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Inhibitors of mammalian DNA topoisomerases
INHIBITORS OF MAMMALIAN D N A TOPOISOMERASES. Giovanni Capranico and France Zunino. Division of Experimental Oncology B, Istituto Nazionale Tumori, 20133 Milan, Italy. DNA topoisomerases are the main targets of important antitumor drugs, including intercalating agents, minor-groove binders and others. These enzymes can be diveded in two mechanistic classes: type I and type II topoisomerases. One type I, and two type II enzymes are present in human cells. The two human topoisomerase II isozymes differ in molecular weight and are encoded by two distinct genes. Their pharmacological features have not been defined, however it is known that expression of the two genes are differentially modulated during cell proliferation and development. One isozyme may potentially represent a more selective target for novel anticancer drugs. Antitumor drugs interfere with topoisomerases by forming ternary DNA-drugenzyme complexes in which DNA strands are broken and proteinlinked. The structural determinants of drug activity have been partially elucidated b y studying the base sequence specificity o f drug action. Moreover, antitumor agents have been shown to act against topoisomerases with a novel mechanism, thus indicating new screening strategies to identify potential anti-tumor drugs. In recent years, x-ray diffraction studies as well as mutant topoisomerases have allowed to progress in the understanding o f the structure and function o f these nuclear proteins. The results will be summarized and discussed. The molecular mechanism o f cell ~eath has been an active area o f research, and published data suggest that tumor suppressor genes and some oncogenes may be involved in the cell death pathway triggered by ternary complex formation.
Lipid modification of low molecular m a s s - G T P binding proteins B.Therese Kinsella'., Erdman R.A., Sheridan K.M.; Maltese W.A. "Department of Biochemistry, University College Dublin, Belfield, Dublin 4. IRELAND. Weis Center for Research, Geisinger Clinic, Danville, PA 17822. USA. The superfamily of ras-related low molecular mass GTP-binding proteins (LMMGP) is involved in a number of specialized cellular functions, including cell differentiation and growth control, actin polymerization and cytoskeleton arrangement, participation i n the superoxide generating NADPH oxidase of phagocytic leukocytes, intracellular vesicular transport. Whereas members of the rasrelated supeffamily of LMMGPs may have different cellular functions, they are mainly found associated with the cytoplasmic side of sp~ecialized cellular membranes where they participate i n their assigned cellular task. The targeting of the LMMGPs to different cellular membranes depends on both their : posttranslational modification by lipids and by the interaction of t specific domains, located within their C-termini, with membrane lipid head groups and/or membrane proteins. LMMGPs can: undergo modification by, one or all, 3 distinct types of lipids : (a) isoprenylation of distinct cysteine residues in distinct "prenylation" motifs located toward the C-terminal amino acid b y ' attachment of C15 trans, trans-famesyl or C20 all transgeranylgeranyl in thioether linkages; (b) carboxyl methylation o f : the ct-carboxyl group of the isoprenylated cysteine residue to from t cysteic acid methyl ester; (c) pahnitoylation (C1610) of one or L more cysteine residues, located in the hypervariable domain l positioned upstream of the prenylated cysteine. Prenylation' substantially increases protein hydrophobicity, and this is enhanced ' by carboxyl methylation of the prenylated cysteines and or by ~ additional pahnitoylation Although prenylation is likely to facilitate protein interaction with membrane lipids, new evidence suggests that it may also mediate protein-protein interactions.
FARNESYLTRANSFERASE AND GERANYLGERANYLTRANSFERASE INHIBITORS
POSTTRANSLATIONAL ISOPRENYLATION OF ONCOGENES IN C E L L P R O L I F E R A T I O N : P H A R M A C O L O G I C A L MODULATION Pierre G. BRAQUET. Bio-Inova DNATech Pharmaceuticals, 48-52 rue de la Gare, F78370 PLAISIR France.
A. Corsini, D. Vcrri, C. Tagliabue,IL Paoletti,R. Fumagalli
Instituteof PharmacologicalSciences,Universityof Milun, Italy.
Membrane transfer of proliferative signal involves a complex series of events including tyrosine kinase receptor activation (eg b-FGF receptor), and transmission of the signal to Grb2 / Sos / Ras / Raf-1 / MAP kinase cascade and transcription. Ras and other proteins as Rab Rho... are synthesized as precursor moieties that require posttranslational processing to become activated. This process allows their membrane localization trough the isoprenylation. The addition of prenyl groups to proteins containing the C-terminal consensus sequence CAAX where C is cysteine, A is usually an aliphatic amino acid and X is another amino acid, is mediated by at least two different specific enzymes, farnesyl protein transferases (FPTase) and geranylgeranyl protein transferases (GGPTase) that respectively catalyzes the transfer of the farnesyl (FPP) and the geranylgeranyl (GGPP) moities to the thiol of the conserved cysteine residue of the protein substrates forming the thioether-linked adduct with the release of inorganic diphosphate. FPTases are metalloenzymes requiring Mg2+ and Zn 2+ for optimal activity. Proteins with Cterminal methionine, scrine, eysteine, alanine, or glutamine residue are good substrates for FPTases. In the ease of Ras, palmitoylation of Cys within the hypervariable region is also required for membrane lncalization. Isoprenylation appears to be a critical modification of Ras and some other oncogenes as Rab, Rho, Rae... and i~s pharmacological modulation may offer new avenues in the treatment of malignant and non malignant proliferative diseases. Lovasta~n a drug that inhibits p21 ras oncoprotein farnesylatinn and membrane association was recently shown to inhibit tumor growth of rastransformed cells. We recently found that lovastatin also inhibits bFGF- and EGF- induced human prostate cell line proliferation (see Paubert-Braquet et al, this meeting). Specifie inhibiters of FPTase have been recently designed. The chemical strategy include several possibilities: 1- competitive inhibition of the binding of FPP to its binding site on the a subunit of the enzyme (isoprenoid-related derivatives); 2- inhibition of the Zn2+ protease of FPTase ~ subunit; and 3- competitive inhibition of the binding of CAAX peptide to its binding site on the ~ subunit (via a peptide/peptidomimetie strategies).
