title: P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays.
abstract: The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an assay 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 the analyte interacting with the immobilised molecule and developed two new improved efficient competition assay methods. Thus, high molecular weight binders were used as amplifiers of the surface plasmon resonance signal. Linkers were attached by a Heck reaction to the para-positions of the P1/P1' benzyloxy groups of a linear C2-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 enzyme and amplifying the signal with an antibody, giving a detection range between 0.1 nM and 10 microM. Immobilisation of the inhibitor resulted in a stable and durable surface but a narrower detection range (1-100 nM). The two competition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors.
authors: Department of Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23, Uppsala, Sweden
authors: M
authors: M
authors: Alterman
authors: H
authors: H
authors: Sjöbom
authors: P
authors: P
authors: Säfsten
authors: P O
authors: PO
authors: Markgren
authors: U H
authors: UH
authors: Danielson
authors: M
authors: M
authors: Hämäläinen
authors: S
authors: S
authors: Löfås
authors: J
authors: J
authors: Hultén
authors: B
authors: B
authors: Classon
authors: B
authors: B
authors: Samuelsson
authors: A
authors: A
authors: Hallberg
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D016333:HIV Protease / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D008968:Molecular Conformation; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D017320:HIV Protease Inhibitors; D016333:HIV Protease