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Chapter 17. Ras Oncogene Directed Approaches in Cancer Chemotherapy
Chapter 17.
Ras Oncogene Directed Approaches in Cancer Chemotherapy Gary L. Bolton, Judith S. Sebolt-Leopold, and John C. Hodges Parke-Davis Pharmaceutical Research Division Warner-LambertCompany Ann Arbor, Michigan 48105
Jntroduction - Traditional approaches to cancer management have involved cytotoxic intervention at the level of DNA replication. While cytotoxic anticancer drugs have shown limited efficacy against rapidly growing tumor cells, there remains a critical need for the development of agents targeted against more refractory, slow growing, solid tumors. The 1990’s have witnessed a revolution in the understanding of signalling pathways that are important to the growth of normal and neoplastic cells. Whether a cell divides or stops dividing depends, to a great extent, on its ability to respond to membrane localized growth stimuli. Among the numerous oncogene or protooncogene encoded proteins that serve as signal transducers in the pathway from the outer membrane to the nucleus, perhaps none are as central as the Ras protein. Ras acts as a common relay point for signals from all of the growth factor receptors examined thus far (1, 2). Furthermore, single base mutations in the gene encoding the 21 kD Ras protein are found in approximately 30% of all human tumors, with the incidence as high as 50 and 90% in colon and pancreatic carcinomas, respectively (3). Comprehensive review of the function and regulation of Ras appears elsewhere (4). This chapter will be devoted to exploring the various strategies for blocking Ras function as well as reviewing progress that has been reported with anti-Ras agents. BlOCHFMlCAl FUNCTION OF RAS IN CELL SlGNAl LING Consistent with their fundamental role in cell proliferation, ras genes are members of a highly conserved and ubiquitous eukaryotic gene family. Mutated alleles of cellular ras genes were first discovered in rat sarcoma virus induced tumors (5, 6). The prevalence of activated or mutated ras alleles in human cancers is well documented (7, 8). All three known mammalian ras genes (H-, K-,and N- ras) encode nearly identical 21 kD proteins, commonly referred to as p21, p 2 l r S or Ras, which belong to a large superfamily of monomeric GTP-binding proteins including those from three other gene subfamilies, rho, rab and arf (9, 10). Collectively these proteins regulate a diverse array of cellular events ranging from cell proliferation and differentiation to cytoskeletal assembly and vesicular trafficking (1 1). A distinguishing biochemical feature of members of the Ras superfamily is their ability to bind guanine nucleotides with high affinity and their ability to hydrolyze bound GTP to GDP and phosphate. Ras serves as a molecular switch for cellular growth and differentiation by cycling between an active, GTP-bound form and an inactive, GDP-bound form. The proportion of active Ras appears to be tightly regulated and is dictated by the presence of GAP (GTPase activating protein) and neurofibromin (product of N F l ) , which ANNUAL REPORTS IN MEDICINAL CHEMISTRY-29
16s
Copyright D 1994 by Academic Press, lnc All rights Of reproductIan m m y form reserved
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serve to accelerate the intrinsic GTPase activity of normal Ras in a negative regulatory fashion. Upon stimulation of membrane receptors, growth factor induced activation of Ras is reflected in an observed increase in cellular Ras-GTP content (1 2,13). The intricacies of signal transduction can be seen in the nature of the protein-protein interactions which serve to transmit signals from growth factor receptors to Ras and subsequently from Ras to the nucleus. Protein interactions of Ras have been the subject of recent reviews (14-20). It is now believed that tyrosine kinases, e.g. EGF receptor, serve to phosphorylate an intermediary protein, the Shc protein, which binds to the SH2 domain of the Grb2 (growth factor receptor binding) protein (21). Subsequent complex formation between Shc and Grb2 allows for membrane recruitment of Sos, which stimulates guanine nucleotide dissociation (22, 23). In this way, Sos promotes GDP release from Ras, allowing it to bind GTP and thereby assume an active conformation. When activated, Ras allows further signal transduction in a process mediated by an effector protein which remains unidentified. While Ras-GAP appears to function in a negative regulatory manner, it does not appear to act alone as the effector complex for cell transformation (24). As a consequence of Ras-effector complex formation, a cascade of protein phosphorylation by cytoplasmic serinelthreonine kinases is set into motion. A complex involving Ras and the Raf-1 protein, which phosphorylates and activates mitogen activated protein kinase kinase (MAPKK or MEK), is the first step in this kinase cascade (25-28). The last kinase results in the phosphorylation of several cellular proteins including the transcription factors Myc and Jun. In this manner activated Ras ultimately results in altered gene expression. Ras does not possess a transmembrane domain; however its ability to play a pivotal role in mitogenic signalling is dependent upon localization at the inner surface of the plasma membrane. Post-translational modifications provide the lipophilicity required for membrane association. The cytoplasmic enzyme protein farnesyltransferase (PFT) recognizes the carboxyl terminus of unprocessed Ras which is characterized by a cysteine residue followed by two aliphatic amino acids and any one of several different amino acids, constituting the so-called CAAX motif. PFT catalyzes the reaction of farnesyl pyrophosphate (FPP) with the CAAX cysteine residue forming a thioether linkage (29-31). Genetically engineered Ras mutants lacking the CAAX sequence do not associate with the plasma membrane and also are not able to transform cells to malignancy (32, 33). Prior to membrane insertion of