Browsing by Subject "Peptide"

Now showing 1 - 10 of 10

Biosynthesis of sulfur containing heterocycles in natural products
(2016-12) Gengler, Jon Peter; Liu, Hung-wen, 1952-
This thesis is a comprehensive review of the biosynthesis of sulfur containing heterocycles in natural metabolites. The review focuses on sulfur incorporation and cyclization of the moieties, with a lesser examination of the role these heterocycles play in the chemistry of their compound's activity.
Characterization and applications of affinity based surface modification of polypyrrole
(2009-12) Nickels, Jonathan D.; Schmidt, Christine E.
I present the characterization and applications of a technique to modify the surface of the conducting polymer, polypyrrole, via a novel, 12-amino acid peptide, THRTSTLDYFVI (T59). This peptide non-covalently binds to the chlorine-doped conducting polymer polypyrrole, allowing it to be used in tethering molecules to polypyrrole for uses such as a scaffold for the treatment of peripheral nerve injury or in surface coatings of neural recording electrodes. I have quantified the binding of this peptide as well as investigating the mechanism of the binding. The equilibrium constant of the binding interaction of PPyCl and the T59 peptide was found through a binding assay to be 92.6 nM, and the off rate was found to be approximately 2.49 s⁻¹, via AFM force spectroscopy. The maximum observed surface density of the peptide was 1.27 +/- 0.42 femtomoles/cm². Furthermore, my studies suggest that the eighth residue, aspartic acid, is the main contributor of the binding, by interacting with the partially positive charge on the backbone of polypyrrole. I have demonstrated practical applications of the technique in the successful modification of a PPyCl surface with the laminin fragment IKVAV, as well as the so-called stealth molecule poly(ethylene glycol) (PEG). A subcutaneous implant study was performed to confirm that the T59 peptide did not induce any significant reaction in vivo. Significantly, the conductivity of a PPyCl surface was unaffected by this surface modification technique.
Development of dry powder inhalation for biological drug modalities
(2020-05) Zhang, Yajie; Williams III, Robert O., III, 1956-; Watts, Alan B.; Smyth, Hugh D.C.; Cui, Zhengrong; Ghosh, Debadyuti
Dry powder inhalers (DPI) have been developed to topically deliver therapeutics to the lung. Generally, a particle size range of 1-5 µm is desirable for the lung deposition of drug aerosols. To date, several particle engineering technologies have been developed and successfully applied to micronize powders of small molecule drugs. However, particle size reduction is challenging for biological materials due to their ductile texture and susceptibility to stresses encountered during processing. In this dissertation, three biological drug modalities (i.e. peptide, protein, and bacteriophage), are micronized using different particle engineering technologies. Chapter One provides a review of formulations and delivery systems used in peptide therapeutic products. Chapter Two describes several stages in the development of an excipient-free peptide inhalation product, including pre-formulation, formulation and processing, stability testing, and animal studies. It was demonstrated that jet milling is effective in manufacturing a stable, excipient-free peptide inhalation powder for the treatment of pulmonary fibrosis. In Chapter Three, inhalable dornase alfa powder is formulated with various excipients and the formulations are processed using thin film freeze-drying (TFFD) technology. The excipient screening was performed by employing design of experiment (DoE). Formulations that were both inhalable and preserve the enzymic activity of the protein were found. In Chapter Four, a review of inactive ingredients used in solid bacteriophage formulations is presented. Finally, Chapter Five explores the feasibility of using TFFD to manufacture inhalable bacteriophage powders. It was proved that TFFD is a suitable technology to produce powders that have desirable properties, including preserved bioactivity of bacteriophage, inhalable size range, and other physical characteristics.
Discovery of blood-brain barrier penetrating peptides by phage display technology to deliver biologics into the brain
(2019-09-19) Peng, Xiujuan; Ghosh, Debadyuti; Williams, Robert O.; Cui, Zhengrong; Baker, Aaron B.
Biologics as macromolecules can’t deliver to the brain without the blood-brain barrier (BBB) transport carriers. A phage display peptide library was employed to screen the BBB shuttling peptides by biopanning against an in vitro BBB model. We identified several novel and efficient BBB permeable peptides. A series of in vivo and in vitro studies were conducted to validate the capability of these peptides to transport the BBB and the brain extracellular matrix (ECM). The findings demonstrated that our peptides, Pep-3 and Pep-9 are able to shuttle across BBB and deliver to the brain parenchyma in vivo. Thereafter, Pep-9 was selected to develop IgG-Pep-9 conjugate formulation by click chemistry. In vivo delivery of IgG-Pep-9 indicated that Pep-9 can carry antibody molecules to transport BBB and deliver to the brain parenchyma.
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
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
Inhalable dosage forms containing difficult-to-formulate drugs : compositions and processing design space
(2020-06-22) Sahakijpijarn, Sawittree; Williams, Robert O., III, 1956-; Cui , Zhengrong; Smyth, Hugh D.C.; Koleng, John J.; Watts , Alan B.
