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. 2022 Jan 18;9(7):2102855. doi: 10.1002/advs.202102855

Figure 1.

Figure 1

Development of a cell‐based assay for multiplexed drug screening. A) Experimental setup. For multiplexed screening of anti‐Parkinson drugs, CaV1.3 (target)‐specific and CaV1.2 (anti‐target)‐specific cell populations—each controlling the expression of a flexibly chosen reporter protein—are placed in the same reaction well to enable simultaneous assessment of inhibitory potency and specificity in potassium chloride (KCl)‐mediated cell depolarization. B) Design of a CCB‐activated (CaB‐A) reporter assay. Synthetic NFAT‐specific promoters control the production of L7Ae, which inhibits the translation of reporter mRNA by binding to specific C/D‐box aptamers in the 5’‐UTR. CCBs activate reporter protein expression by inhibiting depolarization‐dependent L7Ae expression. C,D) Optimization of CaB‐A for use‐dependent CCB analysis. (C) CaV1.2 (pCaV1.2/pKK56)‐ or (D) CaV1.3 (pCaV1.3/pKK56)‐transgenic HEK‐293 cells were co‐transfected with a NFAT‐controlled L7Ae expression vector (pMX125; PNFAT4‐L7Ae‐pA) and different reporter vectors containing one (pMX195; PSV40‐(C/D‐box)1‐SEAP‐pA) or two tandem C/D‐box aptamer repeats (pMX199; PSV40‐(C/D‐box)2‐SEAP‐pA). The cells were depolarized with different levels of KCl (0, 20, and 30 mm) and cultivated for 48 h in the absence or presence (10 µm) of nicardipine. SEAP levels in culture supernatants were scored. Data points are presented as mean ± SD (n = 3 independent experiments). Numerical values displayed on top of each column‐group represent induction‐folds, calculated as the SEAP values resulting from 10 µm nicardipine divided by SEAP values resulting from 0 µm nicardipine. (E, F) Validation of CaB‐A with clinically approved CCBs. E) HEK‐293 cells transfected with CaV1.2 (pCaV1.2/pKK56/pMX125/pMX199)‐ or F) CaV1.3 (pCaV1.3/pKK56/pMX125/pMX199)‐dependent CaB‐A systems were depolarized with 20 mm KCl and immediately seeded into culture wells containing different concentrations of CCBs. Data are mean ± SD of SEAP levels scored at 48 h after exposure to CCBs (n = 3 independent experiments).