farnesylated Ras, the three terminal amino acids are trimmed by proteolytic processing (34-36). This is followed by methyl esterification at the new Cterminal cysteine residue in a step carried out by a protein methyltransferase (37, 38). Posttranslational processing through prenylation, proteolysis and esterification is described in greater detail in several recent reviews (39-42). Lipid association between prenyl groups and membrane lipids may not be the only way in which prenylated proteins bind to membranes. A second hypothesis is that protein prenylation also mediates protein-protein interactions, e.g. specific interactions between membrane receptors and prenylated proteins (43). The exact nature in which farnesylation promotes membrane localization is not well understood at this time. PFT, which is a heterodimer composed of 49 and 46 kD subunits, is ubiquitous among eukaryotic cells. Mammalian PFT is structurally and functionally similar to the yeast enzyme indicative of conservation through evolution (44). PFT shares a common prenyl pyrophosphate binding subunit (alpha subunit) with a second prenylation enzyme, protein geranygeranyltransferasetype I, which unlike PFT, recognizes CAAX motifs that terminate in leucine (45). The beta subunit of PFT is believed to provide CAAX box specificity (46).
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Cloning and expression of both subunits of human PFT have been reported (47, 48). A detailed kinetic analysis of this enzyme indicates a favored, but not obligate, pathway which consists of FPP binding prior to Ras binding, followed by catalysis of thioether formation as the predicted sequence in vivo (49). In addition to the C-terminal CAAX motif, Ras proteins may possess additional structural features promoting membrane association. For example, K-Ras4B contains a stretch of six lysine residues upstream of the CAAX motif. Protonation of this lysine rich region is thought to provide a positively charged region that would be attracted to the negatively charged phosphate surface of membrane bilayers (50). On the other hand, HRas, N-Ras and K-Ras4A all contain cysteine palmitoylation sites upstream of the C A M motif. Mutant proteins lacking these cysteine residues are no longer efficiently localized at the plasma membrane and have impaired ability to transform cells (51, 52). STRATEGIES FOR THERAPEUTIC INTERVENTION Potential strategies for inhibiting the function of Ras in tumors include antisense oligonucleotide approaches, inhibition of the post-translational processing enzymes, and blockade of the numerous Ras-protein interactions. Antisense Ras RNA oligonucleotides have been investigated for their ability to block the proliferative action of oncogenic Ras (53). Similarly, antisense and dominant inhibitory PFT expression plasmids have been shown to reduce colony formation in Ras-transformed cells (54). The feasibility of such antisense approaches remains to be proven through a demonstration of antitumor efficacy in animal models. Alternatively, agents that either disallow formation of Ras-GTP or restore GTPase activity to mutant Ras, e.g. by modulating Ras/GAP interaction, are also theoretical targets for therapeutic intervention. The feasibility of these approaches would depend upon a better understanding of GAP’S role and a more precise knowledge of key effector molecules. Furthermore Ras activity could also be antagonized by agents that interfere with the formation of a number of Ras-protein complexes, e.g. Ras-Sos, that have recently been elucidated. Molecules that inhibit the formation of such complexes have not been reported. Current research has been more highly focused on agents which interfere with the membrane localization of Ras (55, 56). Historically the first inhibitors of Ras membrane localization which exhibited antiproliferative effects were agents that block the biosynthetic pathway to FPP, namely, mevinolin and compactin (57). However, as inhibitors of HMG CoA reductase, these compounds also block steroid biosynthesis and hence are cytotoxic at concentrations which result in anti-Ras effects (58). The search for less toxic agents have more recently centered on inhibitors of the enzymes that catalyze the post-translational modification of Ras. Of these enzymes, PFT has been the most widely studied in terms of mechanism and inhibitors. Potential shortcomings of PFT inhibitors as anti-Ras agents include their degree of selectivity for 1) PFT relative to other prenylation enzymes, 2) Ras relative to other farnesylated substrates, and 3)oncogenic Ras relative to normal Ras. The potential for similar specificity shortcomings would also be expected for inhibitors of the protease, methyltransferase and palmitoyltransferase enzymes that complete Ras processing. Present evidence indicates that geranylgeranyl modified proteins terminate in either CAAL, CC, CXC, or CCXX motifs and as such are processed by distinct protein geranylgeranyltransferases (PGT) (59-61). Thus by designing an agent specific for the
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peptide binding subunit of PFT, it should be possible to circumvent PFT/PGT specificity concerns. In addition to Ras, other proteins are substrates of PFT and would presumably be affected by a PFT inhibitor. Namely, lamin 6,which is required for nuclear envelope integrity, and transducin, which is important in retinal signal transduction, are both substrates for PFT. Inhibition of their farnesylation is thus a potential toxicity concern. Selectivity for oncogenic Ras relative to normal Ras would be a desirable property of a PFT inhibitor since Ras is expressed in virtually all tissues and plays a critical role in normal cellular proliferation. A precedent now exists for farnesylation inhibitors that appear to preferentially inhibit oncogenic Ras function (62, 63). The mechanism of this selectivity remains largely an enigma. Evidence suggesting a reduced affinity of Raf-1 for oncogenic Ras relative to normal Ras may provide a clue (64).
