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Advanced Technologies Accelerate Peptide Drug Discovery and Development

Powerful platforms for candidate identification, and insights into cellular effects and biomolecular interactions

by Sartorius
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Peptides are small signaling molecules that bind cell surface receptors or ion channels and initiate an intracellular effect. Adrenocorticotropic hormone (ACTH) and insulin were the first native peptides isolated from mammalian tissues and exert their effects on the hypothalamic-pituitary axis and glucoregulatory systems, respectively. Since then, thousands of other naturally occurring peptides have been discovered, and many others have been chemically synthesized. Of the many advantages of peptides over existing therapeutics, their specificity makes them particularly attractive molecules for therapeutic development. To date, over 60 therapeutic peptides have been approved for indications ranging from hypertension and diabetes to prostate cancer and Cushing’s disease, and hundreds more are in active clinical development. Recently, a peptide-based binder was investigated for its ability to inhibit the SARS-CoV-2 receptor binding domain and angiotensin-converting enzyme 2 (ACE2) interaction that facilitates viral entry into cells.

Specificity and Other Advantages of Peptides

Peptides have numerous advantages over other types of therapeutics. Unlike antibodies, peptides are small and easily synthesized, and are often associated with lower production costs. They also offer a high degree of specificity and affinity, as many are designed to bind cell surface receptors, such as G protein-coupled receptors (GPCRs) and initiate intracellular effects. Cell-penetrating peptides (CPPs), including TAT and penetration, also overcome some of the challenges associated with gene and protein therapy including toxicity and immunogenicity. CPPs effectively penetrate the membrane to deliver various macromolecules, while preserving their biological activity. Many novel therapeutics, especially in oncology, are peptide-drug conjugates that bind cell surface targets with high affinity to deliver, for example, chemotherapeutic agents. This ensures high local concentration and minimizes exposure throughout the rest of the body to enhance safety.

Given the many advantages over existing therapeutic agents, significant efforts are underway to develop peptide-based drugs. Leveraging advanced technologies including advanced flow cytometry, live-cell analysis, and biolayer interferometry can accelerate the discovery process.

Rapidly Identify Candidates With Advanced Flow Cytometry

Figure 1. Schematic representation of a 384 multiplexed peptide screen with target cells of interest using iQue® advanced flow cytometry to generate plate-based data output, such as heat maps. Changes in cell or well color indicate cell penetration of fluorescently labeled peptide fragments. 

Therapeutic peptide discovery and development necessitates numerous assays to identify candidates with the desired biological outcomes. Assays are designed to determine binding specificity, membrane penetration, and peptide-mediated uptake of macromolecules and drugs, among numerous other parameters. Traditional flow cytometry approaches are complex and time-consuming and create bottlenecks in the discovery process. Advanced high-throughput flow cytometry systems fully automate processing and analysis workflows to provide rapid, actionable answers. The combination of multiplex quantitation capabilities and cellular phenotype and function applications provides deeper, more meaningful biological insights (Figure 1). An advanced platform also supports small volumes and assay miniaturization to conserve valuable samples and reagents.

Insights Into Direct Cellular Effects With Live-Cell Imaging

The cellular effects of peptide drug candidates—including activation or inhibition of enzymes, receptors, or biochemical pathways, among others—must also be assessed. This may be achieved with in vitro techniques to visualize and quantify cell behavior. Traditional methods require cell cultures to be removed from the incubator for imaging, which disrupts the cells and as such, are limited to endpoint analysis. These methods are also laborious and time-consuming. 

Figure 2. HD phase-contrast and fluorescent images of Incucyte® NucLight HT-1080 cells showing morphological changes in response to staurosporine (SSP), camptothecin (CMP) and cyclohexamide (CHX) (A). 96-well microplate graphs showing the kinetic measurement of cell death as determined by Incucyte® Cytotox Green Dye in response to several concentrations of SSP, CMP and CHX in MDA-MB-231 cells and HT-1080 cells (B).  Images and data were acquired and analyzed in an Incucyte® Live-Cell Analysis System.

A live-cell imaging and analysis platform provides insights into the direct cellular effects of peptide candidates from within the incubator, eliminating the need to remove and disrupt cells while acquiring images. With continuous image acquisition and analysis capabilities, quantitative live-cell imaging technology enables users to obtain time-lapsed, kinetic measurements over days or months, ensuring important data points are never missed. The ability to automatically acquire and analyze images in microplate format offers walk-away convenience and enhances productivity. Live-cell imaging platforms also support multiplexed, high-throughput screening to provide information-rich data, and complex assays to obtain deeper, more physiological relevant insights into biological activity (Figure 2). 

Real-Time Insights on Biomolecular Interactions With BLI

 Biolayer interferometry (BLI) is a label-free technique that provides insights into real-time biomolecular interactions including binding affinity, association and dissociation kinetics and specificity. These parameters provide critical insights into the mechanism of interaction between candidate peptides and their molecular targets. BLI is an optical analytical approach to analyze the interference pattern of white light reverberated from two surfaces, the inner reference layer, and the layer of protein settled on the sensor tip. When a ligand immobilized on the biosensor tip binds with an analyte in solution, it produces a wavelength shift that is a direct measure of the change in optical thickness of the biological layer. This technology replaces label-dependent methods and provides rapid, accurate results. BLI also enables users to analyze samples in crude matrix and can analyze up to 96 samples in just minutes to enhance productivity (Figure 3). 

Figure 3. Peptide binding assays on the Octet®. Octet BLI assays can be used for peptide binding characterization and identifying peptide binders from a screen. (A) Potential biosensor binding assay configuration for a peptide binding assay. A ligand is immobilized onto a biosensor and peptide binding levels are measured. Alternatively, depending on the assay, the orientation can be reversed where the peptide can be immobilized. (B) Depiction of BLI kinetic data. Affinity constants (KD ) are measured using the ratio between the dissociation and association rates. (C) The affinity plot shows the relationship between the association (ka ) and dissociation (kd) rate constants and the equilibrium dissociation constant (KD ). Identical affinities can be produced by varying kinetic rates and Octet assays enable differentiation of binders based on kinetics and affinities. 

Since the introduction of insulin in the clinic in the 1920s, and the advent of peptide synthesis in the 1950s, dozens of therapeutic peptides have been developed, and discovery efforts are rapidly increasing. Sartorius offers powerful technologies, including advanced flow cytometry, live-cell imaging, and biolayer interferometry platforms to accelerate therapeutic peptide discovery.

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