Pulmonary drug delivery has recently gained much importance in the health care research area as it enables to target the drug delivery directly to lung both for local and systemic treatment. Despite extensive studies in the last decade, the development of inhaled formulations is still challenging, especially for difficult-to-formulate drugs such as macromolecule drugs and amorphous small molecule drugs. Due to the fragile nature of macromolecules, they are prone to degrade when exposed to physicochemical stress such as temperatures, pH, storage humidity, formulation component, atomization. The first two studies demonstrate the strategies to overcome the stability challenge of proteins and peptide in order to deliver a drug to the lung by nebulization. By the right selection of nebulizer and formulation optimization, the stability of fibrinolysins can be preserved after lyophilization, reconstitution, and nebulization using vibrating mesh nebulizers. Furthermore, the effect of counterion on the stability of peptide was studied in the second study. The formulation and processing were optimized to preserve volatile counterions, thus minimizing the pH change of reconstituted solutions and maintaining the stability of peptide. In addition to macromolecules, amorphous small molecules drug is another type of difficult-to-formulate drugs since they are thermodynamically unstable and thus tend to undergo crystallization during storage, which can affect the drug aerosolization and drug release upon deposition in the airway. The third study aimed to investigate the feasibility of thin film freezing to prepare dry powder for inhalation containing a high potency of amorphous tacrolimus. We found that using ultra-rapid freezing can increase drug loading up to 95% while maintaining good aerosol performance and stability. Finally, a new strategy to overcome the challenge in developing ordered mixture dry powder for inhalation was developed in the last study. We found that powders made by TFF ordered mixing process exhibited superior aerosol performance and less variation in content uniformity, compared to powders made by conventional powder blending. Throughout these studies, we can conclude that the right design of formulation and process can help to overcome the challenges in developing inhaled formulations of difficult-to-formulate drugs
Optimization of force fields for molecular dynamics
(2014-12) Di Pierro, Michele; Elber, Ron
A technology for optimization of potential parameters from condensed phase simulations (POP) is discussed and illustrated. It is based on direct calculations of the derivatives of macroscopic observables with respect to the potential parameters. The derivatives are used in a local minimization scheme, comparing simulated and experimental data. In particular, we show that the Newton Trust-Region protocol allows for accurate and robust optimization. POP is illustrated for a toy problem of alanine dipeptide and is applied to folding of the peptide WAAAH. The helix fraction is highly sensitive to the potential parameters while the slope of the melting curve is not. The sensitivity variations make it difficult to satisfy both observations simultaneously. We conjecture that there is no set of parameters that reproduces experimental melting curves of short peptides that are modeled with the usual functional form of a force field. We then apply the newly developed technology to study the liquid mixture of tert-butanol and water. We are able to obtain, after 4 iterations, the correct phase behavior and accurately predict the value of the Kirkwood Buff (KB) integrals. We further illustrate that a potential that is determined solely by KB information, or the pair correlation function, is not necessarily unique.
Preparation of biomimetic surfaces that facilitate the native cellular process of peptide self-assembly
(2015-11-24) Dugger, Jason Wade; Webb, Lauren J.; Vanden Bout, David; Roberts, Sean; Maynard, Jennifer; Elber, Ron
The ability to maintain or reproduce biomolecular structures on inorganic substrates has the potential to impact diverse fields such as sensing and molecular electronics, as well as the study of biological self-assembly and structure-function relationships. Because the structure and self-assembly of biomolecules are exquisitely sensitive to their local chemical and electrostatic environment, the goal of reproducing or mimicking biological function in an abiological environment, including at a surface, is challenging. However, simple and well-characterized chemical modifications of prepared surfaces can be used to tune surface chemistry, structure, electrostatics, and reactivity of inorganic materials to facilitate biofunctionalization and function. Here, we describe the covalent attachment of 13-residue β-stranded peptides containing alkyne groups to a flat gold surface functionalized with an azide-terminated self-assembled monolayer (SAM) through a Huisgen cycloaddition, or “click,” reaction. The chemical composition and structural morphology of these surfaces were characterized using X-ray photoelectron spectroscopy (XPS), grazing incidence angle reflection-absorption infrared spectroscopy (GRAS-IR), surface circular dichroism (CD), atomic force microscopy v (AFM), and neutron reflectometry (NR). The surface-bound β-strands self-assemble into antiparallel β-sheets to form fibrillar structures 24.9 ± 1.6 nm in diameter and 2.83 ± 0.74 nm in height on the reactive surface. The results herein provide a platform for studying and controlling the self-assembly process of biomolecules into larger supermolecular structures while allowing tunable control through chemical functionalization of the surface. Interest in the mechanisms of formation of fibrillar structures have most commonly been associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s, but fibrils may actually represent the thermodynamic low-energy conformation of a much larger class of peptides and proteins. The protocol developed here is an important step towards uncovering not only the factors that dictate self- assembly, but also the mechanisms by which this fibrillar class of superstructures form.
