A General Method for Discovering Inhibitors of Protein−DNA Interactions Using Photonic Crystal Biosensors

Abstract

Protein-DNA interactions are essential for fundamental cellular processes such as transcription, DNA damage repair, and apoptosis. As such, small molecule disruptors of these interactions could be powerful tools for investigation of these biological processes, and such compounds would have great potential as therapeutics. Unfortunately, there are few methods available for the rapid identification of compounds that disrupt protein-DNA interactions. Here we show that photonic crystal (PC) technology can be utilized to detect protein-DNA interactions, and can be used in a high-throughput screening mode to identify compounds that prevent protein-DNA binding. The PC technology is used to detect binding between protein-DNA interactions that are DNA-sequencedependent (the bacterial toxin-antitoxin system MazEF) and those that are DNA-sequenceindependent (the human apoptosis inducing factor (AIF)). The PC technology was further utilized in a screen for inhibitors of the AIF-DNA interaction, and through this screen aurin tricarboxylic acid was identified as the first in vitro inhibitor of AIF. The generality and simplicity of the photonic crystal method should enable this technology to find broad utility for identification of compounds that inhibit protein-DNA binding.High-throughput screening (HTS) of compound collections is now a staple of modern drug discovery. In the most common incarnation, in vitro enzyme inhibition screens of large (>100,000 members) compound libraries are conducted using substrates that provide an easily quantified chromogenic/ fluorescent readout. Such screens have led to the discovery of many novel enzyme inhibitors and drug leads (1,2). Unfortunately, many potential drug targets are not enzymes, and thus for these systems high-throughput methods are needed that go beyond enzyme inhibition assays and directly report on small molecule-protein binding events.One area in which small molecule ligands for nonenzyme proteins would be useful is in the disruption of protein-macromolecule interactions. The identification of compounds that perturb protein-protein or protein-nucleic acid interactions is extremely challenging (3-6), and this is partly due to the paucity of good high-throughput screens. Successes in modulating protein-protein and protein-nucleic acid interactions with small molecules fall into a few classes: surface receptor-ligand interactions (integrins (7-9), IL-1/2 (10,11), TNFα (12)), (47). Another limitation is the potential for false positives due to fluorescent compounds, which is an inherent limitation of any fluorescence-based HTS method. Given the largely unexplored pharmacological realm that is protein-nucleic acid interactions, HTS assays independent of fluorescent tags would be extremely useful, especially in those cases where fluorescence anisotropy is not possible. In this report we describe the first use of photonic crystal technology for the development of an assay capable of detecting protein-DNA binding and further apply it in a high-throughput screening mode fo...

Nanolithography for oxide nanoarrays and their application in medical devices

“…The grating structure is position on the bottom of a standard 384-well microplate, whereas each well functions as a miniturized test-tube. 43 In light of this paper, protein nanopatterns have been utilized to improve immunodetection sensitivity, whereas the sensor surface is prepared with bioadhesive domains of nanometric dimension in a nonadhesive matrix formed by means of colloidal lithography. 44…”

Optimization of photonic crystal enhanced fluorescence by excitation laser angle scanning

“…Fundamentally, this effect occurs because small changes in optical density on the PC surface result in a shift in reflected wavelength (for a fixed illumination angle) -an effect that has been used effectively for PC-based label-free detection [16][17][18][19][20][21][22][23][24]. When considering the use of PCEF surfaces for multiplexed assays using an array of immobilized biomolecule capture spots, this problem is especially critical because the optical density associated with the capture molecules substantially modifies the resonant conditions on the PCEF surface.…”

Validation of an Optical Microplate Label-Free Platform in the Screening of Chemical Libraries for Direct Binding to a Nuclear Receptor

“…[2][3][4][5][6][7][8][9][10][11][12][13] Current HTS technologies are almost all based on industry-standard microplates. Traditional label-free approaches such as ITC and surface plasmon resonance (SPR) do not comply with this architecture.…”

“…3,[6][7][8] However, the recently commercialized photonic crystal-based biosensors are manufactured in standard microplate formats of 96, 384 and 1,536 wells. [9][10][11][12][13] These microplates can be read in easy-to-use benchtop plate readers, thereby forming a simple sensitive biosensor system, fully compatible with standard liquid and microplate handling automation. Biomass changes at the biosensor surface cause alterations in local refractive index (as with SPR), detected by precisely measurable shifts in the peak wavelength value (PWV) of sensor-specifically reflected light.…”

“…Such applications span from the screening of compound libraries to hit (in)validation, and mechanistic pharmacology of leads and from agonist trafficking to receptor panning. 4,[9][10][11][12][13] ABBREVIATIONS: b-RIP, biotinylated peptide derived from RIP140; CV, coefficient of variation; ERRg, estrogen-related receptor gamma; LBD, ligand-binding domain; PWV, peak-wavelength value; SD, standard deviation; SPR, surface plasmon resonance; DPWV, the change in peak-wavelength value.…”