Automation of a high-performance liquid chromatography-based enzyme assay: evaluation of inhibition constants for human immunodeficiency virus-1 protease inhibitors.
Enzyme-based assays are commonly employed in clinical and pharmaceutical laboratories to aid in quantitation of organic substances. Many enzyme assays are tedious, requiring the addition of reagents at multiple time intervals. The HPLC-based analysis of reaction products requires an additional step of vialing the samples and placing them in the autosampler. Such time-consuming, repetitive procedures are ideally suited for automation. We automated an HIV protease assay for the purposes of screening compounds as inhibitors of HIV protease and determining inhibition constants. Automation was accomplished by interfacing a robotic sample processor from a Gilson Model 232/401 biocompatible automatic sample processor and injector, with a Hewlett Packard HPLC. We used this configuration to automate the following steps: (a) preparation of serial dilutions of inhibitor, (b) enzyme assay setup, and (c) quantitation of products of enzyme assays. The resulting automated method produced inhibition constants that were of comparable accuracy and precision to those determined by manual methods.
Pazhanisamy
S
S
Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139, USA.
Stuver
C M
CM
Livingston
D J
DJ
D000595:Amino Acid Sequence; D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D016333:HIV Protease / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010436:Pepstatins / Q000494:pharmacology; D015203:Reproducibility of Results; D013379:Substrate Specificity
D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D016333:HIV Protease