Identifying protein-ligand binding interactions is a key step during early-stage drug discovery. Existing screening techniques are often associated with drawbacks such as low throughput, high sample consumption, and dynamic range limitations. The increasing use of fragment-based drug discovery (FBDD) demands that these techniques also detect very weak interactions (mM K(D) values). This paper presents the development and validation of a fully automated screen by mass spectrometry, capable of detecting fragment binding into the millimolar K(D) range. Low sample consumption, high throughput, and wide dynamic range make this a highly attractive, orthogonal approach. The method was applied to screen 157 compounds in 6 h against the anti-apoptotic protein target Bcl-x(L). Mass spectrometry results were validated using STD-NMR, HSQC-NMR, and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for screening.

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Ball oder Röhre: Flexible und amorphe Nanoröhren bilden sich aus einem Palladiumsalz und einem mehrzähnigen Liganden mit tetraedrischer Struktur (rechts). Dagegen führen Liganden mit zweidimensionaler Geometrie unabhängig von der Zahl der Metallkoordinationsstellen zur Bildung von Kugeln und deren Aggregaten (links).

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1. The aim was to identify the major metabolites of saquinavir (SQV) from human hepatic microsomal incubations and the CYP isoform(s) responsible. 2. Ten fractions containing various metabolites were separated by isocratic reversed-phase HPLC and characterized by HPLC, mass spectrometry and NMR. 3. Metabolites were either mono- or di-hydroxylated derivatives of SQV. Fast-atom bombardment and electrospray MS showed that hydroxylation was predominantly situated on the decahydroisoquinoline ring. A major metabolite (M4) was rigorously identified as 6-equatorial-hydroxy SQV. 4. Metabolism of saquinavir to all metabolites was inhibited by the CYP3A4-selective inhibitor ketoconazole (IC50 = 0.55 +/- 0.12 microM). Other isoform-selective inhibitors were non-inhibitory. The protease inhibitors ritonavir, indinavir and nelfinavir potently inhibited SQV metabolism in hepatic microsomes with IC50 = 0.025 +/- 0.004, 0.82 +/- 0.26 and 0. 58 +/- 0.14 microM, respectively. 5. Saquinavir metabolism correlated with immunochemically determined CYP3A4 levels and testosterone 6beta-hydroxylation, but it failed to correlate with either immunochemically determined CYPIA2 levels or marker activities for CYP1A2, 2C9 or 2E1. 6. Heterologously expressed CYP3A4 metabolized saquinavir with a similar metabolic profile to that of human liver microsomes. 7. Km, and Vmax for total SQV metabolism were 0.61 +/- 0.19 microM and 1.82 +/- 1.13 nmol mg(-1) min(-1), respectively. 8. The extensive involvement of hepatic CYP3A4 in the metabolism of saquinavir predicts high intrinsic clearance of saquinavir. Inhibitors of CYP3A4 such as other protease inhibitors will substantially increase the bioavailability of saquinavir.

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