5$PROTFIN F
NHI IT
-
CAAX Analogs Initial investigations following the isolation and purification of PFT demonstrated that the full Ras protein was not required for enzyme activity (65). The enzyme farnesylates a number of CA,A2X tetrapeptides, including CVlM (66-71). The preferred C-terminal (X) residues are serine, methionine, glutamine, alanine, or cysteine, which result in PFT binding. In general, A, and A, can be any aliphatic amino acid. However, substitution at the A, position has a critical effect on the ability of the tetrapeptide to function as a substrate. The tetrapeptide CVFM (I), containing an aromatic residue at A, is a potent inhibitor (IC, = 25 nM) of PFT, and does not undergo farnesylation (67). A further requirement for this behavior of CVFM is a free N-terminus, in that acylation restores the ability for farnesylation (68). The liabilities of these tetrapeptides as potential drug candidates, which include their inefficient cellular uptake and rapid proteolytic degradation, have resulted in little further development. However, these compounds have served as a foundation for the design and synthesis of several classes of peptidomimetic derivatives of the C A M sequence. A number of potent inhibitors of PFT of this type have been reported recent I y .
The pseudo-tetrapeptideL-731,735 (2) was designed as a CllM analog in which the two N-terminal amide bonds were reduced and methionine was replaced with homoserine (62). These modifications resulted in a potent in vitro (IC5,=18 nM) inhibitor of PFT. The was also an effective inhibitor (IC,,= 282 corresponding lactone derivative L-731,734 (3) nM), and selectively inhibited both Ras processing in a transformed cell line and growth in soft agar. Systematic modification of 1 has also led to the identification of 8581 (4), which showed a twofold increase in activity vs 1 in virro (72). This analog was found to selectively inhibit the processing of farnesylated proteins rather geranylgeranylated proteins in a Rastransformed cell line, but failed to discriminate between farnesyiation of H-Ras and lamin A. Microinjection of 4 into frog oocytes also inhibited Ras-dependent maturation.
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2
Others have also prepared a number of analogs of 1 to systematically examine the effect of backbone modification on activity and to further define substrate-inhibitor patterns (73). A number of modifications, including N-methylation and amide bond replacement, were investigated but these changes gave little insight to the substratehhibitor backbone conformation that is preferred by PFT. Replacement of the two aliphatic residues in the CAAX motif with a benzodiazepine derived mimic of a dipeptide turn led to an extremely potent inhibitor BZA-26 (6)with an ICm of 0.85 nM (63). This compound was designed to allow both the N-terminal cysteine and the C-terminal methionine to coordinate the Zn*+ ion that is present at the peptide binding site (74, 75). Recent proton NMR studies with an enzyme-bound heptapeptide substrate (KTKCVFM) have shown that the C-terminal tetrapeptide portion is directly involved in binding to the enzyme via an induced type I p-turn conformation (76). Although less potent in vifro, the corresponding methyl ester, BZAdB (S), restored a normal growth pattern to Ras-transformed cells. Incorporation of a 3-aminomethylbenzoic acid (AMBA) moiety as a replacement for the two internal aliphatic residues led to Cys-AMBA-Met (Z), an analog containing no peptidic linkages (77). This compound was a more potent inhibitor of human PFT than CVIM, but no cellular activity was reported.
Farnesvl PvroDhosDhate Analoas - Synthetic analogs of farnesyl pyrophosphate (FPP) have received less attention as potential inhibitors of PFT, perhaps because of specificity concerns due to its involvement in other biological pathways. However, two hydrolytically stable analogs of FPP have shown potent inhibition of PFT (78). Analogs and 9 were found to be competitive inhibitors of FPP, with Ki values of 5 nM and 830 nM, respectively. g was
a
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shown previously to be a potent inhibitor of squalene synthetase (79). A slight inhibition of Ras processing in a Ras-transformed cell line was noted with at 1 uM (80). This effect, while modest, was the first demonstration that the cellular processing of Ras could be moderated by a synthetic PFT inhibitor. However, it could not be determined if higher concentrations of would lead to further inhibition of Ras processing, due to the observation of cellular toxicity. Farnesylamine and other related long chain aliphatic amines have also been shown to inhibit the farnesylation and growth of Ras-transformed cells at high micromolar concentrations (81).
