| Literature DB >> 26435848 |
Jeong Heon Lee1, Kai Bao1, John V Frangioni2, Hak Soo Choi3.
Abstract
The screening of living cells using high-throughput microarrays is technically challenging. Great care must be taken in the chemical presentation of potential ligands and the number of collisions that cells make with them. To overcome these issues, we have developed a glass slide-based microarray system to discover small molecule ligands that preferentially bind to one cell type over another, including when the cells differ by only a single receptor. Chemical spots of 300 ± 10 μm in diameter are conjugated covalently to glass slides using an arraying robot, and novel near-infrared fluorophores with peak emission at 700 nm and 800 nm are used to label two different cell types. By carefully optimizing incubation conditions, including cell density, motion, kinetics, detection, etc. we demonstrate that cell-ligand binding occurs, and that the number of cells bound per chemical spot correlates with ligand affinity and specificity. This screening system lays the foundation for high-throughput discovery of novel ligands to the cell surface.Entities:
Keywords: cell-based assay; drug discovery; high-throughput screening; live cells; microarrays; small molecules
Year: 2015 PMID: 26435848 PMCID: PMC4589137 DOI: 10.3390/microarrays4010053
Source DB: PubMed Journal: Microarrays (Basel) ISSN: 2076-3905
Figure 1Dual-channel screening strategy and controls. (A) Living integrin αvβ3-positive M21 cells (target cells; stained with ESNF10 and pseudo-colored in red) and integrin αvβ3‑negative M21-L cells (control cells; stained with IR786 and pseudo-colored in green) prior to dissociation from their respective plates. Scale bars = 100 μm. (B) The same cells mixed together and panned over PAAm positive control spots (top row) or cRGDyK ligand spots (bottom row). The yellow color indicates co-localized M21 and M21-L cells. Scale bars = 300 μm.
Figure 2Optimization of screening parameters: (A) Maximizing sensitivity through the effect of motion, incubation time, ligand spotting concentration, and the number of panned cells using a cRGDyK array and a mixture of M21 and M21-L cells. Shown are mean ± SD for each data point from 4 randomly chosen spots on the slide. (B) Maximizing specificity through the use of serum or competing receptor-negative cells.
Figure 3Robustness of the SMM Screening Assay: Three different model systems, varying in ligand chemical structure, cell type, receptor transmembrane topology, Bmax, and ligand affinity were tested as described in the text. Shown are mean ± SD for each data point from four randomly chosen spots on the slide. Receptor-positive and receptor-negative cells were labeled with 700 nm and 800 nm NIR fluorophores and pseudo-colored red and green, respectively, during microscopy. Scale bars = 200 μm.
Selected small molecule ligands and cell lines used for SMM screening.
| Compound | M.W.(Da) | Affinity ( | Specificity (Receptor) | Tested Cells (Positive/Negative) | Ref. |
|---|---|---|---|---|---|
| β-AG | 248.19 | 2 µM | PSMA | LNCaP/PC3 | [ |
| β-AG trimer | 1213.20 | 60 nM | PSMA | LNCaP/PC3 | [ |
| GPI | 311.23 | 9 nM | PSMA | LNCaP/PC3 | [ |
| GPI trimer | 1360.23 | 0.7 nM | PSMA | LNCaP/PC3 | [ |
| KUE | 319.31 | 15 nM | PSMA | LNCaP/PC3 | [ |
| cRGDyK | 619.67 | 50 nM | Integrin αvβ3 | M21/M21-L | [ |
| α-MSH | 1664.88 | 0.4 nM | MC1R | B16/LNCaP | [ |
| PAAm | ~70,000 | N.A. | Nonspecific | All Cells | [ |
β-AG, beta Ala-Gly; cRGDyk, cyclo Arg-Gly-Asp-D-Tyr-Lys; GPI, 2[(3-amino-3-carboxypropyl)(hydroxy)(phosphinyl)-methyl]pentane-1,5-dioic acid; KUE, 2-(3-(5-amino-1-carboxypentyl)ureido)pentanedioic acid; MC1R, melanocortin 1 receptors; α-MSH, α-melanocyte stimulating hormone; M.W., molecular weight; N.A., not applicable; PAAm, polyallrylamine; PSMA, prostate-specific membrane antigen.
Figure 4The effect of affinity on cell-ligand spot binding: PSMA-positive LNCaP cells and PSMA-negative PC3 cells were labeled with 700 nm and 800 nm NIR fluorophores and pseudo-colored in red and green, respectively. (A) Chemical structures of targeting ligands employed, (B) 4× microscopy images, and (C) 20× microscopy images. Scale bars = 200 μm.