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Claims  |
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What is claimed is:
1. A method to identify a compound that inhibits proteolytic cleavage by a
dibasic amino acid processing endoprotease, said method comprising:
(a) contacting a yeast strain containing a precursor protein having a
dibasic amino acid processing site with a putative inhibitory compound
under conditions in which, in the absence of said compound, said yeast
strain is capable of effecting cleavage of said precursor protein into
cleavage products; and
(b) assaying for production of at least one of said cleavage products,
wherein production of a reduced amount of said at least one of said
cleavage products in the presence of said putative inhibitory compound
compared to in the absence of said putative inhibitory compound indicates
that said compound is able to inhibit proteolytic cleavage by said
endoprotease.
2. The method of claim 1, wherein said yeast strain is of a genus selected
from the group consisting of Saccharomyces, Pichia, Hansenula,
Schizosaccharomyces, Kluyveromyces, Yarrowia and Candida.
3. The method of claim 1, wherein said yeast strain is of the species
Saccharomyces cerevisiae.
4. The method of claim 1, wherein said cleavage of said precursor protein
in step (a) is accomplished by a yeast Kex2 endoprotease or functional
equivalent thereof, said functional equivalent being capable of cleaving a
yeast .alpha.-factor precursor protein in an .alpha.-factor zone clearing
assay.
5. The method of claim 1, wherein said cleavage of said precursor protein
in step (a) is accomplished by a dibasic amino acid processing
endoprotease heterologous to said yeast strain, said heterologous
endoprotease being produced by said yeast strain.
6. The method of claim 5, wherein said yeast strain is Kex2
endoprotease-deficient.
7. The method of claim 6, wherein said yeast strain is deficient in at
least one soluble vacuolar protease.
8. The method of claim 1, wherein said yeast strain comprises a Kex2
endoprotease-deficient Saccharomyces cerevisiae strain capable of
producing a heterologous dibasic amino acid processing endoprotease.
9. The method of claim 8, wherein said yeast strain comprises Saccharomyces
cerevisiae kex2.DELTA..
10. The method of claim 1, wherein said endoprotease comprises a cellular
endoprotease selected from the group consisting of animal endoproteases
and plant endoproteases.
11. The method of claim 1, wherein said endoprotease comprises a cellular
endoprotease selected from the group consisting of mammalian, avian, fish
and insect cellular endoproteases.
12. The method of claim 1, wherein said endoprotease is selected from the
group consisting of human, simian, feline, canine, bovine and rodent
cellular endoproteases.
13. The method of claim 1, wherein said endoprotease is derived from a cell
type that is capable of producing infectious virus upon infection by an
enveloped virus.
14. The method of claim 1, wherein said endoprotease is capable of
effecting cleavage of at least one envelope protein of an enveloped virus.
15. The method of claim 1, wherein said endoprotease is derived from a cell
type that is capable of producing infectious virus upon infection by a
human immunodeficiency virus.
16. The method of claim 1, wherein said endoprotease is capable of
effecting cleavage of human immunodeficiency virus gp160.
17. The method of claim 1, wherein said endoprotease comprises a human CD4+
T-lymphocyte dibasic amino acid processing endoprotease.
18. The method of claim 1, wherein said precursor protein comprises a yeast
precursor protein.
19. The method of claim 1, wherein said precursor protein is selected from
the group consisting of yeast .alpha.-factor precursor proteins and yeast
killer toxin precursor proteins.
20. The method of claim 1, wherein said precursor protein comprises a
precursor protein heterologous to said yeast strain, said yeast strain
being capable of producing said heterologous precursor protein.
21. The method of claim 1, wherein inhibition of said cleavage of said
precursor protein into at least one cleavage protein reduces the
infectivity of an infectious agent.
22. The method of claim 1, wherein said precursor protein comprises a
polyprotein.
23. The method of claim 1, wherein said precursor protein is selected from
the group consisting of viral, bacterial, fungal, plant and animal
precursor proteins.
24. The method of claim 1, wherein said precursor protein comprises at
least one precursor viral envelope protein.
25. The method of claim 1, wherein said precursor protein is a precursor
envelope protein of a virus selected from the group consisting of
retroviruses, herpes viruses, hepadnaviruses, pox viruses,
orthomyxoviruses, paramyxoviruses, rhabdoviruses, togaviruses, arena
viruses, bunyaviruses and coronaviruses.
26. The method of claim 1, wherein said precursor protein is selected from
the group consisting of a precursor retrovirus envelope protein, a
precursor hepatitis virus envelope protein, and a precursor herpes virus
envelope protein.
27. The method of claim 1, wherein said precursor protein comprises a
precursor lentivirus envelope protein or a precursor lymphotrophic virus
envelope protein.
28. The method of claim 1, wherein said precursor protein is selected from
the group consisting of HIV-1 gp160, HIV-2 gp160, HTLV-I gp69, HTLV-II
gp69, and functional equivalents thereof, said functional equivalents
being capable, upon cleavage by a dibasic amino acid processing
endoprotease, of functioning as viral envelope proteins.