m
Covalent attachment of isoprendd lipids (famesol or g~unylgffuniol) is an obligatory ix~ranslational modification of many proteins involved in cell physiology and proliferation. For this reason, the two classes of enz]anes that catalize the addition of a prenyl group to proteins, named farnesyltransfcrase (FTase) and geranylgezanyltransferase(GGTase I and n), have gained attention as a novel targets for the developmentof agents aimed at controlling abnormal cell growth such as my0cyte proliferation under atherogenic conditions or tumor development Among the prenylatedproteins, much attention has been focussedon p21 ras for its key role in the pathophysiology of cell proliferation. Because farnes3,1afionof p21 ms is required for its mitogenic activity, intensive search for FTase inhibitors is under way. Several types of protein famesylation inhibitors were investigated in cultured smmoth muscle cells (SMC). One type includes agents that indirectlyinhibit the biosynthesisof famesyI-PPsuch as inhibitotsof 3hydroxy-3-methylglutaryl-connzymeA fflMG-CoA) reductase, the rate limiting enzyme in isopreaoids synthesis. Fluvastatin (F) and simvastatin (S), specific competitiveinhibitorsof the enzyme, decreased human and rat SMC proliferation at the reported therapeuticconcentrations.The inhibitionof cell proliferation(70% decrease) inducedby 3.5 mM F and S was completelyprevented by the addition of 1110 mM mevalonate, partially (70-85%) by 10 mM farnesol and by 5 mM geranylgeraniol. These results indicate the involvement of specificisopreaoids meml~ites, probablyfamesylatedor geranylgeranylatedproteins, in the control of cell proliferation. Another class of agents that directly inhibit FTase consists of short peptides which mimics the carboxy-terminiof famesylatedproteins. Among them, the benzodiazepine peptidomimetic BZA-5B was shown to inhibit SMC proliferation in a dose- and time-dependent mann~. The inhibitory effect of 50 mM BZA-5B (60% d~res~) was partially prevented by mevalenate (80%) and geranyigeraniol(65%) but not by famesol. On the other hand, SMC prolifexation was not affectedby the closelyrelated compound BZA-7B, which does not inhibit Frase. Taken together,these findings imply that Frase inhibitorscould representa new class of agents potentially active in the control of one of the major event involvedin the pathogenesisof athorosclerosis.
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Lipid modification of low molecular m a s s - G T P binding proteins B.Therese Kinsella'., Erdman R.A., Sheridan K.M.; Maltese W.A. "Department of Biochemistry, University College Dublin, Belfield, Dublin 4. IRELAND. Weis Center for Research, Geisinger Clinic, Danville, PA 17822. USA. The superfamily of ras-related low molecular mass GTP-binding proteins (LMMGP) is involved in a number of specialized cellular functions, including cell differentiation and growth control, actin polymerization and cytoskeleton arrangement, participation i n the superoxide generating NADPH oxidase of phagocytic leukocytes, intracellular vesicular transport. Whereas members of the rasrelated supeffamily of LMMGPs may have different cellular functions, they are mainly found associated with the cytoplasmic side of sp~ecialized cellular membranes where they participate i n their assigned cellular task. The targeting of the LMMGPs to different cellular membranes depends on both their : posttranslational modification by lipids and by the interaction of t specific domains, located within their C-termini, with membrane lipid head groups and/or membrane proteins. LMMGPs can: undergo modification by, one or all, 3 distinct types of lipids : (a) isoprenylation of distinct cysteine residues in distinct "prenylation" motifs located toward the C-terminal amino acid b y ' attachment of C15 trans, trans-famesyl or C20 all transgeranylgeranyl in thioether linkages; (b) carboxyl methylation o f : the ct-carboxyl group of the isoprenylated cysteine residue to from t cysteic acid methyl ester; (c) pahnitoylation (C1610) of one or L more cysteine residues, located in the hypervariable domain l positioned upstream of the prenylated cysteine. Prenylation' substantially increases protein hydrophobicity, and this is enhanced ' by carboxyl methylation of the prenylated cysteines and or by ~ additional pahnitoylation Although prenylation is likely to facilitate protein interaction with membrane lipids, new evidence suggests that it may also mediate protein-protein interactions.
