Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-06-01
2003-03-18
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S005000, C435S006120, C435S069200, C536S023100, C536S023720
Reexamination Certificate
active
06534310
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to compositions and methods for inhibiting activity of multimeric enzymes. In particular, it relates to inhibition of proteases by formation of dysfunctional protease multimers.
Protein structure is typically discussed in terms of four levels. The primary structure is the amino acid sequence; the secondary structure is any regular local structure of linear segment, such as an &agr;-helix; the tertiary structure is the overall topology of the folded polypeptide chain; and the quaternary structure is the aggregation of single polypeptides or subunits to form a functional molecule.
Many proteins exist as assemblies of two or more polypeptide chains, which may be identical or different. Complex interactions between the subunits are required to produce a functional protein. For example, multimeric enzymes can be rendered inactive, if the interaction of the monomers is disrupted.
Multimeric enzymes of particular interest to the present invention are multimeric proteases. Inhibition of protease activity may be useful in a number of contexts. For example, inhibition of retroviral proteases, which are critical to retroviral maturation and infectivity, can be used to inhibit retroviral infection.
Retroviruses are those viruses which have a single stranded RNA genome, a lipid envelope, and encode an RNA-dependent deoxyribonucleic acid (DNA) polymerase, known as reverse transcriptase. During their life cycle, the RNA genome is reverse transcribed into a DNA copy which is integrated into the genome of the host cell. A number of retroviruses cause disease states in humans. These include the lentiviruses, human immunodeficiency viruses (HIV-1 and HIV-2), which cause acquired immune deficiency syndrome (AIDS), and the oncoviruses, human T-cell lymphotrophic viruses I and II which cause T cell leukemias.
Retroviruses, such as HIV-1, encode aspartic proteases that process polyprotein precursors into viral structural proteins and replicative enzymes that are essential for viral proliferation. Autoprocessing of the protease from the gag/pol polyprotein precursor results in the release of the protease and the generation of mature structural and enzymatic proteins derived from the gag and gag/pol polyproteins.
Studies of the crystal structure of HIV proteases (Navia, et al,
Nature
337:615-620 (1989); Wlodawer, et al.,
Science
245:616-621 (1989)) have confirmed the homodimeric nature of these enzymes. Assembly of the two HIV protease monomers results in a dimer of approximately 22KD and generates an active site at the interface of the subunits.
These X-ray crystallographic studies have also defined regions of interaction between the monomers. Each monomer contributes half of the active site, which includes two catalytic aspartic acids as well as threonine/serine and glycine residues which are conserved among all aspartyl proteases for their structural role in maintaining active site geometry. The two N-termini and two C-termini of the individual monomers form &bgr;-strands that interdigitate to create a four-stranded anti-parallel &bgr;-sheet. These interactions appear to be a major stabilizing force in the enzyme and contribute over 50% of the inter-subunit contacts and hydrogen bonds. Dimer formation generates not only the catalytic center, but also the extended substrate binding pocket.
Since viral proteolytic activity is essential for the generation of infectious virus particles in HIV and related retroviruses, therapeutic intervention for HIV-1 and HIV-2 has targeted the HIV protease. Small molecules have been developed as inhibitors and are currently undergoing clinical trials as antiviral agents. In clinical trials however, resistance to these inhibitors is being observed. Thus, new approaches for inhibiting retroviral proteases, in particular HIV-1 protease, are an important therapeutic goal in the treatment of retroviral infections. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods of inhibiting multimeric enzymes, in particular proteases, both in vivo and in vitro. The methods comprise contacting a first protease monomer with a second, defective protease monomer, such that the defective monomer and the first monomer form a dysfunctional multimeric protease. If the target protease is associated with a disease state, the step of contacting may be carried out by administering the defective monomer to a patient. For example, a gene encoding the defective monomer may be administered in a retroviral vector suitable for gene therapy.
In some embodiments, the defective monomer is a defective human immunodeficiency virus (HIV) protease monomer. For example, a defective HIV protease monomer may be one in which an active site aspartic acid is replaced by a second amino acid residue, such as arginine, lysine, asparagine and the like. Alternatively the defective HIV protease monomer may be a monomer fragment, e.g., residues 6-99 of the HIV protease monomer.
Thus, the invention also provides methods of inhibiting HIV replication in a mammalian cell. The method comprises introducing into the cell a recombinant construct comprising a promoter sequence operably linked to a polynucleotide encoding a defective HIV protease monomer. The target cell may be a human hematopoietic stem cell.
The invention further provides recombinant constructs comprising a promoter sequence operably linked to a polynucleotide encoding a defective protease monomer, such as a defective HIV protease monomer. The promoter sequence may be, for example, from an HIV LTR.
The constructs may be incorporated in retroviral vectors capable of infecting mammalian cells. The invention thus provides mammalian cells (e.g., human T cells) comprising a recombinant expression cassette including promoter sequences operably linked to a polynucleotide encoding a defective protease monomer.
Definitions
As used herein a “protease monomer” refers to a subunit of a multimeric protease that, when aggregated in the proper quaternary structure, results in the formation of an active protease.
As used herein the term “HIV protease monomer” refers to a monomer of approximately 99 amino acid residues, which results from autoprocessing of the HIV gag and gag/pol polyprotein precursors. The amino acid sequence and three-dimensional structure of an HIV protease monomer is described in Wlodawer et al.
Science
245:616-621 (1989).
A “defective protease monomer” is a mutated form of a protease monomer that is capable of forming a multimeric protease with one or more wild type monomers. The wild type monomers to which the defective monomer binds may be from the same or different proteases. Thus, the monomers of the invention can be used to inhibit the activity of a single protease or a family of related proteases. The multimeric protease so formed is dysfunctional, such that enzyme activity as determined in a standard assay for the particular protease is reduced by at least 50%, usually 75%, and preferably 90% or more, as compared to the wild-type multimeric enzyme.
A “defective HIV protease monomer” of the invention refers to an HIV protease monomer which, when present in an HIV-infected cell, forms a heterodimeric or homodimeric dysfunctional HIV protease and which substantially inhibits HIV protease activity in an infected cell. HIV protease activity can be determined using an in vitro assay that measures the rate of specific hydrolysis of the decapeptide ATLNFPISPW, which corresponds to the sequence of the natural HIV-1 gag-pol polyprotein sequence from amino acid 151-160 and represents the junction between the protease and the reverse transcriptase. Cleavage occurs at the F—P bond. A suitable protocol for carrying out this assay is described in Babe et al.
Biochem.
30:106-111 (1991). HIV protease activity is substantially inhibited if activity as measured in this assay is reduced by at least 50%, preferably 75%, and most preferably 95% or more.
As used herein a “polynucleotide encoding a defective protease monomer” is one which, w
Babe Lilia M.
Craik Charles S.
Rosé Jason
Park Hankyel T.
Regents of the University of California
Townsend and Townsend / and Crew LLP
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