29. The method of claim 1, wherein said precursor protein further comprises
at least one protein segment that enhances correct processing of said
precursor protein in the Golgi apparatus of said yeast strain.
30. The method of claim 29, wherein said segment enhances export of said
precursor protein through the secretory pathway of said yeast strain.
31. The method of claim 29, wherein said segment is selected from the group
consisting of leader sequences, dibasic amino acid processing sites, and
combinations thereof.
32. The method of claim 29, wherein said segment comprises a yeast
.alpha.-factor mating pheromone leader sequence joined to a yeast
.alpha.-factor dibasic amino acid processing site.
33. The method of claim 1, wherein said yeast strain comprises a Kex2
endoprotease-deficient Saccharomyces cerevisiae strain capable of
producing a precursor envelope protein selected from the group consisting
of HIV-1 gp160, HIV-2 gp160, HTLV-I gp69, HTLV-II gp69, and functional
equivalents thereof, said functional equivalents being capable, upon
cleavage by a dibasic amino acid processing endoprotease, of functioning
as viral envelope proteins, and capable of producing a human CD4+
T-lymphocyte dibasic amino acid processing endoprotease capable of
cleaving said precursor envelope protein.
34. The method of claim 1, wherein said step of assaying comprises testing
for dibasic amino acid processing endoprotease activity by an
.alpha.-factor zone clearing assay.
35. The method of claim 1, wherein said step of assaying comprises testing
for said cleavage of said precursor protein by measuring the amount of
cleavage protein produced from said precursor protein.
36. The method of claim 1, wherein said inhibitory compound is capable of
being endocytosed.
37. The method of claim 1, wherein said inhibitory compound is selected
from the group consisting of peptides, mimetopes, and mixtures thereof.
38. The method of claim 1, wherein said inhibitory compound is capable of
chemically inactivating said endoprotease.
39. A method to identify an inhibitory compound that reduces the
infectivity of an infectious agent, comprising:
(a) contacting a yeast strain containing a precursor protein having a
dibasic amino acid processing site with a putative inhibitory compound
under conditions in which, in the absence of the compound, said yeast
strain is capable of effecting cleavage of said precursor protein into
cleavage products; and
(b) assaying for production of said cleavage products, wherein production
of a reduced amount of said cleavage products in the presence of said
putative inhibitory compound compared to in the absence of said putative
inhibitory compound indicates that said compound is able to inhibit
proteolytic cleavage by said endoprotease, the ability of said compound to
inhibit said cleavage being indicative of the ability of said compound to
reduce the spread of said infectious agent in an organism infected by said
infectious agent.
40. The method of claim 39, wherein said yeast strain is capable of
producing a precursor protein of said infectious agent.
41. The method of claim 39, wherein said yeast strain is a Kex2
endoprotease-deficient yeast strain capable of producing a dibasic amino
acid processing endoprotease, said endoprotease being derived from a cell
type capable of being infected by said infectious agent.
42. The method of claim 39, wherein said infectious agent comprises an
enveloped virus.
43. A test kit to identify a compound capable of inhibiting a dibasic amino
acid processing endoprotease, said kit comprising a yeast strain capable
of producing a precursor protein selected from the group consisting of
yeast and heterologous precursor proteins, said yeast strain being capable
of effecting cleavage of said precursor protein into cleavage products,
and a means for determining the extent of cleavage by said yeast strain in
the presence of a putative inhibitory compound, said means for determining
comprising means for assaying for production of said cleavage products,
wherein production of a reduced amount of said cleavage products in the
presence of said putative inhibitory compound compared to in the absence
of said putative inhibitory compound indicates that said compound is able
to inhibit proteolytic cleavage by said endoprotease.
44. The kit of claim 43, wherein said yeast strain comprises a Kex2
endoprotease-deficient yeast strain capable of producing a heterologous
dibasic amino acid processing endoprotease.
45. The kit of claim 43, wherein inhibition of said cleavage of said
precursor protein into at least one cleavage protein reduces the
infectivity of an infectious agent.
46. The kit of claim 43, wherein said means for determining comprises an
.alpha.-factor zone clearing assay.
47. A method to identify a compound capable of inhibiting an animal or
plant dibasic amino acid processing endoprotease, said method comprising:
(a) contacting a putative inhibitory compound with a secreted soluble
dibasic amino acid processing endoprotease protein fragment in the
presence of a precursor protein having a dibasic amino acid processing
site under conditions in which, in the absence of said compound, said
protein fragment is capable of effecting cleavage of said precursor
protein into cleavage products; and
(b) assaying for production of said cleavage products, wherein production
of a reduced amount of said cleavage products in the presence of said
putative inhibitory compound compared to in the absence of said putative
inhibitory compound indicates that said compound is able to inhibit
proteolytic cleavage by said endoprotease.
48. The method of claim 47, wherein said protein fragment comprises a
secreted soluble yeast Kex2 protein fragment.
