Determining selectivity of drugs by quantitative two-dimensional gel analysis

In vitro pharmacologic measures of drug specificity are well established, i.e, drug interaction with a specific target such as an enzyme, receptor, or ion channel. However, in vitro measures of drug selectivity, defined as effects on secondary targets, are lacking. Two-dimensional gel electrophoresis (2-D gel) was examined as a measure of drug selectivity by comparing the effects of three drugs, tenidap, piroxicam, and dexamethasone, on the synthesis of intracellular proteins in lipopolysaccharide (LPS) stimulated murine macrophages. A set of 902 S-35-methionine-labeled proteins were separated consistently, identified by their coordinates of apparent isoelectric point and molecular weight, and quantified. LPS altered the concentrations of 45 proteins. Tenidap, at 10 mu M, affected a total of five proteins (suppressed three; stimulated two), whereas piroxicam, at 10 mu M, suppressed two proteins. Dexamethasone at 0.01 mu M suppressed eight proteins and stimulated one. Thus, none of the drugs reversed the LPS-induced changes. Two of the eight proteins suppressed by dexamethasone were also suppressed by tenidap and were identified as proIL-1 alpha and proIL-1 beta. Since the subset of affected proteins provided a unique protein "fingerprint" for each drug, the three drugs were mechanistically differential-ed by 2-D gel analysis. Compared to LPS (5% affected proteins), ail three drugs were selective (less than or equal to 1% affected) with piroxicam > tenidap > dexamethasone. With identification of affected proteins, this technique can provide a useful in vitro assessment of drug selectivity.