Synthesis of model nanoparticle-peptide conjugates and development of an electrochemical strategy to regulate microdroplet pH in microfluidics
(2021-12-02) Wilder, Logan Michael; Crooks, Richard M. (Richard McConnell); Webb, Lauren J; Brodbelt, Jennifer S; Hoffman, David W
Described herein is the synthesis and characterization of model nanoparticle (NP)-peptide conjugates intended for biorecognition applications. Also described is the development of an electrochemical strategy for regulating microdroplet pH in microfluidics. These works present new tools for control of biological and biochemical systems. NP-biomolecule interactions are difficult to control. This limits the application of NPs in biochemistry and medicine. Here, we present the synthesis of water-soluble AuNPs with attached peptides which are conformationally α-helical. Specifically, ~5 nm AuNPs with attached, azide-terminated alkylthiol-oligoethylene glycol ligands were synthesized. The AuNPs were then conjugated to peptides using Cu-catalyzed azide-alkyne cycloaddition, and the AuNP-conjugated peptides were demonstrated to be helical using circular dichroism spectroscopy. These AuNP-peptide conjugates are useful models for designing AuNP-peptide conjugates for biorecognition applications. We also present a study of NP-peptide interactions which determined that surface layers of oligoethylene glycol on NPs can play a role in directing NP-attached peptide structure. This insight is useful for understanding NP-peptide and NP-protein interactions. Droplet microfluidics enables high-throughput, individually controlled, and physically isolated experiments in nano-to-picoliter volumes. Regulation of the contents of droplets in microfluidic channels is challenging but attractive for some applications. Here, we present a technique to regulate the pH of a microdroplet in motion through a microfluidic channel using electrochemistry. Specifically, water electrolysis is used to alter pH in different regions of a microdroplet while the microdroplet is simultaneously split into descendant droplets. This produces descendant droplets with predictably changed pH. This technique enables temporal and quantitative control of pH in droplet microfluidics. Starting with a droplet pH of 6.8, a pH range of 5.9 to 7.7 can be achieved. Further, when the final droplet pH is in the range of 6.5 to 7.7, it is within ±0.1 pH units of prediction. Finally, this technique achieves the difficult task of isolating the electrolysis products generated during droplet electrochemistry and thus can likely be applied to the generation of other reagents within microdroplets.
Ultrafast 2D IR spectroscopy of membrane peptide systems
(2020-05-06) Flanagan, Jennifer Catherine; Baiz, Carlos R.; Brodbelt, Jennifer S; Rosales, Adrianne M; Brenna, James T; Webb, Lauren J
Plasma membranes are the main liaisons between the intercellular and extracellular environment, playing a critical role in numerous biological processes. Recent research has challenged the long-standing "fluid mosaic model," representing membranes as densely packed, heterogeneous environments. Within these complex membranes are transmembrane proteins which comprise up to 50% of the membrane mass, and are themselves diverse in sequence, structure, and function. Combining two-dimensional infrared spectroscopy (2D IR) and molecular dynamics simulation (MD), this thesis explores membrane complexity from two perspectives: first, it addresses the sequence heterogeneity in transmembrane peptides; and second, it explores the effect this crowded environment has on the lipids themselves and the implications this has on future membrane studies. Site-specific hydration of transmembrane peptides was probed using singly isotope-labeled pH (Low) Insertion Peptides, or pHLIPs. These peptides are a class of small transmembrane helices containing ~30% polar residues. By including a single-residue ¹³C=¹⁸O isotope label on the pHLIP backbone, IR experiments effectively produce a single-residue spectrum separate from the main peptide peak. With computational models to connect atomistic structure from MD to infrared frequency shifts, these site-specific spectra reveal local hydration as far as 1 nm into the hydrophobic membrane core. Crowding experiments probed dynamics at the lipid-water interface of model membranes as a function of transmembrane peptide concentration. These dynamics, drawn from time-dependent 2D IR, trend non-monotonically with peptide concentration, revealing three dynamical regimes: a pure lipid-like, a bulk-like, and a crowded regime. Through similar computational methods, these dynamics were linked to water structure at the lipid-water interface, which is perturbed by peptide insertion. Finally, preliminary work has been carried out in developing transient 2D IR methods for applications to protein folding. The first pH-jump 2D IR experiment has been performed by implementing an ultraviolet pump laser to a photoacid-containing sample. Pumping the sample with UV dissociates the photoacid, causing an instantaneous, local pH drop, and the effect on the sample is probed by 2D IR. This new method extends the picosecond-scale 2D IR experiment to a micro-to-millisecond timescale, and has potential for studying pH-initiated conformational transitions such as protein folding and polymerization.