49. The method of claim 47, wherein said protein fragment comprises a
soluble secreted mammalian dibasic amino acid processing endoprotease
protein fragment. |
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Claims |
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Description |
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FIELD OF THE INVENTION
The present invention relates to a yeast-based assay to identify compounds
that inhibit dibasic amino acid processing endoproteases. Such compounds
can be used, for example, to treat infectious diseases in which dibasic
amino acid processing endoprotease cleavage is involved in disease
progression. In particular, the assay can be used to identify antiviral
drugs, including drugs that reduce the spread of HIV and that retard or
reverse the onset of the acquired immunodeficiency syndrome (AIDS). The
present invention is also related to a method to isolate dibasic amino
acid processing endoprotease genes.
BACKGROUND OF THE INVENTION
A number of enveloped viruses, including retroviruses, hepatitis viruses,
herpes viruses, orthomyxoviruses and paramyxoviruses, produce precursor
envelope glycoproteins that require cleavage by a cellular dibasic amino
acid processing endoprotease as one step in the process of envelope
glycoprotein maturation. As precursor envelope glycoproteins are being
synthesized, they are directed into the host cell secretory pathway for
transport to the cell surface. As the precursor proteins move through the
pathway, they are subjected to a variety of post-translational events
including glycosylation and proteolytic cleavage (see, for example, Stein
et al., pp. 2640-2649, 1990, J. Biol. Chem., vol. 265). The precursor
human immunodeficiency virus (HIV) envelope protein gp160, for example, is
co-translationally glycosylated and subsequently cleaved into gp120 and
gp41 by a cellular dibasic amino acid processing endoprotease that
apparently is localized in the Golgi apparatus. The gp120 and gp41
proteins are further glycosylated prior to reaching the infected cell
surface. Cleavage of the HIV gp160 protein has been shown to be necessary
for membrane fusion, syncytium formation and viral infectivity (see, for
example, McCune et al., pp. 55-67, 1988, Cell, vol. 53; Kowalski et al.,
pp. 1351-1355, 1987, Science, vol. 237). The inventor, however, is unaware
of antiviral drugs that have been designed to block cleavage of precursor
envelope proteins by cellular dibasic amino acid processing endoproteases.
Although the genes encoding human furin (also called PACE), murine furin,
murine PC1 (also called PC3), human PC2, human PACE4, and human PACE 4.1
dibasic amino acid processing endoproteases have been isolated (for
reviews, see Barr, pp. 1-3, 1991, Cell, vol. 66; Kiefer et al., pp.
757-769, 1991, DNA and Cell Biology, vol. 10), a number of cellular
dibasic amino acid processing endoproteases remain to be identified,
including the human CD4+ T-lymphocyte dibasic amino acid processing
endoprotease responsible for cleaving HIV gp160 into gp120 and gp41.
Nucleoside analogs are a common type of antiviral drug, particularly for
treating retroviral infections as the analogs can inhibit the ability of
the retroviral reverse transcriptase enzyme to make a DNA copy of the
incoming viral RNA. For example, HIV infections are being treated with AZT
(3'-azidothymidine), ddI (2'3'-dideoxyinosine), ddC
(2'3'-dideoxycytidine), and d4T (didehydrothymidine). Nucleoside analogs,
however, have short half-lives and can exhibit substantial side effects.
In addition, viruses resistant to the nucleoside analog being administered
often develop within a relatively short period of time.
Non-nucleoside inhibitors of HIV reverse transcriptase, such as TIBO
(tetrahydro-imidazo(4,5,1-jk)(1,4)-benzodiazepin-2(1H)-one), BI-RG-587
(11-cyclopropyl-7-methyl-dipyrido-(2,3-b:3'3'-f)1,4-diazepin-6H-5-one),
pyridones, and bis(heteroaryl)piperazines, are also being developed and
tested. Since these compounds are highly selective for the HIV reverse
transcriptase enzyme, they apparently cause less severe side effects than
do nucleoside analogs. Decreased sensitivity of HIV to these agents,
however, develops rapidly.
The HIV-encoded aspartyl protease that processes the gag and gag/pol
polyproteins to yield the mature structural proteins and enzymes required
for virion formation (p24, p17, p15, reverse transcriptase) has also been
targeted as an enzyme against which to design antiviral agents. HIV
protease inhibitors, at least theoretically, can inhibit HIV production by
chronically infected cells and, as such, have an advantage over reverse
transcriptase inhibitors that apparently can only block replication if
added to cells before HIV infection. Peptide-based substrate analogs are
being prepared and tested. One drawback of HIV protease inhibitors is the
development of HIV strains that are resistant to the inhibitor being
administered.
