Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2002-07-25
2004-11-02
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007100, C435S007900, C536S023720
Reexamination Certificate
active
06811970
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a new assay to measure the process of HIV-1 reverse transcriptase (RT) dimerization. This invention particularly relates to a new method suitable for adaptation to high-throughput screening for inhibitors of this process.
BACKGROUND OF THE INVENTION
Reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) plays a key role in the replication of HIV by converting single-stranded genomic RNA into double-stranded proviral DNA and represents one of the main targets for the development of AIDS therapy. Most inhibitors of RT described in the past years, whether nucleoside analogues or non-nucleoside inhibitors target the polymerase activity of RT but present some limitations including toxicity and the emergence of resistant strains.
The biologically relevant and active form of HIV RT found in infectious virions is a heterodimer containing two polypeptides, p66 and p51; the latter derived from the former by proteolytic cleavage of its C-terminal domain during viral maturation. The two subunits of 66 and 51 kDa form are present in a 1 to 1 ratio. This heterodimeric RT is produced in a two-step dimerization process, the kinetics of which involve the rapid association of the two subunits into an immature dimer, followed by a slow conformational change yielding the fully active form (p66+p51→p66/p51 immature→p66/p51 active; Divita et al., 1995, J. Mol. Biol. 245, 508-52112, 13).
The DNA polymerase and RNase H activities of HIV-1 RT are dependent on the dimeric structure of the enzyme (Restle et al., 1990, J. Biol. Chem. 265, 8986-8988; Restle et al., 1992, FEBS Letters 300, 97-100). Because dimerization of these subunits is required for enzymatic activity, interference with the dimerization of HIV-1 RT could constitute an appropriate target for the development of anti-HIV compounds. A case in point: synthetic peptides from the thumb domain of the p51 subunit of RT inhibit the “maturation” process (Morris et al., 1999, Biochemistry 38, 15097-15103). Compounds that interfere with the formation and/or stability of the RT dimer may therefore represent a novel class of antiviral compounds.
Several publications disclose association-dissociation assays for measuring the kinetics of the p51-p66 dimerization process (Cabodevilla et al., 2001, Eur. J. Biochem. 2681163-172; Morris et al., 1999, J.Biol. Chem. 274(35), 2491-24946; Divita et al, 1995, Biochemistry 34, 16337-16346; Divita et al., 1994, J. Biol. Chem. 269(18), 13080-13083; Divita et al., 1993, FEBS Letters 324(2), 153-158; Becerra et al., 1991, Biochemistry 30, 11707-11719). They all proceed to measure the dimerization by either: size-exclusion HPLC; measuring the RNA-dependent DNA polymerase activity of the sample; immunoprecipitation; or by monitoring intrinsic fluorescence emission of the protein. None of these publications disclose a binding assay suitable for HTS format.
Tachedjian et al., 2000 (PNAS 97(12) 6334-6339) disclose a yeast 2-hybrid system to study the association-dissociation process of RT dimerization. However, this system is not easily amenable to a high throughput assay for assessing large numbers of potential inhibitors.
Howard et al., 1991, J. Biol. Chem. 266(34), 23003-23009 suggest an assay system to monitor therapeutic agents that act by preventing dimer formation. Again, this publication does not disclose a binding assay amenable for high throughput screening suitable for the identification of potential inhibitors of heterodimerization.
SUMMARY OF THE INVENTION
The present invention provides a high-throughput assay suitable for assessing large numbers of compounds for their activity against the dimerization of HIV RT. The general principle of the assay of the present invention involves dissociating a p66/p66 RT homodimer (appropriately labeled with a detectable moiety and/or affinity tagged) in the presence of limiting concentrations of a dissociation agent (i.e. a denaturant such as urea), contacting it with the p51 RT subunit and incubating the mixture in the presence of an excess denaturant-free (or denaturant reduced) buffer to allow re-association of the RT subunits, with subsequent affinity capture of any reconstituted p51/p66 RT heterodimer.
After washing to remove unbound material, the amount of affinity-associated material is assessed by measuring the level of a detectable moiety (or alternatively, by measuring the reconstituted RT polymerase activity in which case the p66 subunit does not necessarily require labeling), and is proportional to the amount of labeled p66/p51 RT heterodimer bound by affinity. This assay is performed in the presence or absence of a test compound whereby a modulation (decrease/increase) in binding of p66 monomer subunit to p51 subunit in the presence of the test compound compared to the control is indicative that the test compound is a modulator of RT dimerization.
In a first aspect of the present invention, there is provided a method for measuring heterodimerization of HIV RT, which comprises the steps of:
a) providing a first solution comprising p66 subunit homodimers in the presence of a dissociation agent;
b) contacting said first solution with p51 RT subunits and incubating in the presence of a reassociation buffer to allow association of a complex of p66/p51 RT subunits, wherein one of said subunits comprises an affinity tag and the other of said subunits comprises a detectable label;
c) contacting the incubate of step b) with an affinity medium under conditions that enable the p66/p51 complex to bind to said affinity medium; and
d) determining the amount of complex formed by measuring the level of detectable label bound to the affinity medium (or by measuring the reconstituted RT polymerase activity).
In a second aspect of the present invention, there is provided a method for identifying compounds capable of modulating the HIV RT heterodimerization, comprising:
carrying out steps a) to d) described above in the presence or absence of a test compound; and
e) comparing the test compound sample to a control sample lacking said compound, whereby modulated p66/p51 complex formation in the test compound sample is indicative of the ability of said compound to modulate heterodimerization.
In a third aspect of the present invention, there is provided a method for identifying compounds capable of interfering with the HIV RT heterodimerization, comprising:
carrying out steps a) to d) described above in the presence or absence of a test compound; and
e) comparing the test compound sample to a control sample lacking said compound, whereby decreased p66/p51 complex formation in the test compound sample is indicative of the ability of said compound to inhibit heterodimerization.
In a fourth aspect of the present invention, there is provided a method for identifying compounds capable of enhancing the HIV RT heterodimerization, comprising:
carrying out steps a) to d) described above in the presence or absence of a test compound; and
e) comparing the test compound sample to a control sample lacking said compound, whereby increased p66/p51 complex formation in the test compound sample is indicative of the ability of said compound to enhance heterodimerization.
According to a fifth aspect of the present invention, there is provided a method for measuring homodimerization of HIV RT, which comprises the steps of:
a) providing a first solution comprising first p66 subunit homodimers in the presence of a dissociation agent;
b) contacting said first solution with second p66 subunits homodimers, in the presence of said dissociation agent, and incubating in the presence of a reassociation buffer to allow association of a complex of p66/p66 RT subunits, wherein one of said subunits comprises an affinity tag and the other of said subunits comprises a detectable label;
c) contacting the incubate of step b) with an affinity medium under conditions that enable the p66/p66 complex to bind to said affinity medium; and
d) determining the amount of complex formed by measuring the level of detectable label bound t
Parniak Michael
Pelletier Alex
Sluis-Cremer Nicolas
Boehringer Ingelheim (Canada) Ltd.
Dow David A.
Morris Michael
Raymond Robert P.
Stucker Jeffrey
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