties or biophysical characteristics. The optimized variants possess a wider range of potential applications compared to their parent molecules, and we detail engineering methods likely to be useful in the engineering of many other protein-based therapeutics.Item Identification of novel allosteric modulators of the glycine receptor using phage display technology(2011-08) Tipps, Megan Elizabeth; Mihic, S. John; Aldrich, Richard W.; Harris, Adron; Iverson, Brent L.; Zakon, Harold H.The glycine receptor (GlyR) is a ligand-gated ion channel and a member of the cys-loop receptor family. Like other members of this family, the GlyR is a target for many drugs of abuse, including alcohol. While the effects of alcohol on these receptors have been well-characterized, the contribution of each receptor subtype to the overall physiological and behavioral effects of alcohol use are unclear. This is partially due to the limited pharmacology of the GlyR, which limits the ability to isolate GlyR function within a complex system. One method for identifying compounds that bind to and modulate a given target is phage display. This approach uses bacteriophage to screen a large number of peptide sequences for affinity at a given target. We developed a phage selection protocol to identify peptides that bind to the GlyR. These peptides were then tested for functional effects at the GlyR using two-electrode voltage clamp physiology. We identified several peptides that were able to modulate GlyR function. Peptide D12-116 showed specificity for the GlyR over two closely related γ-aminobutyric acid (GABA) channels. In addition, this method is easily adapted for the selection of peptides that bind to any cell-expressed target, increasing the utility of phage display in the neurobiology field. Another shortcoming in GlyR pharmacology is the lack of modulators with specificity for a single GlyR subtype. We next adjusted our selection protocol to search for peptides that can distinguish between the different Gly R α subtypes. We identified several promising lead peptides that show subtype preference. Finally, we found that trifluoroacetic acid (TFA), a common peptide contaminant, also modulates GlyR function. This finding has important implications for both previously reported peptide modulators and the pharmacology of several volatile anesthetics, for which TFA is the major metabolite