Other strategies for inhibiting HIV infection that are being pursued
include inhibition of other HIV-encoded proteins such as Tat, Rev, and
integrase; blocking entry of the virus into the cell by, for example,
soluble CD4 receptor molecules; targeted delivery of toxins to
HIV-infected cells; inhibition of viral functions using antisense
technology; and immune constitution protocols. Although several of these
technologies are at the early stages of development, clinical trials
conducted using some of these technologies have been disappointing. For a
recent review of present and future strategies to treat HIV infection, see
Johnston et al., pp. 1286-1293, 1993, Science, vol. 260.
Most assays used to test antiviral drugs are either in vitro or mammalian
cell culture assays, many relying on the use of infectious virus.
Mammalian cell culture assays are usually costly, complex, time-consuming,
and potentially dangerous if infectious virus is used. Recently, a
Drosophila cell-based assay was developed for screening inhibitors of the
HIV Rev protein. For a review of methods to identify HIV inhibitors, see
Johnston et al., 1993, Science, ibid.
Thus, there remains a need to identify antiviral drugs with improved
efficacy that have fewer side effects than known drugs and against which
an infected host is less likely to develop resistance. A preferred class
of inhibitors to identify are those that can be used to treat infectious
diseases, such as HIV infections, in which proliferation of the infectious
agent depends on dibasic amino acid processing endoprotease cleavage. In
order to identify such drugs in a rapid and straightforward manner, an
improved assay is required that is less complex, less expensive, less
time-consuming, and more selective than currently used methods. There is
also a need for a method to identify the cellular dibasic amino acid
processing endoproteases that effect cleavage of such infectious agents in
vivo, such as the human CD4+ T-lymphocyte dibasic amino acid processing
endoprotease that cleaves HIV gp160, in order to identify specific
inhibitors having greater selectivity and, hence, fewer side effects.
SUMMARY OF THE INVENTION
The inventor has discovered that yeast strains having a functional Kex2
endoprotease are also able to properly process precursor proteins of other
organisms (i.e., heterologous precursor proteins), such as mammalian
precursor proteins, that require cleavage by a dibasic amino acid
processing endoprotease in order to become mature proteins. Based on this
finding, the present invention involves the use of yeast strains to
identify compounds that inhibit a dibasic amino acid processing
endoprotease from cleaving a heterologous precursor protein into one or
more cleavage proteins. Such inhibitory compounds can reduce the
infectivity of an infectious agent by interfering with the production of
one or more cleavage proteins required in the production of an infectious
agent. For example, many if not all envelope (or enveloped) viruses
produce precursor envelope proteins that require cleavage by dibasic amino
acid processing endoproteases in order to propagate infectious virus. One
such precursor envelope protein is the HIV gp160. The present invention
includes the use of a yeast-based assay to identify drugs capable of
reducing the spread of HIV and, thus to retard or reverse the onset of
AIDS, because the drugs are capable of blocking the cleavage of gp160 into
gp120 and gp41 in T lymphocytes.
Furthermore, in light of the aforementioned discovery, yeast strains
lacking a functional Kex2 endoprotease can be used to identify genes
encoding other dibasic amino acid processing endoproteases that cleave
specific precursor proteins in vivo. One example of such an endoprotease
is the human CD4+ T-lymphocyte endoprotease(s) responsible for cleaving
the precursor HIV envelope protein gp160 into the mature gp120 and gp41
glycoproteins, a cleavage that is required to form infectious virus and to
promote fusion between HIV-infected and non-infected cells leading to
immunodeficiency.
One embodiment of the present invention is a method to identify a compound
that inhibits proteolytic cleavage by a dibasic amino acid processing
endoprotease that includes the steps of (a) contacting a yeast strain with
a putative inhibitory compound under conditions in which, in the absence
of the compound, the yeast strain is capable of cleaving a precursor
protein having a dibasic amino acid processing site and (b) determining if
the putative inhibitory compound inhibits cleavage of the precursor
protein. A number of yeast strains can be used, including Saccharomyces
cerevisiae.
Cleavage can be monitored in a yeast strain that produces an active Kex2
endoprotease, or functional equivalent thereof. Alternatively, cleavage
can be monitored in a Kex2 endoprotease-deficient yeast strain that can
express a heterologous dibasic amino acid processing endoprotease, such as
an animal or plant dibasic amino acid processing endoprotease (yeast Kex2
endoprotease-deficient yeast strains are viable as are Chinese hamster
ovary cells that lack a functional dibasic amino acid processing
endoprotease). Preferably the dibasic amino acid processing endoprotease
is capable of effecting cleavage of at least one envelope protein of an
enveloped virus. Precursor proteins can be either yeast precursor proteins
or precursor proteins that are heterologous to the yeast strain that
produces them. Preferred heterologous precursor proteins are proteins that
when cleaved promote the propagation and/or infectivity of an infectious
agent, such as the precursor envelope proteins of retroviruses and other
enveloped viruses. Heterologous precursor proteins can include protein
segments that enhance correct export and processing of the precursor
protein. Putative inhibitory compounds can include peptides, mimetopes,
and mixtures thereof. Cleavage inhibition can be detected using a variety
of techniques including, for example, the .alpha.-factor zone clearing, or
halo, assay.