FARNESYLTRANSFERASE AND GERANYLGERANYLTRANSFERASE INHIBITORS
POSTTRANSLATIONAL ISOPRENYLATION OF ONCOGENES IN C E L L P R O L I F E R A T I O N : P H A R M A C O L O G I C A L MODULATION Pierre G. BRAQUET. Bio-Inova DNATech Pharmaceuticals, 48-52 rue de la Gare, F78370 PLAISIR France.
A. Corsini, D. Vcrri, C. Tagliabue,IL Paoletti,R. Fumagalli
Instituteof PharmacologicalSciences,Universityof Milun, Italy.
Membrane transfer of proliferative signal involves a complex series of events including tyrosine kinase receptor activation (eg b-FGF receptor), and transmission of the signal to Grb2 / Sos / Ras / Raf-1 / MAP kinase cascade and transcription. Ras and other proteins as Rab Rho... are synthesized as precursor moieties that require posttranslational processing to become activated. This process allows their membrane localization trough the isoprenylation. The addition of prenyl groups to proteins containing the C-terminal consensus sequence CAAX where C is cysteine, A is usually an aliphatic amino acid and X is another amino acid, is mediated by at least two different specific enzymes, farnesyl protein transferases (FPTase) and geranylgeranyl protein transferases (GGPTase) that respectively catalyzes the transfer of the farnesyl (FPP) and the geranylgeranyl (GGPP) moities to the thiol of the conserved cysteine residue of the protein substrates forming the thioether-linked adduct with the release of inorganic diphosphate. FPTases are metalloenzymes requiring Mg2+ and Zn 2+ for optimal activity. Proteins with Cterminal methionine, scrine, eysteine, alanine, or glutamine residue are good substrates for FPTases. In the ease of Ras, palmitoylation of Cys within the hypervariable region is also required for membrane lncalization. Isoprenylation appears to be a critical modification of Ras and some other oncogenes as Rab, Rho, Rae... and i~s pharmacological modulation may offer new avenues in the treatment of malignant and non malignant proliferative diseases. Lovasta~n a drug that inhibits p21 ras oncoprotein farnesylatinn and membrane association was recently shown to inhibit tumor growth of rastransformed cells. We recently found that lovastatin also inhibits bFGF- and EGF- induced human prostate cell line proliferation (see Paubert-Braquet et al, this meeting). Specifie inhibiters of FPTase have been recently designed. The chemical strategy include several possibilities: 1- competitive inhibition of the binding of FPP to its binding site on the a subunit of the enzyme (isoprenoid-related derivatives); 2- inhibition of the Zn2+ protease of FPTase ~ subunit; and 3- competitive inhibition of the binding of CAAX peptide to its binding site on the ~ subunit (via a peptide/peptidomimetie strategies).
m
Covalent attachment of isoprendd lipids (famesol or g~unylgffuniol) is an obligatory ix~ranslational modification of many proteins involved in cell physiology and proliferation. For this reason, the two classes of enz]anes that catalize the addition of a prenyl group to proteins, named farnesyltransfcrase (FTase) and geranylgezanyltransferase(GGTase I and n), have gained attention as a novel targets for the developmentof agents aimed at controlling abnormal cell growth such as my0cyte proliferation under atherogenic conditions or tumor development Among the prenylatedproteins, much attention has been focussedon p21 ras for its key role in the pathophysiology of cell proliferation. Because farnes3,1afionof p21 ms is required for its mitogenic activity, intensive search for FTase inhibitors is under way. Several types of protein famesylation inhibitors were investigated in cultured smmoth muscle cells (SMC). One type includes agents that indirectlyinhibit the biosynthesisof famesyI-PPsuch as inhibitotsof 3hydroxy-3-methylglutaryl-connzymeA fflMG-CoA) reductase, the rate limiting enzyme in isopreaoids synthesis. Fluvastatin (F) and simvastatin (S), specific competitiveinhibitorsof the enzyme, decreased human and rat SMC proliferation at the reported therapeuticconcentrations.The inhibitionof cell proliferation(70% decrease) inducedby 3.5 mM F and S was completelyprevented by the addition of 1110 mM mevalonate, partially (70-85%) by 10 mM farnesol and by 5 mM geranylgeraniol. These results indicate the involvement of specificisopreaoids meml~ites, probablyfamesylatedor geranylgeranylatedproteins, in the control of cell proliferation. Another class of agents that directly inhibit FTase consists of short peptides which mimics the carboxy-terminiof famesylatedproteins. Among them, the benzodiazepine peptidomimetic BZA-5B was shown to inhibit SMC proliferation in a dose- and time-dependent mann~. The inhibitory effect of 50 mM BZA-5B (60% d~res~) was partially prevented by mevalenate (80%) and geranyigeraniol(65%) but not by famesol. On the other hand, SMC prolifexation was not affectedby the closelyrelated compound BZA-7B, which does not inhibit Frase. Taken together,these findings imply that Frase inhibitorscould representa new class of agents potentially active in the control of one of the major event involvedin the pathogenesisof athorosclerosis.
24
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