Natural Products - The use of PFT in high volume screening has led to the identification of a variety of natural products of microbial origin which inhibit PFT (82). 10'-Desmethoxystreptonigrin (lQ), an analog of streptonigrin (11) was found to be a weak inhibitor (IC,= 21 uM) of the enzyme, and also was cytotoxic to several cell lines (83). Interestingly, streptonigrin itself was approximately threefold less potent in vim.
Other antibiotics which show activity are derivatives of the manumycin family (84). UCF1 -C also known as manumycin, was the most potent inhibitor of this class (ICm= 5 uM). Kinetic analysis demonstrated that was a competitive inhibitor (Ki = 1.2 uM) with respect to FPP, and noncompetitive with respect to protein substrate. The reduced (dithiol) form of the fungal epipolydithiodiketopiperazine toxin, gliotoxin (U),was also a modest (lCs0= 1.1 uM) inhibitor of PFT (85). This compound was found to be a noncompetitive inhibitor with respect to the Ras protein.
(u),
Two novel dicarboxylic acid derivatives, chaetomellic acid A
(14)and chaetomellic
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acid B (X), were isolated from a fermentation extract and found to be potent inhibitors of PFT, with C I , values of 55 nM and 185 nM, respectively (80, 86, 87). These compounds were shown to be effective mimics of FPP by kinetic analysis and by computer modeling of their overlapping steric and electrostatic regions.
Zaragozic acid A (U),previously disclosed as a potent inhibitor of squalene synthetase, has also been identified as a PFT inhibitor (IC, = 216 nM) (80). A synthetic analog (U) showed increased PFT inhibition (IC, = 12 nM) and selectivity versus PGT. These compounds were competitive inhibitors with respect to FPP, and noncompetitive inhibitors with respect to the Ras protein. They had no effect on Ras processing in a Ras transformed cell line, presumably due to poor cell permeability,
0
The pepticinnamins, a novel series of six related pentapeptides, were also isolated by the screening of various fermentation broths (88). Pepticinnamin E (M), the major component of the mixture and only member whose structure was elucidated, was found to be a good inhibitor (IC, = 300 nM) of PFT (89). Limonene and related metabolites perillic acid and dihydroperillic acid, have also been demonstrated to inhibit protein isoprenylation and cell growth (90, 91). The antibiotic patulin (19) inhibited protein prenylation in a mouse cell line, and exhibited weak (I&= 290 uM) inhibition of PFT (92).
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PROTFASF AND MFTHYl TRANSFFRASF INHIBITORS Protease lnhib ' itors - Compared to the rapidly growing number of recent publications surrounding PFT and its inhibitors, the literature available on the subsequent C-terminal Ras processing enzymes is small. Two microsomal enzymes that cleave AAX from a farnesylCAAX motif have been reported. The first is a bovine liver endoprotease which cleaves the C-terminal tripeptide from farnesylated proteins (35). The second is a rat brain carboxypeptidase which sequentially removes X, A2 and A, (36). Both enzymes show high affinity for a farnesyl-CAAX motif compared to its CAAX precursor. Farnesyl-CAAX analogs with isosteric replacements for the scissile peptide bond have been shown to be inhibitors of isoprenylated protein endoprotease (93). The most potent of these are 2p (Ki = 86 nM) and fi (Ki = 64 nM). In principle, these molecules could also be inhibitors of the carboxypeptidase although no data are currently available.
BocHN
-
a X=CH2 21 X = CH(OH)CH2CO
SMe
Methvltransferase Inhibitors - Microsomal enzyme preparations that catalyze the methyl esterification of farnesylcysteine residues located at the C-terminus of peptides have also been described (94-96). To date a number of weak inhibitors (Ki =. 25 uM) have been reported including S-farnesyl mercaptopropionic acids such as MFPT and FPA (B) (96).
(a)
SUMMARY AND FUTURE DIRFCTlONS Because of the central signalling role of Ras in cell division and because of the frequency of mutant ras genes in human tumors, inhibition of Ras is likely to continue to be a popular target for cancer chemotherapy research. To date, the most promising strategy is to block Ras function by inhibiting its membrane localization, more specifically by preventing C-terminal processing. The bulk of current research is focused on PFT inhibitors. There are numerous CAAX analogs, FPP analogs and natural products which are inhibitors of PFT in vitro that provide excellent lead structures from which future drug candidates may be derived. Although several structural types have been shown to inhibit Ras processing and Ras dependant transformations in cell culture assays, the major hurdle of demonstrating the antineoplastic efficacy of a PFT inhibitor in vivo remains to be cleared.