Another embodiment of the present invention is a method to identify an
inhibitory compound that reduces the infectivity of an infectious agent
that includes the steps of (a) contacting a yeast strain with a putative
inhibitory compound under conditions in which, in the absence of the
compound, the yeast strain is capable of cleaving a precursor protein
having a dibasic amino acid processing site and (b) determining if the
putative inhibitory compound inhibits cleavage of the precursor protein.
The ability of the compound to inhibit cleavage is indicative of (i.e.,
positively correlates with) the ability of the compound to reduce the
spread of the infectious agent in an organism infected by the infectious
agent.
Yet another embodiment of the present invention is a method to identify a
compound capable of inhibiting an animal or plant dibasic amino acid
processing endoprotease that includes (a) contacting a putative inhibitory
compound with a secreted soluble dibasic amino acid processing
endoprotease protein fragment in the presence of a precursor protein and
(b) determining if the putative inhibitory compound is capable of
inhibiting cleavage of the precursor protein by the protein fragment. The
protein fragment can be a secreted soluble yeast Kex2 protein fragment or
a soluble secreted fragment of an animal or plant dibasic amino acid
processing endoprotease.
One embodiment of the present invention is a test kit to identify a
compound capable of inhibiting a dibasic amino acid processing
endoprotease that includes a yeast strain that is capable both of
producing a yeast or heterologous precursor protein and of cleaving the
precursor protein. The kit also includes a means for determining the
extent of cleavage by the yeast strain in the presence of a putative
inhibitory compound.
The present invention also includes yeast strains capable of producing a
heterologous precursor protein having a dibasic amino acid processing site
that are capable of correctly processing the precursor protein into at
least one cleavage protein. Such strains include Kex2
endoprotease-deficient yeast strains capable of producing a heterologous
dibasic amino acid processing endoprotease capable of cleaving the
precursor protein. Particularly preferred yeast strains are of the species
Saccharomyces cerevisiae.
Additional yeast strains of the present invention include Kex2
endoprotease-deficient yeast strains capable of producing a heterologous
precursor protein but that are essentially incapable of correctly
processing the precursor protein into at least one cleavage protein, such
as Saccharomyces cerevisiae kex2.DELTA. and progeny and mutants thereof,
that are Kex2 endoprotease-deficient.
Another embodiment of the present invention includes compounds that inhibit
dibasic amino acid processing endoproteases. Such compounds can be
identified according to the heretofore disclosed methods and/or by using
the heretofore disclosed test kits and/or yeast strains. Particularly
useful inhibitory compounds of the present invention are compounds that
are capable of inhibiting dibasic amino acid processing endoprotease
cleavage of a precursor protein into at least one cleavage protein by at
least about 50 percent when the compound is contacted with the
endoprotease at a compound concentration of less than or about 100
micromolar, such that treatment with the inhibitory compound reduces the
infectivity of an infectious agent, such as of a virus. Inhibitory
compounds of the present invention can include a component that targets
the compound to the desired cell type. Inhibitory compounds preferably
enter cells by endocytosis.
Yet another embodiment of the present invention is a method to identify a
gene encoding an animal or plant dibasic amino acid processing
endoprotease that includes (a) transforming a Kex2 endoprotease-deficient
yeast strain with a cDNA library prepared from RNA isolated from a cell
type capable of producing the dibasic amino acid processing endoprotease;
(b) isolating a transformed yeast strain capable of expressing a
functional dibasic amino acid processing endoprotease as determined by the
ability of the transformed yeast strain to form a clear zone, or halo, in
an .alpha.-factor zone-clearing assay; and (c) recovering a cDNA encoding
the dibasic amino acid processing endoprotease from the isolated
transformed yeast strain.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic representation of recombinant molecule p.alpha./env.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes a novel method to identify compounds that
inhibit dibasic amino acid processing endoproteases, yeast strains that
are useful in such a method, and assay kits based on such a method. The
present invention can also be used to isolate dibasic amino acid
processing endoprotease genes.
The present invention includes the use of yeast strain assay systems, and
particularly the use of the protein secretory apparatus of yeast, to
identify compounds that inhibit a variety of organisms' dibasic amino acid
processing endoproteases. Yeast strains possess a dibasic amino acid
processing endoprotease located in the Golgi apparatus called Kex2
endoprotease (see, for example, Julius et al. pp. 1075-1089, 1984, Cell,
vol. 37). Kex2 endoprotease is capable of processing (i.e., cleaving)
yeast precursor proteins having dibasic amino acid processing sites, such
as precursor proteins for .alpha.-factor mating pheromones and killer
toxins. Yeast strains lacking a functional Kex2 endoprotease can grow
normally; such strains, however, are unable to mate and show reduced
functions at low growth temperatures (i.e., at less than about 14.degree.
