3.3. Screening of Diverse Chemical and Cellular Interactions
To explore the usefulness of our SMM screening system, we tested three cell-ligand interactions, which all differed in terms of cell type, receptor type, ligand type, and Bmax (i.e., the number of receptors per cell) and are shown in Figure 3 and summarized in Table 1. M21 cells have approximately 5 × 104 integrin αvβ3 receptors per cell (i.e., Bmax) on their surface, while M21-L cells have no detectable integrin αvβ3 receptors (1 × 103) [27]. Integrin αvβ3 has a type I transmembrane topology and binds the cyclic peptide ligand cRGDyK with an affinity of approximately 50 nM. LNCaP cells have approximately 2 × 105 prostate-specific membrane antigen (PSMA) receptors per cell on their surface, while PC3 cells have no detectable PSMA [28]. PSMA has a type II transmembrane topology and binds the small molecule KUE with an affinity of approximately 15 nM. B16 cells have approximately 7 × 103 melanocortin 1 receptors (MC1R) receptors per cell on their surface, while LNCaP cells have no detectable MC1R [29]. MC1R is a G protein-coupled receptor with 7 transmembrane domains and binds the peptide α-melanocyte stimulating hormone (α-MSH) with an affinity of approximately 0.4 nM. Using the optimized parameters from Figure 2 (no motion, 60 min incubation time, 1 mM ligand spotting concentration, and 4 × 106 panned cells per slide), all three cell-ligand interactions are detectable with our SMM screening system with relatively high specificity and with sensitivity proportional to Bmax.
It should be noted, however, that many of the system parameters are inter-dependent and should be re-optimized for a particular model system. For example, in the presence of extremely high affinity and Bmax, motion might not only be possible but could improve specificity by reducing non-specific interactions. Similarly, we only explored the presence or absence of 10% serum, but in some model systems, a higher or lower concentration could be optimal.
microarrays-04-00053-t001_Table 1 Table 1  Selected small molecule ligands and cell lines used for SMM screening.   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.

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