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Ras Oncogene Directed Approaches in Cancer Chemotherapy Gary L. Bolton, Judith S. Sebolt-Leopold, and John C. Hodges Parke-Davis Pharmaceutical Research Division Warner-LambertCompany Ann Arbor, Michigan 48105
Jntroduction - Traditional approaches to cancer management have involved cytotoxic intervention at the level of DNA replication. While cytotoxic anticancer drugs have shown limited efficacy against rapidly growing tumor cells, there remains a critical need for the development of agents targeted against more refractory, slow growing, solid tumors. The 1990’s have witnessed a revolution in the understanding of signalling pathways that are important to the growth of normal and neoplastic cells. Whether a cell divides or stops dividing depends, to a great extent, on its ability to respond to membrane localized growth stimuli. Among the numerous oncogene or protooncogene encoded proteins that serve as signal transducers in the pathway from the outer membrane to the nucleus, perhaps none are as central as the Ras protein. Ras acts as a common relay point for signals from all of the growth factor receptors examined thus far (1, 2). Furthermore, single base mutations in the gene encoding the 21 kD Ras protein are found in approximately 30% of all human tumors, with the incidence as high as 50 and 90% in colon and pancreatic carcinomas, respectively (3). Comprehensive review of the function and regulation of Ras appears elsewhere (4). This chapter will be devoted to exploring the various strategies for blocking Ras function as well as reviewing progress that has been reported with anti-Ras agents. BlOCHFMlCAl FUNCTION OF RAS IN CELL SlGNAl LING Consistent with their fundamental role in cell proliferation, ras genes are members of a highly conserved and ubiquitous eukaryotic gene family. Mutated alleles of cellular ras genes were first discovered in rat sarcoma virus induced tumors (5, 6). The prevalence of activated or mutated ras alleles in human cancers is well documented (7, 8). All three known mammalian ras genes (H-, K-,and N- ras) encode nearly identical 21 kD proteins, commonly referred to as p21, p 2 l r S or Ras, which belong to a large superfamily of monomeric GTP-binding proteins including those from three other gene subfamilies, rho, rab and arf (9, 10). Collectively these proteins regulate a diverse array of cellular events ranging from cell proliferation and differentiation to cytoskeletal assembly and vesicular trafficking (1 1). A distinguishing biochemical feature of members of the Ras superfamily is their ability to bind guanine nucleotides with high affinity and their ability to hydrolyze bound GTP to GDP and phosphate. Ras serves as a molecular switch for cellular growth and differentiation by cycling between an active, GTP-bound form and an inactive, GDP-bound form. The proportion of active Ras appears to be tightly regulated and is dictated by the presence of GAP (GTPase activating protein) and neurofibromin (product of N F l ) , which ANNUAL REPORTS IN MEDICINAL CHEMISTRY-29
16s
Copyright D 1994 by Academic Press, lnc All rights Of reproductIan m m y form reserved
Be
Section In- Chemotherapeutic Agents
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serve to accelerate the intrinsic GTPase activity of normal Ras in a negative regulatory fashion. Upon stimulation of membrane receptors, growth factor induced activation of Ras is reflected in an observed increase in cellular Ras-GTP content (1 2,13). The intricacies of signal transduction can be seen in the nature of the protein-protein interactions which serve to transmit signals from growth factor receptors to Ras and subsequently from Ras to the nucleus. Protein interactions of Ras have been the subject of recent reviews (14-20). It is now believed that tyrosine kinases, e.g. EGF receptor, serve to phosphorylate an intermediary protein, the Shc protein, which binds to the SH2 domain of the Grb2 (growth factor receptor binding) protein (21). Subsequent complex formation between Shc and Grb2 allows for membrane recruitment of Sos, which stimulates guanine nucleotide dissociation (22, 23). In this way, Sos promotes GDP release from Ras, allowing it to bind GTP and thereby assume an active conformation. When activated, Ras allows further signal transduction in a process mediated by an effector protein which remains unidentified. While Ras-GAP appears to function in a negative regulatory manner, it does not appear to act alone as the effector complex for cell transformation (24). As a consequence of Ras-effector complex formation, a cascade of protein phosphorylation by cytoplasmic serinelthreonine kinases is set into motion. A complex involving Ras and the Raf-1 protein, which phosphorylates and activates mitogen activated protein kinase kinase (MAPKK or MEK), is the first step in this kinase cascade (25-28). The last kinase results in the phosphorylation of several cellular proteins including the transcription factors Myc and Jun. In this manner activated Ras ultimately results in altered gene expression. Ras does not possess a transmembrane domain; however its ability to play a pivotal role in mitogenic signalling is dependent upon localization at the inner surface of the plasma membrane. Post-translational modifications provide the lipophilicity required for membrane association. The cytoplasmic enzyme protein farnesyltransferase (PFT) recognizes the carboxyl terminus of unprocessed Ras which is characterized by a cysteine residue followed by two aliphatic amino acids and any one of several different amino acids, constituting the so-called CAAX motif. PFT catalyzes the reaction of farnesyl pyrophosphate (FPP) with the CAAX cysteine residue forming a thioether linkage (29-31). Genetically engineered Ras mutants lacking the CAAX sequence do not associate with the plasma membrane and also are not able to transform cells to malignancy (32, 33). Prior to membrane insertion of farnesylated Ras, the three terminal amino acids are trimmed by proteolytic processing (34-36). This is followed by methyl esterification at the new Cterminal cysteine residue in a step carried out by a protein methyltransferase (37, 38). Posttranslational processing through prenylation, proteolysis and esterification is described in greater detail in several recent reviews (39-42). Lipid association between prenyl groups and membrane lipids may not be the only way in which prenylated proteins bind to membranes. A second hypothesis is that protein prenylation also mediates protein-protein interactions, e.g. specific interactions between membrane receptors and prenylated proteins (43). The exact nature in which farnesylation promotes membrane localization is not well understood at this time. PFT, which is a heterodimer composed of 49 and 46 kD subunits, is ubiquitous among eukaryotic cells. Mammalian PFT is structurally and functionally similar to the yeast enzyme indicative of conservation through evolution (44). PFT shares a common prenyl pyrophosphate binding subunit (alpha subunit) with a second prenylation enzyme, protein geranygeranyltransferasetype I, which unlike PFT, recognizes CAAX motifs that terminate in leucine (45). The beta subunit of PFT is believed to provide CAAX box specificity (46).
