Small molecule and peptide therapeutic drugs are highly sought after in most areas of disease research due to their desirable pharmacological properties and low propensity for immunogenicity. In small molecule drug discovery, the path to lead molecule can stem from a number of sources or starting points including fragment screening, HTS, de novo structural design, etc. The determination and evaluation of the affinity of small molecule binding to a therapeutic target is a significant component of drug discovery and lead optimization.
The Hit-to-Lead (H2L) and Lead Optimization process is important to accurately determine biological potency in vitro so that structure activity relationships (SAR) can be used for efficient structural design. Highly sensitive biophysical methods are often required for this step in the pipeline as protein target MW can be large compared to the small molecules being characterized.
Pioneer FE System: Measure Binding Interactions of Small Molecules to Large MW Proteins or Targets with Low Activity
Pioneer FE has sufficient sensitivity to measure and characterize binding interactions of small molecules to large MW proteins or protein targets with low activity. A well-established model system1, 2, the binding of carbonic anhydrase inhibitors, was examined to assess the Pioneer system's ability to determine kinetic constants for small molecules (172–341 Da). Biotinylated Carbonic anhydrase was captured onto a COOH5 biosensor functionalized with NeutrAvidin™. Kinetic assays with six sulfonamide inhibitors were performed in duplicate and the results shown in Figure 1.
Figure 1: Analysis of sulfonamide inhibitors binding Carbonic anhydrase II with the Pioneer system. Two-fold dilution series of Sulpiride (top left), Furosemide (top right), CBS (middle left), Sulfanilamide (middle right), Benzenesulfonamide (bottom). Data were analyzed with the two compartment (mass transport limited) 1:1 kinetics model.
The data in Figure 1 show the Pioneer system’s capabilities for measuring and characterizing kinetics/affinity for small molecule inhibitors with conventional kinetic methods. The Pioneer systems have a key advantage for kinetic characterization when using OneStep® Injections, which can characterize an interaction for kinetics and affinity in a single gradient injection. Figure 2 shows the same sulfonamide inhibitors plus methylsulfonamide (95 Da) each characterized using OneStep injections.
Figure 2: OneStep kinetic analysis of sulfonamide inhibitors binding darbonic anhydrase II with the Pioneer system. Each OneStep Injection represents a sigmoidal concentration gradient of analyte which encodes kinetic rate constants of binding. Data were analyzed with the two compartment (mass transport limited) 1:1 kinetics model.
Pioneer FE is the recommended solution for analysis of small molecule — target interactions when therapeutic targets are large or partially inactive, and when analytes are low MW. Pioneer’s OneStep technology offers a full kinetic and affinity characterization of binding events in a single injection. Small molecule screening applications with OneStep Injections therefore determine both whether an analyte binds target and the affinity of target binding. The ability of OneStep Injections to capture kinetic information in one inject also benefits assays where target activity is unstable or target cannot be regenerated.
Octet RED96e, RED384 and HTX Systems: Detection of Small Molecules and Peptides Against Smaller MW Therapeutic Targets
The Octet RED96e, Octet RED384 and HTX systems are designed with rapid data acquisition to enable the detection of small molecules and peptides against smaller MW Pioneer's OneStep technology offers a full kinetic and affinity characterization of binding events in a single injection. Small molecule screening applications with OneStep Injections therefore determine both whether an analyte binds target and the affinity of target binding. OneStep Injection ability to capture kinetic information in one inject also benefits assays where target activity is unstable or target cannot be regenerated.
Label-free analysis of the association and dissociation of a small molecule with the target protein of interest results in the determination of kinetic constants including the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD).
The biotinylated protein targets (carbonic anhydrase II) were immobilized onto Super Streptavidin Biosensors, and the association and dissociation of a dilution series of benzenesulfonamide was monitored in parallel to minimize time to results.
Figure 3: Analysis of benzenesulfonamide binding carbonic anhydrase at 100-0.39 µM benzenesulfonsemide (4X dilution series, N=5) in PBS with 0.5% DMSO.
The data in Figure 3 demonstrate the Octet RED96e system’s ability to generate high-quality kinetic data on a small molecule (157 Da) interaction with excellent reproducibility (N=5). The Octet systems enable simple and fast characterization of small molecule:protein interactions for smaller MW protein targets.
1. Papalia et al., Analytical Biochem, 359 (2006), 94-105.
2. Myszka, Analytical Biochem, 329 (2004), 316-323.