Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2000-09-19
2004-09-14
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S069200, C435S193000
Reexamination Certificate
active
06790633
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the identification and characterization of an enzyme involved in expression of the cancer phenotype, as well as to the identification and selection of compounds for its inhibition. In particular aspects, the invention relates to farnesyl protein transferase enzymes which are involved in, among other things, the transfer of farnesyl groups to oncogenic ras protein.
2. Description of the Related Art
In recent years, some progress has been made in the elucidation of cellular events lending to the development or progression of various types of cancers. A great amount of research has centered on identifying genes which are altered or mutated in cancer relative to normal cells. In fact, genetic research has led to the identification of a variety of gene families in which mutations can lead to the development of a wide variety of tumors. The ras gene family is a family of closely related genes that frequently contain mutations involved in many human tumors, including tumors of virtually every tumor group (see, e.g., ref. 1 for a review). In fact, altered ras genes are the most frequently identified oncogenes in human tumors (2).
The ras gene family comprises three genes, H-ras, Kras and N-ras, which encode similar proteins with molecular weights of about 21,000 (2). These proteins, often termed. P21
ras
,comprise a family of GTP-binding and hydrolyzing proteins that regulate cell growth when bound to the inner surface of the plasma membrane (3,4). Overproduction of P21
ras
proteins or mutations that abolish their GTP-ase activity lead to uncontrolled cell division (5). However, the transforming activity of ras is dependent on the localization of the protein to membranes, a property thought to be conferred by the addition of farnesyl groups (3,6).
A precedent for the covalent isoprenylation of proteins had been established about a decade ago when peptide mating factors secreted by several fungi were shown to contain a farnesyl group attached in thioether linkage to the C-terminal cysteine (7-9). Subsequent studies with the mating a-factor from
Saccharomyces cerevisiae
and farnesylated proteins from animal cells have clarified the mechanism of farnesylation. In each of these proteins the farnesylated cysteine is initially the fourth residue from the C terminus (see refs. 3, 4 and 10). Immediately after translation, in a sequence of events whose order is not yet totally established, a farnesyl group is attached to this cysteine, the protein is cleaved on the C-terminal side of this residue, and the free COOH group of the cysteine is methylated (3, 10, 11, 12). All of these reactions are required for the secretion of active a-factor in Saccharomyces (4).
Most, if not all, of the known p21
ras
proteins contain the cysteine prerequisite, which is processed by farnesylation, proteolysis and COOH-methylation, just as with the yeast mating factor (3, 4, 10, 11, 12). The farnesylated p21
ras
binds loosely to the plasma membrane, from which most of it can be released with salt (3). After binding to the membrane, some P21
ras
proteins are further modified by the addition of palmitate in thioester linkage to cysteines near the farnesylated C-terminal cysteine (3). Palmitylation renders the protein even more hydrophobic and anchors it more tightly to the plasma membrane.
However, although it appears to be clear that farnesylation is a key event in ras-related cancer development, prior to now, the nature of this event has remained obscure. Nothing has been known previously, for example, of the nature of the enzyme or enzymes which may be involved in ras tumorigenesis or required by the tumor cell to achieve farnesylation. If the mechanisms that underlie farnesylation of cancer-related proteins such as P21
ras
could be elucidated, then procedures and perhaps even pharmacologic agents could be developed in an attempt to control or inhibit expression of the oncogenic phenotype in a wide variety of cancers. It goes without saying that such discoveries would be of pioneering proportions in cancer therapy.
SUMMARY OF THE INVENTION
The present invention addresses one or more shortcomings in the prior art through the identification and characterization of an enzyme, termed farnesyl:protein transferase, involved in the oncogenic process through the transfer of farnesyl groups to various proteins, including oncogenic ras proteins. Further, the present invention provides novel compounds, including proteins and peptides, that are capable of inhibiting the farnesyl:protein transferase enzyme.
It is therefore an object of the present invention to provide ready means for obtaining farnesyl transferase enzymes from tissues of choice using techniques which are proposed to be generally applicable to all such farnesyl protein transferases.
It is an additional object of the invention to provide means for obtaining these enzymes in a relatively purified form, allowing their use in predictive assays for identifying compounds having the ability to reduce the activity of or inhibit the farnesyl transferase activity, particularly in the context of p21
ras
proteins.
It is a still further object of the invention to identify classes of compounds which demonstrate farnesyl transferase inhibiting activity, along with a potential application of these compounds in the treatment of cancer, particularly ras -related cancers.
Farnesyl:Protein Transferase Enzyme
Accordingly, in certain embodiments, the present invention relates to compositions which include a purified farnesyl protein transferase enzyme, characterized as follows:
a) capable of catalyzing the transfer of farnesyl to a protein or peptide having a farnesyl acceptor moiety;
b) capable of binding to an affinity chromatography medium comprised of TKCVIM coupled to a suitable matrix;
c) exhibiting a molecular weight of between about 70,000 and about 100,000 upon gel filtration chromatography; and
d) having a farnesyl transferase activity that is capable of being inhibited by one of the following peptides:
i) TKCVIM;
ii) CVIM; or
iii) KKSKTKCVIM.
As used herein, the phrase “capable of catalyzing the transfer of farnesol to a protein or peptide having a farnesyl acceptor moiety,” is intended to refer to the functional attributes of farnesyl transferase enzymes of the present invention, which catalyze the transfer of farnesol, typically in the form of all-trans farnesol, from all-trans farnesyl pyrophosphate to proteins which have a sequence recognized by the enzyme for attachment of the farnesyl moieties. Thus, the term “farnesyl acceptor moiety” is intended to refer to any sequence, typically a short amino acid recognition sequence, which is recognized by the enzyme and to which a farnesyl group will be attached by such an enzyme.
Farnesyl acceptor moieties have been characterized by others in various proteins as a four amino acid sequence found at the carboxy terminus of target proteins. This four amino acid sequence has been characterized as -C-A-A-X, wherein “C” is a cysteine residue, “A ” refers to any aliphatic amino acid, and “X” refers to any amino acid. Of course, the term “aliphatic amino acid” is well-known in the art to mean any amino acid having an aliphatic side chain, such as, for example, leucine, isoleucine, alanine, methionine, valine, etc. While the most preferred aliphatic amino acids, for the purposes of the present invention include valine and isoleucine, it is believed that virtually any aliphatic amino acids in the designated position can be recognized within the farnesyl acceptor moiety. In addition, the enzyme has been shown to recognize a peptide containing a hydroxylated amino acid (serine) in place of an aliphatic amino acid (CSIM). Of course, principal examples of proteins or peptides having a farnesyl acceptor moiety, for the purposes of the present invention, will be the p21
ras
proteins, including p21
H-ras
p21
K-rasA,
p21
rasB
and p21
N-ra
. Thus, in light of the present disclosure, a wide variety of peptidyl sequences having a farnesyl acceptor moiety
Brown Michael S.
Goldstein Joseph L.
Reiss Yuval
Fulbright & Jaworski LLP
Gitomer Ralph
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