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Cloning and expression of both subunits of human PFT have been reported (47, 48). A detailed kinetic analysis of this enzyme indicates a favored, but not obligate, pathway which consists of FPP binding prior to Ras binding, followed by catalysis of thioether formation as the predicted sequence in vivo (49). In addition to the C-terminal CAAX motif, Ras proteins may possess additional structural features promoting membrane association. For example, K-Ras4B contains a stretch of six lysine residues upstream of the CAAX motif. Protonation of this lysine rich region is thought to provide a positively charged region that would be attracted to the negatively charged phosphate surface of membrane bilayers (50). On the other hand, HRas, N-Ras and K-Ras4A all contain cysteine palmitoylation sites upstream of the C A M motif. Mutant proteins lacking these cysteine residues are no longer efficiently localized at the plasma membrane and have impaired ability to transform cells (51, 52). STRATEGIES FOR THERAPEUTIC INTERVENTION Potential strategies for inhibiting the function of Ras in tumors include antisense oligonucleotide approaches, inhibition of the post-translational processing enzymes, and blockade of the numerous Ras-protein interactions. Antisense Ras RNA oligonucleotides have been investigated for their ability to block the proliferative action of oncogenic Ras (53). Similarly, antisense and dominant inhibitory PFT expression plasmids have been shown to reduce colony formation in Ras-transformed cells (54). The feasibility of such antisense approaches remains to be proven through a demonstration of antitumor efficacy in animal models. Alternatively, agents that either disallow formation of Ras-GTP or restore GTPase activity to mutant Ras, e.g. by modulating Ras/GAP interaction, are also theoretical targets for therapeutic intervention. The feasibility of these approaches would depend upon a better understanding of GAP’S role and a more precise knowledge of key effector molecules. Furthermore Ras activity could also be antagonized by agents that interfere with the formation of a number of Ras-protein complexes, e.g. Ras-Sos, that have recently been elucidated. Molecules that inhibit the formation of such complexes have not been reported. Current research has been more highly focused on agents which interfere with the membrane localization of Ras (55, 56). Historically the first inhibitors of Ras membrane localization which exhibited antiproliferative effects were agents that block the biosynthetic pathway to FPP, namely, mevinolin and compactin (57). However, as inhibitors of HMG CoA reductase, these compounds also block steroid biosynthesis and hence are cytotoxic at concentrations which result in anti-Ras effects (58). The search for less toxic agents have more recently centered on inhibitors of the enzymes that catalyze the post-translational modification of Ras. Of these enzymes, PFT has been the most widely studied in terms of mechanism and inhibitors. Potential shortcomings of PFT inhibitors as anti-Ras agents include their degree of selectivity for 1) PFT relative to other prenylation enzymes, 2) Ras relative to other farnesylated substrates, and 3)oncogenic Ras relative to normal Ras. The potential for similar specificity shortcomings would also be expected for inhibitors of the protease, methyltransferase and palmitoyltransferase enzymes that complete Ras processing. Present evidence indicates that geranylgeranyl modified proteins terminate in either CAAL, CC, CXC, or CCXX motifs and as such are processed by distinct protein geranylgeranyltransferases (PGT) (59-61). Thus by designing an agent specific for the
Section In-Chemotherapeutle Agents
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peptide binding subunit of PFT, it should be possible to circumvent PFT/PGT specificity concerns. In addition to Ras, other proteins are substrates of PFT and would presumably be affected by a PFT inhibitor. Namely, lamin 6,which is required for nuclear envelope integrity, and transducin, which is important in retinal signal transduction, are both substrates for PFT. Inhibition of their farnesylation is thus a potential toxicity concern. Selectivity for oncogenic Ras relative to normal Ras would be a desirable property of a PFT inhibitor since Ras is expressed in virtually all tissues and plays a critical role in normal cellular proliferation. A precedent now exists for farnesylation inhibitors that appear to preferentially inhibit oncogenic Ras function (62, 63). The mechanism of this selectivity remains largely an enigma. Evidence suggesting a reduced affinity of Raf-1 for oncogenic Ras relative to normal Ras may provide a clue (64).