C.). Apparently all wild-type yeast strains, regardless of genus or
species, have a Kex2 endoprotease, or functional equivalent thereof, since
all wild-type yeast strains apparently are capable of mating. As used
herein, a functional equivalent of a Kex2 endoprotease is a yeast dibasic
amino acid processing endoprotease that has a similar proteolytic activity
to Kex2 and, as such, can produce, for example, mature .alpha.-factor
mating pheromones. As used herein, the phrases a "yeast strain lacking a
functional Kex2 endoprotease" and a "Kex2 endoprotease-deficient yeast
strain" each refer to a yeast strain in which the Kex2 endoprotease is
either absent or modified such that the enzyme has essentially no
proteolytic activity (i.e., less than about 10 percent, preferably less
than about 5 percent, and more preferably less than about 1 percent of
wild-type Kex2 endoprotease activity). As such, a Kex2
endoprotease-deficient strain is essentially unable to produce mature
.alpha.-factor mating pheromones unless the strain is supplemented with a
functional dibasic amino acid processing endoprotease, for example, by
transforming the strain with a gene encoding a functional dibasic amino
acid processing endoprotease.
One embodiment of the present invention is a method to identify a compound
that inhibits a dibasic amino acid processing endoprotease from cleaving a
precursor protein heterologous to a yeast precursor protein that includes
the steps of (a) contacting a yeast strain with a putative inhibitory
compound under conditions in which, in the absence of the compound, the
yeast strain is able to cleave either a yeast or heterologous precursor
protein having a dibasic amino acid processing site, and (b) determining
whether the putative inhibitory compound inhibits the ability of the yeast
strain to cleave such a yeast or heterologous precursor protein. In the
instance of a system based on cleavage of a yeast precursor protein, the
ability of the putative inhibitory compound to inhibit the cleavage of the
yeast precursor protein is indicative of (positively correlates with) the
ability of the putative inhibitory compound to inhibit the cleavage of a
heterologous precursor protein. Such a correlation is based on the finding
that yeast Kex2 endoproteases are capable of cleaving heterologous
precursor proteins. Since yeast Kex2 endoproteases can cleave heterologous
precursor proteins naturally cleaved by other dibasic amino acid
processing endoproteases, albeit possibly not with equivalent affinity or
specific activity, it has been found that compounds that inhibit Kex2
endoprotease can inhibit heterologous dibasic amino acid processing
endoproteases. The heterologous precursor protein is preferably a protein,
the cleavage of which is instrumental in the formation of an infectious
agent and, as such, inhibition of the cleavage reduces the infectivity of
such an agent.
According to the aforementioned method, cleavage of the yeast or
heterologous precursor protein can be accomplished by the yeast strain's
endogenous Kex2 endoprotease or functional equivalent thereof. An
advantage of using a yeast strain expressing its own Kex2 endoprotease is
the ability to easily screen a number of compounds for potential dibasic
amino acid processing endoprotease inhibitory activity. Alternatively, a
yeast strain lacking a functional yeast Kex2 endoprotease (i.e., a Kex2
endoprotease-deficient yeast strain), can be transformed with a gene
encoding a heterologous dibasic amino acid processing endoprotease in such
a manner that the yeast strain is able to produce (i.e., express) the
heterologous dibasic amino acid processing endoprotease. Preferably, the
heterologous dibasic amino acid processing endoprotease is the protease
that naturally cleaves the heterologous precursor protein. An advantage of
using a Kex2 endoprotease-deficient strain expressing a heterologous
dibasic amino acid processing endoprotease is that such a method
identifies compounds that interact with the heterologous dibasic amino
acid processing endoprotease with high affinity and specificity without
affecting cell viability. A preferred yeast strain to use to identify
compounds that inhibit HIV infection is a Kex2 endoprotease-deficient
Saccharomyces cerevisiae strain that expresses a human CD4+ T-lymphocyte
dibasic amino acid processing endoprotease responsible for cleaving an HIV
gp 160, such as HIV-1 gp160, HIV-2 gp160, or functional equivalents
thereof.
The term dibasic amino acid processing endoprotease refers to any
proteolytic enzyme that cleaves a precursor protein (also referred to as a
proprotein) at a dibasic amino acid processing site within the precursor
protein. Dibasic amino acid processing endoproteases are typically serine
proteases of the subtilisin family, such as those described by Barr, pp.
1-3, 1991, Cell, Vol. 66. Dibasic amino acid processing endoproteases of
the present invention can be of any species, including viral, bacterial,
fungal, plant, and animal dibasic amino acid processing endoproteases.
Preferred dibasic amino acid processing endoproteases are cellular dibasic
amino acid processing endoproteases that cleave precursor proteins into
cleavage proteins that enable the propagation of an infectious agent.
