ISOLATION, IDENTIFICATION AND IMMUNOSUPPRESSIVE ACTIVITY OF A NEW IMM-125 METABOLITE FROM HUMAN LIVER-MICROSOMES - IDENTIFICATION OF ITS CYCLOPHILIN-A IMM-125 METABOLITE COMPLEX BY NANOSPRAY TANDEM MASS-SPECTROMETRY

The isolation from human liver microsomes and identification by electr ospray mass spectrometry and tandem mass spectrometry of a neat metabo lite of IMM-125 resulting from the biotransformation of the amino acid I vinylic methyl group to a carboxylic acid, called the IMM-125-COOH metabolite, is described. It was found that the complex of this new me tabolite with cyclophilin A is formed less easily than the correspondi ng cyclophilin A-IMM-125-CH2OH main metabolite and cyclophilin A-IMM-1 25 complexes. However, when formed, the IMM-125-COOH metabolite-cyclop hilin A complex requires more collision-induced dissociation (CID) to dissociate the complex than the complexes formed with the two other li gands. The nanospray tandem mass spectrum of the IMM-125-COOH metaboli te-cyclophilin A complex (m/z 1755) gives rise to cyclophiIin A-ligand complexes of m/z 1751 by elimination of CO2 and of m/z 1749 by loss o f CO2 and H2O or glycerol. Since immunosuppressive activity is known t o be dependent on the formation of a binary complex between cyclophili n A and the drug and since the target for the binary complex was found to be the calcium- and calmodulin-dependent protein phosphatase, calc ineuria, it could be interesting to measure for structurally related i mmunosuppressive drugs the CID energy necessary to dissociate the bina ry complexes in order to evaluate whether a correlation with the phosp hatase activity could be derived. (C) 1998 John Wiley & Sons, Ltd.