Label-free primary screening and affinity ranking of fragment libraries using parallel analysis of protein panels.

The authors present fragment screening data obtained using a label-free parallel analysis approach where the binding of fragment library compounds to 4 different target proteins can be screened simultaneously using surface plasmon resonance detection. They suggest this method as a first step in fragment screening to identify and select binders, reducing the demanding requirements on subsequent X-ray or nuclear magnetic resonance studies, and as a valuable "clean-up" tool to eliminate unwanted promiscuous binders from libraries. A small directed fragment library of known thrombin binders and a general 500-compound fragment library were used in this study. Thrombin, blocked thrombin, carbonic anhydrase, and glutathione-S-transferase were immobilized on the sensor chip surface, and the direct binding of the fragments was studied in real time. Only 12 microg of each protein is needed for screening of a 3000-compound fragment library. For screening, a binding site-blocked target as reference facilitates the identification of binding site-selective hits and the signals from other reference proteins for the elimination of false positives. The scope and limitations of this screening approach are discussed for both target-directed and general fragment libraries.

Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors.

HIV-1 protease is an important target for treatment of AIDS, and efficient drugs have been developed. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. In this study, nine C-terminally duplicated HIV-1 protease inhibitors were cocrystallised with the enzyme, the crystal structures analysed at 1.8-2.3 A resolution, and the inhibitory activity of the compounds characterized in order to evaluate the effects of the individual modifications. These compounds comprise two central hydroxy groups that mimic the geminal hydroxy groups of a cleavage-reaction intermediate. One of the hydroxy groups is located between the delta-oxygen atoms of the two catalytic aspartic acid residues, and the other in the gauche position relative to the first. The asymmetric binding of the two central inhibitory hydroxyls induced a small deviation from exact C2 symmetry in the whole enzyme-inhibitor complex. The study shows that the protease molecule could accommodate its structure to different sizes of the P2/P2' groups. The structural alterations were, however, relatively conservative and limited. The binding capacity of the S3/S3' sites was exploited by elongation of the compounds with groups in the P3/P3' positions or by extension of the P1/P1' groups. Furthermore, water molecules were shown to be important binding links between the protease and the inhibitors. This study produced a number of inhibitors with Ki values in the 100 picomolar range.