Cellular dibasic amino acid processing endoproteases are preferred over
enzyme targets inherent to the infectious agent (e.g., polymerases,
regulatory factors, surface antigens, or proteases encoded by the
infectious agent) because it is believed that over time, drug-resistant
infectious agents are likely to develop much more rapidly than are
drug-resistant cellular proteases. Cellular dibasic amino acid processing
endoproteases are also attractive targets for inhibitory drug therapy
because the cellular location of dibasic amino acid processing
endoproteases in the secretory pathway (often in or near the Golgi
apparatus) causes dibasic amino acid processing endoproteases to be
susceptible to compounds that are endocytosed by cells. As such,
inhibitory drug compounds can be of any substance capable of being
endocytosed including compounds that are at least partially, and
preferably essentially completely, soluble in an aqueous (hydrophilic)
solution. That is, inhibitory compounds of the present invention do not
need to be lipophilic as the compounds need not cross cell membranes if
"delivered" by endocytosis. Furthermore, inhibitors of cellular dibasic
amino acid processing endoproteases are less likely to cause severe side
effects since reductions in cellular dibasic amino acid processing
endoprotease activity apparently are not significantly harmful to the
cell, as demonstrated, for yeast Kex2 endoprotease-deficient strains (see,
for example, Julius et al., 1984, Cell, ibid.) and Chinese hamster ovary
cell mutants that apparently lack a functional dibasic amino acid
processing endoprotease as they are unable to cleave the precursor
envelope proteins of Sindbis virus or Newcastle disease virus (see, for
example, Moehring et al., pp. 2590-2594, 1993, J. Biol. Chem., vol. 268;
Inocencio et al., pp. 593-595, 1993, J. Virology, vol. 67).
Preferred cellular dibasic amino acid processing endoproteases include
animal and plant dibasic amino acid processing endoproteases, with
mammalian, avian, fish, and insect cellular dibasic amino acid processing
endoproteases being more preferred, the dibasic amino acid processing
endoproteases of humans, livestock and pets being even more preferred, and
human, simian, feline, canine, bovine and rodent cellular dibasic amino
acid processing endoproteases being even more preferred. Particularly
preferred dibasic amino acid processing endoproteases to target are human
dibasic amino acid processing endoproteases.
Preferred dibasic amino acid processing endoproteases include endoproteases
that naturally are found in (i.e., the cellular source of which is) cell
types that are capable of producing infectious viruses upon infection by
an enveloped virus or cell types that produce hormones. Examples of such
cell types include, but are not limited to, CD4+ T-lymphocytes (natural
source of the dibasic amino acid processing endoprotease that cleaves HIV
gp160; also the natural source of several lymphokines), macrophages, liver
cells (natural source of furin and of the dibasic amino acid processing
endoprotease that cleaves precursor hepatitis envelope; the liver is also
the source of a number of prohormones that are processed by dibasic amino
acid processing endoproteases), pancreatic cells (source of insulin),
kidney cells (source of renin), dendritic cells, pituitary cells (source
of PC1/PC3 and PC2) and neurons as well as other immune and/or brain
cells. More preferred dibasic amino acid processing endoproteases include
CD4+ T-lymphocyte dibasic amino acid processing endoproteases, furin, PC1
(same as PC3), PC2, PACE4, and PACE 4.1.
Dibasic amino acid processing endoproteases that are able to effect
cleavage of at least one precursor envelope protein of an enveloped virus
are particularly preferred, with dibasic amino acid processing
endoproteases being able to cleave an HIV gp160 being more preferred.
Dibasic amino acid processing endoproteases that are naturally found in
cell types that are capable of producing infectious virus upon infection
by a lentivirus are particularly preferred, and particularly CD4+
T-lymphocyte dibasic amino acid processing endoproteases capable of
cleaving an HIV gp160 or HTLV gp69 precursor protein.
The phrase dibasic amino acid processing site refers to a site on the
precursor protein that can be cleaved by a dibasic amino acid processing
endoprotease. Dibasic amino acid processing sites usually include at least
one pair of basic amino acid residues that are substantially adjacent to
each other. Suitable sites include, but are not limited to, Lys-Arg,
Arg-Arg, Lys-Lys, Pro-Arg, Lys/Arg-X-Lys/Arg, Lys/Arg-X-X-Lys/Arg, (i.e.,
Lys-Xaa-Xaa-Lys (SEQ ID NO: 1), Lys-Xaa-Xaa-Arg (SEQ ID NO: 2),
Arg-Xaa-Xaa-Lys (SEQ ID NO: 3), and Arg-Xaa-Xaa-Arg (SEQ ID NO: 4) ) where
"Lys" is lysine, "Arg" is arginine, "Pro" is proline and "X" is any amino
acid. A particularly preferred dibasic amino acid processing site is the
Arg-Glu-Lys-Arg (SEQ ID NO: 5) site found in HIV gp160 precursor proteins,
wherein "Glu" is glutamic acid.