5$PROTFIN F
NHI IT
-
CAAX Analogs Initial investigations following the isolation and purification of PFT demonstrated that the full Ras protein was not required for enzyme activity (65). The enzyme farnesylates a number of CA,A2X tetrapeptides, including CVlM (66-71). The preferred C-terminal (X) residues are serine, methionine, glutamine, alanine, or cysteine, which result in PFT binding. In general, A, and A, can be any aliphatic amino acid. However, substitution at the A, position has a critical effect on the ability of the tetrapeptide to function as a substrate. The tetrapeptide CVFM (I), containing an aromatic residue at A, is a potent inhibitor (IC, = 25 nM) of PFT, and does not undergo farnesylation (67). A further requirement for this behavior of CVFM is a free N-terminus, in that acylation restores the ability for farnesylation (68). The liabilities of these tetrapeptides as potential drug candidates, which include their inefficient cellular uptake and rapid proteolytic degradation, have resulted in little further development. However, these compounds have served as a foundation for the design and synthesis of several classes of peptidomimetic derivatives of the C A M sequence. A number of potent inhibitors of PFT of this type have been reported recent I y .
The pseudo-tetrapeptideL-731,735 (2) was designed as a CllM analog in which the two N-terminal amide bonds were reduced and methionine was replaced with homoserine (62). These modifications resulted in a potent in vitro (IC5,=18 nM) inhibitor of PFT. The was also an effective inhibitor (IC,,= 282 corresponding lactone derivative L-731,734 (3) nM), and selectively inhibited both Ras processing in a transformed cell line and growth in soft agar. Systematic modification of 1 has also led to the identification of 8581 (4), which showed a twofold increase in activity vs 1 in virro (72). This analog was found to selectively inhibit the processing of farnesylated proteins rather geranylgeranylated proteins in a Rastransformed cell line, but failed to discriminate between farnesyiation of H-Ras and lamin A. Microinjection of 4 into frog oocytes also inhibited Ras-dependent maturation.
Ras Oncogene
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2
Others have also prepared a number of analogs of 1 to systematically examine the effect of backbone modification on activity and to further define substrate-inhibitor patterns (73). A number of modifications, including N-methylation and amide bond replacement, were investigated but these changes gave little insight to the substratehhibitor backbone conformation that is preferred by PFT. Replacement of the two aliphatic residues in the CAAX motif with a benzodiazepine derived mimic of a dipeptide turn led to an extremely potent inhibitor BZA-26 (6)with an ICm of 0.85 nM (63). This compound was designed to allow both the N-terminal cysteine and the C-terminal methionine to coordinate the Zn*+ ion that is present at the peptide binding site (74, 75). Recent proton NMR studies with an enzyme-bound heptapeptide substrate (KTKCVFM) have shown that the C-terminal tetrapeptide portion is directly involved in binding to the enzyme via an induced type I p-turn conformation (76). Although less potent in vifro, the corresponding methyl ester, BZAdB (S), restored a normal growth pattern to Ras-transformed cells. Incorporation of a 3-aminomethylbenzoic acid (AMBA) moiety as a replacement for the two internal aliphatic residues led to Cys-AMBA-Met (Z), an analog containing no peptidic linkages (77). This compound was a more potent inhibitor of human PFT than CVIM, but no cellular activity was reported.
Farnesvl PvroDhosDhate Analoas - Synthetic analogs of farnesyl pyrophosphate (FPP) have received less attention as potential inhibitors of PFT, perhaps because of specificity concerns due to its involvement in other biological pathways. However, two hydrolytically stable analogs of FPP have shown potent inhibition of PFT (78). Analogs and 9 were found to be competitive inhibitors of FPP, with Ki values of 5 nM and 830 nM, respectively. g was
a
m
sectmn in-chernotherweutic Agents
Plattner, Ed
shown previously to be a potent inhibitor of squalene synthetase (79). A slight inhibition of Ras processing in a Ras-transformed cell line was noted with at 1 uM (80). This effect, while modest, was the first demonstration that the cellular processing of Ras could be moderated by a synthetic PFT inhibitor. However, it could not be determined if higher concentrations of would lead to further inhibition of Ras processing, due to the observation of cellular toxicity. Farnesylamine and other related long chain aliphatic amines have also been shown to inhibit the farnesylation and growth of Ras-transformed cells at high micromolar concentrations (81).
