Quantitative Evaluation of Viral Protein Binding to Phosphoinositide Receptors and Pharmacological Inhibition

Abstract

There is significant interest in developing analytical methods to characterize molecular recognition events between proteins and phosphoinositides, which are a medically important class of carbohydrate-functionalized lipids. Within this scope, one area of high priority involves quantitatively evaluating drug candidates that pharmacologically inhibit protein–phosphoinositide interactions. As full-length proteins are often difficult to produce, establishing methods to study these interactions with shorter, bioactive peptides would be advantageous. Herein, we report an atomic force microscopy (AFM)-based force spectroscopic approach to detect the specific interaction between an amphipathic, α-helical (AH) peptide derived from the hepatitis C virus NS5A protein and its biological target, the phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] phosphoinositide receptor. After optimization of the peptide tethering strategy and measurement parameters, the binding specificity of AH peptide for PI(4,5)P2 receptors was comparatively evaluated across a panel of phosphoinositides and the influence of ionic strength on AH–PI(4,5)P2 binding strength was tested. Importantly, these capabilities were translated into the development of a novel experimental methodology to determine the inhibitory activity of a small-molecule drug candidate acting against the AH–PI(4,5)P2 interaction, and extracted kinetic parameters agree well with literature values obtained by conventional biochemical methods. Taken together, our findings provide a nanomechanical basis for explaining the high binding specificity of the NS5A AH to PI(4,5)P2 receptors, in turn establishing an analytical framework to study phosphoinositide-binding viral peptides and proteins as well as a broadly applicable approach to evaluate candidate inhibitors of protein–phosphoinositide interactions.

Exploring Interactions of Aptamers with Aβ40 Amyloid Aggregates and Its Application: Detection of Amyloid Aggregates

“…For each experiment, 3000 force curves were measured with several newly functionalized AFM tips in different scan areas (5 × 5 μm 2 ) of the freshly modified substrates. Furthermore, the analysis of rupture force distribution through these obtained force–distance curves and Gaussian fitting was conducted. , Subsequently, the most probable interaction force could be obtained and was presented as the mean ± standard error of the mean.…”

“…Furthermore, the analysis of rupture force distribution through these obtained force−distance curves and Gaussian fitting was conducted. 33,34 Subsequently, the most probable interaction force could be obtained and was presented as the mean ± standard error of the mean.…”

Characterization of Hepatitis C Virus Core Protein Dimerization by Atomic Force Microscopy

“…where τ D denotes the diffusive relaxation time, τ D = 10 −9 s. The most probable interaction forces were calculated by the Gaussian fitting of the force distribution histograms, as the rupture force measured by AFM was affected by the orientation of biomolecules. 26,28 By averaging the values of the most probable interaction forces obtained from three independent experiments, the interaction force was determined. The data were presented as mean ± standard error of the mean (SEM).…”

“…Atomic force microscopy (AFM) is a rapidly developing analytical technique to image macromolecules with high resolution and to measure bimolecular interaction force at the single-molecule level under physiological conditions. − Especially, AFM singlemolecular force spectroscopy (SMFS) has been widely used to study protein unfolding − and binding of ligand–receptor, − antibody–antigen, − DNA–protein, aptamer–protein, , and DNA–DNA. , In this work, we demonstrated, for the first time, the investigation of protein dimerization by quantifying the intermolecular binding forces from SMFS. In contrast to the observation of one peak in the normal rupture force histogram from previous reports, we found two forces in single-molecule force measurement between HCV core protein and its antibody.…”

Biomimetic Nanomaterial Strategies for Virus Targeting: Antiviral Therapies and Vaccines

“…[ 69 ] To date, most of these measurement strategies have been employed for understanding the fundamental role of multivalency in viral protein–glycan interactions while there remains tremendous potential to utilize these approaches for evaluating the functional performance of new nanomaterial inhibitors as well as to test drug inhibitor candidates that prevent such interactions. [ 70 ]…”