The term precursor protein refers to a protein that undergoes
post-translational modification during maturation, a process that includes
at least one step of cleavage by a dibasic amino acid processing
endoprotease at a dibasic amino acid processing site within the precursor
protein to form at least one cleavage protein. The terms cleavage protein,
cleaved protein, cleavage product, and cleaved product each refer to a
protein that has been produced by proteolytic cleavage of a precursor
protein, the cleavage being required, but not necessarily sufficient, for
the protein to become mature and bioactive. It should be understood that
cleavage proteins of the present invention can undergo additional
post-translational maturation steps prior and/or subsequent to dibasic
amino acid processing endoprotease cleavage. A precursor protein of the
present invention can be a polyprotein such that the precursor protein
contains more than one cleavage protein which can be separated by cleavage
with a dibasic amino acid processing endoprotease. Both yeast and
heterologous precursor proteins can be useful in the present invention.
The term yeast precursor protein refers to a precursor protein of the same
species as the yeast strain used in the identification of inhibitory
compounds in accordance with the present invention. Yeast precursor
proteins are preferably produced endogenously (i.e., naturally) by the
yeast strain. Any yeast precursor protein having a dibasic amino acid
processing site, the cleavage of which can be detected, can be monitored
to determine whether the putative inhibitory compound can inhibit the
ability of a dibasic amino acid processing endoprotease to cleave a
heterologous precursor protein. Suitable yeast precursor proteins include,
but are not limited to precursor proteins of .alpha.-factor mating
pheromones and killer toxins. A preferred yeast precursor protein to
monitor is a precursor .alpha.-factor protein.
The phrases a precursor protein heterologous to a yeast precursor protein
and a heterologous precursor protein each refer to a precursor protein
that is naturally produced in a cell type other than the yeast strain used
in the identification of inhibitory compounds in accordance with the
present invention. The heterologous precursor protein can be, for example,
a precursor protein of an infectious agent or a labeled precursor protein
that can be used as a marker in the method to identify compounds that
inhibit dibasic amino acid processing endoproteases. A heterologous
precursor protein can be produced by a yeast strain of the present
invention by genetically engineering the yeast strain to produce the
protein, using recombinant techniques known to those skilled in the art to
insert the gene encoding the protein into the yeast strain in a manner
such that the yeast strain is capable of expressing (i.e., producing) the
precursor protein (see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Labs Press, 1989; Pichuantes et al.,
in Principles and Practice of Protein Engineering, Hanser, 1993, in press,
Cleland and Craik, eds.).
Heterologous precursor proteins can be from any species, including viral,
bacterial, fungal, plant, and animal, including human, precursor proteins,
with viral, bacterial, and parasite precursor proteins being preferred,
and viral precursor proteins being more preferred. Preferred precursor
proteins include precursor proteins, the cleavage products (i.e., cleavage
proteins) of which are important in, and often critical for, the
production of an infectious agent. As such, preferred heterologous
precursor proteins include precursor viral envelope proteins, such as the
precursor envelope proteins of enveloped viruses such as retroviruses,
herpes viruses, hepadnaviruses, pox viruses, orthomyxoviruses,
paramyxoviruses, rhabdoviruses, togaviruses, arena viruses, bunyaviruses
and coronaviruses, with precursor envelope proteins of retroviruses,
herpes viruses and hepatitis viruses being more preferred. Particularly
preferred precursor proteins are precursor envelope proteins of
T-lymphotrophic viruses, such as human T-cell lymphotrophic virus (HTLV),
bovine leukemia virus (BLV) and feline leukemia virus (FLV), with HTLV-I
gp69, HTLV-II gp69, and functional equivalents thereof being more
preferred lymphotrophic precursor proteins. Also particularly preferred
are the precursor proteins of lentiviruses, such as simian (SIV), feline
(FIV), canine (CIV), and human (HIV) immunodeficiency viruses, with HIV-1
gp160, HIV-2 gp160, and functional equivalents thereof, being particularly
preferred lentivirus precursor proteins.
One preferred class of heterologous precursor proteins is a precursor
protein that includes at least one protein segment that enhances correct
processing of the precursor protein in the yeast Golgi apparatus and/or
export (e.g., proper folding, other post-translational modifications and
migration) through the yeast secretory pathway. Without being bound by
theory, it is believed that a yeast protein segment, such as a natural
"pro" or "leader" sequence of a proprotein and/or a dibasic amino acid
processing site, joined to a heterologous precursor protein may improve
the likelihood of efficient maturation (e.g., export and processing) of
the precursor protein. It has been found that attachment of yeast leader
segments, such as .alpha.-factor, invertase and carboxypeptidase Y leader
segments, to otherwise mature heterologous proteins promotes proper
folding and migration of the proteins through the secretory pathway (see,
for example, Graham et al., pp. 209-218, 1991, J. Cell Biology, vol. 114).
Preferred yeast segments for use in the present invention include yeast
.alpha.-factor mating pheromone leader sequences, yeast .alpha.-factor
dibasic amino acid processing sites and yeast .alpha.-factor mating
pheromone leader sequences joined to yeast .alpha.-factor dibasic amino
acid processing sites. A particularly preferred precursor protein of the
present invention is an .alpha.-factor mating pheromone leader and
.alpha.-factor dibasic amino acid processing site joined to an HIV
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