APRIL 2008 VOLUME 1 ISSUE 1

Application Highlights: Small Molecule

As part of this issue's focus on small molecule research, this Application Highlight features analyses performed on the Octet RED System.

- Matthew Kirtley, Product Manager, ForteBio

A substantial task of drug discovery and lead optimization is determining and evaluating the affinity of small molecules binding to a prospective therapeutic target. Speed is particularly essential during early drug discovery stages, when there may be thousands of potential lead compounds to be investigated. The Octet RED System in conjunction with Super Streptavidin Biosensors is designed to enable the detection of small molecules and peptides, with rapid data acquisition and sensitivity in a simple, user-friendly workflow.

Protein: Small Molecule Detection

By confirming target hits and identifying unsuitable compounds earlier in secondary screening, biomolecular interaction analysis helps better characterize leads by providing comprehensive characterization of kinetic parameters such as on/off rates, affinity constants, and steady state analysis.

The binding of inhibitors to carbonic anhydrase, a well-known model system^1,2, was assayed to demonstrate the Octet RED System's ability to accurately measure the kinetic constants of low molecular weight compounds. Biotinylated carbonic anhydrase was immobilized onto Super Streptavidin biosensors, followed by the association and dissociation of sulpiride and benzenesulfonamide, which represent a low affinity inhibitor and a low molecular weight compound, respectively. Non-specific binding of the compound to Super Streptavidin biosensors blocked with biocytin was not observed in the concentration range examined. Analysis was performed by globally fitting the data (Figures 1 and 2).

Figure 1. Analysis of a weak binding interaction. Sulpiride (341 Da):carbonic anhydrase binding data at 500-63 mM sulpiride (2X dilution series) in PBS with 1% DMSO.

Figure 2. Analysis of a low molecular weight compound. Benzenesulfonamide (157 Da):carbonic anhydrase binding data at 100-0.4 mM benzenesulfonamide (3X dilution series) in PBS with 0.5% DMSO

In a separate experiment (Figure 3), furosemide binding data was also analyzed using global fitting data from a series of concentrations and the resulting kinetic constants k_a, k_d, K_D of 0.56 X 10⁵ M^-1s^-1, 0.050 s^-1, and 890 nM, respectively, are also in close agreement with the values of 9.7 X 10⁴ M^-1s^-1, 0.050 s^-1, and 513 nM previously reported for analysis with surface plasmon resonance².

Protein:Peptide Detection

Evaluation of the kinetic profiles of your protein: peptide interactions will help detect the small changes in affinity that occur through iterations of optimization, providing information that regular endpoint assays leave out.

The data shown in Figure 4 represent the binding of varying concentrations of His-tagged ubiquitin (10, 3.3, 1, 0.33, 0.1mM) to a biotinylated anti-HA antibody that has been immobilized onto Streptavidin High Binding Biosensors. Calculations of the kinetic parameters are then automatically carried out by the Octet RED System software.

Figure 3. Analysis of furosemide:carbonic anhydrase binding data at 30-0.12 mM furosemide (3X dilution series, N=5) in PBS with 0.5% DMSO.

Figure 4. Titration series of HA-ubiquitin (8 kDa) binding to anti-HA-antibody.

Fast Protein Quantitation

The ability to perform quantitation of antibody preparations is critical to help select the preparations that are the best producers. Once the level of production has been evaluated, proceeding with the optimal preparation is quick and easy.

Assays for determining protein concentration on the Octet RED System are based on the binding rate of the protein of interest to the biosensor surface. Different protein concentrations result in different binding rates.

The Octet RED System software calculates the binding rates from standards with known values to generate a standard curve - the binding rate of each standard is proportional to its concentration (Figure 5).

Octet RED System vs. Surface Plasmon Resonance: Blind Study

In a blind study, ten compounds (162-330 Daltons) were analyzed using the Octet RED System, and the results were compared to results obtained using a Biacore SPR system.

The K_D values obtained using the Octet RED System and SPR are listed in Table 1. For each compound, the K_D determined by the two methods is generally within 2-3X.

Figure 5. Octet software screen capture showing lgG protein quantitation.

Table 1. Accuracy of Octet RED System K_D determination compared to SPR K_D determination

Reproducibility

While accuracy is important, it doesn't matter what results you get if they can't be repeated in subsequent experiments. The Octet RED System produces robust, accurate data that is also reproducible.

To demonstrate the reproducibility of results obtained from the Octet RED System, two different inhibitors binding to carbonic anhydrase were assayed. Kinetic constants for multiple analyses agree within <30% for benzenesulfonamide and within <17% for furosemide (Tables 2 and 3).

Tables 2 & 3. Reproducibility data for furosemide (upper table) and benzenesufonamide (lower table).

Additional detail and data analyses are available in our SBS 2008 poster Label-Free Determination of Kinetic Constants for Small Molecule Binding to Proteins Using ForteBio's Octet RED Multi-Channel Platform (available for download from our web site). You can also learn more about the Octet RED System at www.fortebio.com, or at one of our presentations (see page 6 for a list of upcoming presentations).

REFERENCES

1 Papalia et al, Analytical Biochem 359
(2006), 94-105

2 Myszka et al, Analytical Biochem 329
(2004), 316-323

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