Natural Products - The use of PFT in high volume screening has led to the identification of a variety of natural products of microbial origin which inhibit PFT (82). 10'-Desmethoxystreptonigrin (lQ), an analog of streptonigrin (11) was found to be a weak inhibitor (IC,= 21 uM) of the enzyme, and also was cytotoxic to several cell lines (83). Interestingly, streptonigrin itself was approximately threefold less potent in vim.
Other antibiotics which show activity are derivatives of the manumycin family (84). UCF1 -C also known as manumycin, was the most potent inhibitor of this class (ICm= 5 uM). Kinetic analysis demonstrated that was a competitive inhibitor (Ki = 1.2 uM) with respect to FPP, and noncompetitive with respect to protein substrate. The reduced (dithiol) form of the fungal epipolydithiodiketopiperazine toxin, gliotoxin (U),was also a modest (lCs0= 1.1 uM) inhibitor of PFT (85). This compound was found to be a noncompetitive inhibitor with respect to the Ras protein.
(u),
Two novel dicarboxylic acid derivatives, chaetomellic acid A
(14)and chaetomellic
Chap. 17
Ras Oncogene
Bolton, Sebalt-Leopold.Hodges
acid B (X), were isolated from a fermentation extract and found to be potent inhibitors of PFT, with C I , values of 55 nM and 185 nM, respectively (80, 86, 87). These compounds were shown to be effective mimics of FPP by kinetic analysis and by computer modeling of their overlapping steric and electrostatic regions.
Zaragozic acid A (U),previously disclosed as a potent inhibitor of squalene synthetase, has also been identified as a PFT inhibitor (IC, = 216 nM) (80). A synthetic analog (U) showed increased PFT inhibition (IC, = 12 nM) and selectivity versus PGT. These compounds were competitive inhibitors with respect to FPP, and noncompetitive inhibitors with respect to the Ras protein. They had no effect on Ras processing in a Ras transformed cell line, presumably due to poor cell permeability,
0
The pepticinnamins, a novel series of six related pentapeptides, were also isolated by the screening of various fermentation broths (88). Pepticinnamin E (M), the major component of the mixture and only member whose structure was elucidated, was found to be a good inhibitor (IC, = 300 nM) of PFT (89). Limonene and related metabolites perillic acid and dihydroperillic acid, have also been demonstrated to inhibit protein isoprenylation and cell growth (90, 91). The antibiotic patulin (19) inhibited protein prenylation in a mouse cell line, and exhibited weak (I&= 290 uM) inhibition of PFT (92).
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122
PROTFASF AND MFTHYl TRANSFFRASF INHIBITORS Protease lnhib ' itors - Compared to the rapidly growing number of recent publications surrounding PFT and its inhibitors, the literature available on the subsequent C-terminal Ras processing enzymes is small. Two microsomal enzymes that cleave AAX from a farnesylCAAX motif have been reported. The first is a bovine liver endoprotease which cleaves the C-terminal tripeptide from farnesylated proteins (35). The second is a rat brain carboxypeptidase which sequentially removes X, A2 and A, (36). Both enzymes show high affinity for a farnesyl-CAAX motif compared to its CAAX precursor. Farnesyl-CAAX analogs with isosteric replacements for the scissile peptide bond have been shown to be inhibitors of isoprenylated protein endoprotease (93). The most potent of these are 2p (Ki = 86 nM) and fi (Ki = 64 nM). In principle, these molecules could also be inhibitors of the carboxypeptidase although no data are currently available.
BocHN
-
a X=CH2 21 X = CH(OH)CH2CO
SMe
Methvltransferase Inhibitors - Microsomal enzyme preparations that catalyze the methyl esterification of farnesylcysteine residues located at the C-terminus of peptides have also been described (94-96). To date a number of weak inhibitors (Ki =. 25 uM) have been reported including S-farnesyl mercaptopropionic acids such as MFPT and FPA (B) (96).
(a)
SUMMARY AND FUTURE DIRFCTlONS Because of the central signalling role of Ras in cell division and because of the frequency of mutant ras genes in human tumors, inhibition of Ras is likely to continue to be a popular target for cancer chemotherapy research. To date, the most promising strategy is to block Ras function by inhibiting its membrane localization, more specifically by preventing C-terminal processing. The bulk of current research is focused on PFT inhibitors. There are numerous CAAX analogs, FPP analogs and natural products which are inhibitors of PFT in vitro that provide excellent lead structures from which future drug candidates may be derived. Although several structural types have been shown to inhibit Ras processing and Ras dependant transformations in cell culture assays, the major hurdle of demonstrating the antineoplastic efficacy of a PFT inhibitor in vivo remains to be cleared.
Ras Oncogene
Chap 17
Boltan, Sebolt Leopold. Hodges
m
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