Fishing – trapping – and vermin destroying – Fishing – Nets
Reexamination Certificate
1998-03-06
2001-06-12
Ceperley, Mary E. (Department: 1641)
Fishing, trapping, and vermin destroying
Fishing
Nets
C435S006120, C435S007100, C435S007200, C435S007230, C435S007500, C435S007720, C435S007800, C435S007920, C435S023000, C435S024000, C435S184000, C435S219000, C436S172000, C436S800000, C436S805000, C436S537000
Reexamination Certificate
active
06243980
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to the use of chemiluminescent 1,2-dioxetanes in homogenous or heterogeneous assays to detect proteases inhibitors.
2. Discussion of the Background
The identification of novel therapeutics that block or inhibit inimical proteases, or proteases that mediate disease conditions, such as the 11-kd protease encoded by the human immunodeficiency virus 1(HIV-1) is a key step in slowing the disease process of AIDS. Retroviral proteases are essential in the process of viral gag-pol polyproteins of the HIV-1 and HIV-2 viruses. There are a few highly conserved consensus sequences in retroviral polyproteins, one of which consists of a pentapeptide (Ser/Thr)-X-X′-(Tyr/Phe)-Pro (SEQ ID No:1). Cleavage occurs between the Tyr or Phe and Pro residues. Blocking activity of these proteases will interfere with the progression of HIV infection. Although potent drugs which block HIV protease activity have been found, there is an ongoing need to find and develop novel inhibitors.
Current methods utilized in rapid screening of protease inhibitors are subject to many interferences from a variety of sources. The most common non-isotopic approach is a fluorescent assay. In one case, such as in the detection of HIV protease, the fluorescent substrate is labeled with a fluorescent dansyl group on one end of a peptide and a quencher on the other. An increase in fluorescence signal occurs upon cleavage of the protease due to the fact that the emitter and the quencher are separated as described in Matayoshi et al., 1990, Science 247: 954-958. Fluorescent substrates for other proteases can be designed with a terminal fluorophore which emits a fluorescent signal upon cleavage by the enzyme.
Both of the above assay approaches are commonly used as high throughput assays for screening large chemical, natural product and combinatorial libraries. These assays tend to have problems related to autofluorescence of biological components due to the nature of the molecules which are screened. Many of the compounds and natural product extracts are colored or fluorescent and are present in the solution when the assay signal is monitored. This results in an assay interference which limits the detection sensitivity and the dynamic range of the assay. This interference can easily be interpreted as an inhibition of the enzyme, making it difficult to determine true positive inhibition, thus, requiring extensive follow-up assays to distinguish true positives from the false positives.
U.S. Pat. No. 5,591,591, assigned to Tropix, describes assays for the detection of proteases wherein a dioxetane compound bearing a proteolytic enzyme-specific amino acid or peptide, is added to a sample suspected of containing the protease, and the amino acid is removed by enzymatic reaction by the protease, causing the dioxetane to decompose and chemiluminescence.
SUMMARY OF THE INVENTION
An alternative approach to the above homogeneous approaches is to use homogenous or heterogeneous assays which are not subject to interference. The present inventors have developed highly sensitive assays using a chemiluminescent 1,2-dioxetane substrate for high throughput screening of HIV-1 protease activity. The present invention provides the advantage of an assay that it is not subject to interferences from colored or fluorescent compounds, and therefore is more sensitive and exhibits a greater dynamic range compared to a direct, fluorescent enzyme assay. This assay, adapted for the detection of HIV protease inhibitors, utilizes a synthetic peptide substrate I (Fam-spacer-Ser-Gln-Asn-Tyr-Pro-lle-Val-Gln-spacer-(Biotin)-NH2) (SEQ ID No:2) whose sequence is derived from the native cleavage site of the Gag polyprotein. (Fam is used herein to indicate fluorescein). Many high throughput HIV screening methodologies exist which utilize large quantities of reagents and involve more laborious manipulations. The assay of the present invention may be advantageously formatted as a simple single plate endpoint assay which is sensitive down to fmoles of captured peptide which is particularly useful for high throughout screening, although it may also be presented as a conventional two-step transfer and dilation capture assay. The assay uses a chemiluminescent 1,2-dioxetane alkaline phosphatase substrate in an immunoassay format for the sensitive detection of cleaved peptide. The uncleaved peptide is recognized by an anti-fluorescein alkaline phosphatase conjugated antibody. Capture conditions have been optimized to assure a linear response to cleaved peptide concentration. This response correlates well to HPLC analysis of cleaved product. The HIV assay has been validated for acetyl-pepstatin, a known HIV-1 inhibitor. This robust assay is amenable to automation and can be used to screen large numbers of compounds in a cost effective 96 well format.
In an alternative embodiment, a homogenous assay less sensitive to color or fluorescence interference than prior art assays is used. In this assay, the same peptide is employed, but labeled at one terminus with fluorescein or other fluorescing energy acceptor, and at the other end by a 1,2-dioxetane label covalently attached to the peptide terminus. After admixture of the target compound, the dioxetane is caused to decompose by addition of a chemical (enzymatic or non-enzymatic) or another trigger, such as application of heat or change in pH. If the peptide has not been cleaved, the dioxetane is in close physical relationship with the fluorescent energy acceptor, and upon triggering, energy transfer assisted fluorescence is observed. The light has a characteristic color dependent on the fluorescent emitter, such as green for fluorescein. If the peptide has been cleaved, the chemiluminescent light of the dioxetane itself, typically a bluer light, is observed.
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Bronstein Irena
Palmer Michelle
Tillotson Bonnie
Voyta John
Ceperley Mary E.
Gabel Gailene R.
Kelber Steven B.
Piper Marbury Rudnick & Wolfe LLP
Tropix, Inc.
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