Probing inhibitors binding to human urokinase crystals by Raman microscopy: Implications for compound screening

Inhibition of urokinase activity represents a promising target for antimeta static therapy for several types of tumor. The present study sets out to in vestigate the potential of Raman spectroscopy for defining the molecular de tails of inhibitor binding to this enzyme, with emphasis on single crystal studies. It is demonstrated that high quality Raman spectra from a series o f five inhibitors bound individually to the active site of human urokinase can be obtained in situ from urokinase single crystals in hanging drops by using a Raman microscope. After recording the spectrum of the free crystal, a solution of inhibitor containing an amidine functional group on a naphth alene ring was added, and the spectrum of the crystal-inhibitor complex was obtained. The resulting difference Raman spectrum contained only vibration al modes due to bound inhibitor, originating from the protonated group, i.e ., the amidinium moiety, as well as naphthalene ring modes and features fro m other functionalities that made up each inhibitor. The identification of the amidinium modes was placed on a quantitative basis by experimental and theoretical work on naphthamidine compounds. For the protonated group, -C-( NH2)(2)(+), the symmetric stretch occurs near 1520 cm(-1), and a less inten se antisymmetric mode appears in the Raman spectra near 1680 cm(-1). The pr esence of vibrational modes near 1520 cm-1 in each of the Raman difference spectra of the five complexes examined unambiguously identifies the protona ted form of the amidinium group in the active site. Several advantages were found for single crystal experiments over solution studies of inhibitor-en zyme complexes, and these are discussed. The use of single crystals permits competitive binding experiments that cannot be undertaken in solution in a ny kind of homogeneous assay format. The Raman difference spectrum for a si ngle crystal that had been exposed to equimolar amounts of all five inhibit ors in the hanging drop showed only the Raman signature of the compound wit h the lowest K-i. These findings suggest that the Raman approach may offer a route in the screening of compounds in drug design applications as well a s an adjunct to crystallographic analysis.