P1/P1 ' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays

The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an as say based on surface plasmon resonance (SPR) was introduced. We have taken advantage of the fact that the SPR signal is proportional to the mass of th e analyte interacting with the immobilised molecule and developed two new i mproved efficient competition assay methods. Thus. high molecular weight bi nders were used as amplifiers of the surface plasmon resonance signal. Link ers were attached by a Heck reaction to the para-positions of the P1/P1' be nzyloxy groups of a linear C-2-symmetric C-terminal duplicated inhibitor to enable (a) biotin labelling or (b) direct immobilisation of the inhibitor to the biosensor surface matrix. The interaction properties of a series of 17 structurally diverse inhibitors was assessed and compared to previously reported data. The most sensitive assay was obtained by immobilising the en zyme and amplifying the signal with an antibody, giving a detection range b etween 0.1 nM and 10 muM Immobilisation of the inhibitor resulted in a stab le and durable surface but a narrower detection range (1-100 nM). The two c ompetition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors. (C) 2001 Elsevier Science B.V. All rights reserv ed.