NOVEL PHARMACEUTICALLY ACTIVE COMPOUNDS INTERACTING WITH GTP-BINDING PROTEINS

Abstract: The present invention relates to compounds that bind to the surface of normal or mutated GTP binding proteins and reduce the activation energy of the GTPase activity thereof by introduction of at least one functional group into the active site of said proteins. Said compounds are of synthetic or semi-synthetic origin. Preferably, the functional group referred to above is an arginine finger or a derivative thereof, a compound that mimics the activity of an arginine finger that is obtained, for example by peptidomimetics or a compound comprising or consisting of an aromatic amine, preferably a 4-amino-benzophenone-derivative. The invention further relates to a pharmaceutical composition comprising the compound of the invention as well as to methods for treating patients suffering from oncogenic activations of Ras genes, from tumors caused by malfunction of G proteins or from neurofibromatosis. Additionally, the present invention relates to kits comprising the compound of the invention as well as to methods for devising said compound. Finally, the invention relates to methods for devising lead compounds for the development of the compounds of the invention.

Description: NOVEL PHARMACEUTICALLY ACTIVE COMPOUNDS INTERACTING WITH GTP-BINDING PROTEINS Background of the invention Throughout this specification, various publications are referenced either directly or by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of the publications in their entireties are hereby incorporated by references into this specification in order to more fully describe the state of the art to which this invention pertains. The coupling of membrane receptors to effector molecules triggering a cellular response to an external signal has been an area of intensive research in recent years. It has been demonstrated that, for example, G proteins couple to a variety of such signal transduction pathways. Said G proteins comprise heterotrimeric G proteins which trigger the downstream cascade of a variety of reactions by alternating between an active and an inactive state. In the inactive state, these G proteins are heterotrimers in which GDP is bound to an alpha chain which is tightly associated with the beta gamma subunits. When the receptor is activated, GDP is replaced by GTP followed by the dissociation of the alpha subunit from the beta gamma subunits. The intrinsic GTPase activity of the alpha chain leads to the cleavage of bound GTP. As a consequence, the G protein returns to its inactive, GDP-bound state. It has been demonstrated that mutations in the DNA encoding the heterotrimeric G proteins can lead to oncogenic transformations. For example, mutations that constitutively activate the alpha s chains of the G s proteins have been assumed to be the causative agent of pituitary tumors. These mutations most often affect residues Arg-207 and Glu-227. Replacement of these amino acids causes impairment of the intrinsic GTPase reaction and blocks the alpha chain in its activated GTP-bound status (for a review, see (57)). Similarly, the Gj 0 proteins are also comprised of three subunits α, β, γ . Mutations in the alpha i 0 subunit have been associated with human tumors of the ovary, adrenal cortex or the pituitary gland (57). Another class of GTP-binding proteins are the Ras proteins. The Ras proteins H-, K- and N-Ras are also central regulators of cellular signal transduction processes leading to cell growth and differentiation. Similar to the heterotrimeric G proteins, they are molecular switches which in the resting cell are in the GDP-bound 'OFF' state. They become GTP-bound and thus activated by the action of receptor protein tyrosine kinases or other growth factor dependent stimuli. In the GTP- bound 'ON' state they interact with downstream targets which transmit the signal to molecules located further downstream in the signaling cascade. GTP hydrolysis mediated by Ras completes what is called the GTPase cycle (1 ). The conformational state of Ras is regulated by two kinds of proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) (2). GEFs such as Sos or Cdc25 increase the dissociation rate of guanine nucleotides thereby facilitating loading of Ras with GTP. GAPs downregulate the switch by accelerating the slow intrinsic rate of GTP hydrolysis by several orders of magnitude. Five mammalian GAPs for Ras have been described (2), the most thoroughly studied of which are p120GAP, the prototype for this class of proteins (3), and neurofibromin, the product of the type I neurofibromatosis (NF1 ) gene (4, 5). Oncogenic mutants of Ras are found in about 25-30% of human tumors. Most of them have an impaired intrinsic GTPase, are insensitive to GAPs and thus unable to switch off the transmitted signal. Due to the action of the negative regulator GAP, normal Ras is found in the inactive GDP-bound state in unstimulated cells, whereas oncogenic Ras remains GTP-bound (1). To understand the molecular basis of Ras mediated oncogenic transformation and to possibly interfere with the processes involved it is of crucial importance to know the details of the mechanism by which Ras hydrolyzes GTP and how it is affected by GAPs. The mechanism by which GAPs accelerate the GTPase reaction has been a matter of considerable debate (6, 7). In one model Ras itself is an efficient GTPase but in order to achieve the fast GTPase reaction GAP favors the attainment of a GTPase competent conformation by catalyzing the rate-limiting isomerization reaction. Conflicting evidence for such an isomerization reaction catalyzed by GAP has been presented (8). In another model the actual chemical cleavage step is modified by GAP, most likely by GAP supplying residues for GTP hydrolysis on Ras and thereby stabilizing the transition state of the reaction. In support of this model we have recently shown by the use of fluorescence spectroscopy that RasGAPs stabilize binding of aluminum fluoride to Ras » GDP, which based on evidence from G α proteins is believed to mimic the transition state of the GTPase reaction (9), and that stoichiometric amounts of GAP are needed for this effect (10). Our recent structure determination of a RasGAP has revealed the catalytic fragment of p120GAP, GAP-334, as a purely helical molecule consisting of 2 domains the larger of which contains all residues conserved among RasGAPs (11 ). From this study and further prior art studies, it was, however, still unclear how the molecular mechanism that triggers the GTPase activation works. Without a detailed knowledge of this mechanism, it is not possible to devise compounds with pharmaceutical applicability that interfere with the generation of human disorders or which halt or reverse such disorders. Accordingly, the technical problem underlying the present invention was to find out the details of GAP stimulated GTP hydrolysis on GTP binding proteins such as Ras. It was of further interest to investigate how oncogenic mutations block GAP action on Ras. It is generally assumed that the oncogenic versions of Ras with point mutations in glycine 12 or glutamine 61 have an intrinsic defect that renders then unable to hydrolyze GTP. However, detailed knowledge of the mechanism by which GAP activates the GTPase reaction and why it cannot do so in oncogenic mutants might enable one to artificially induce GTP hydrolysis an either wild type or mutant Ras or other GTP-binding proteins. These would give the chance of devising compounds that would induce GTP hydrolysis in various pathological situations. The solution to said technical problem is achieved by providing the embodiments characterized in the claims. Brief summary of the invention The present invention relates to compounds that bind to the surface of normal or mutated GTP binding proteins and reduce the activation energy of the GTPase activity thereof by introduction of at least one functional group into the active site of said proteins. Said compounds are of synthetic or semi-synthetic origin. Preferably, the functional group referred to above is an arginine finger or a derivative thereof, a compound that mimics the activity of an arginine finger that is obtained, for example, by peptidomimetics or a compound comprising or consisting of an aromatic amine, preferably a 4-amino-benzophenone-derivative. The invention further relates to a pharmaceutical composition comprising the compound of the invention as well as to methods for treating patients suffering from oncogenic activations of Ras genes, from tumors caused by malfunction of G proteins or from neurofibromatosis. Additionally, the present invention relates to kits comprising the compound of the invention as well as to methods for devising said compound. Finally, the invention relates to methods for devising lead compounds for the development of the compounds of the invention. Detailed description of the invention In accordance with the foregoing, the present invention relates to a compound characterized in that it (a) binds with a sufficient affinity to the surface of a normal or mutated GTP- binding protein to allow a contact with the active site thereof, wherein the mutation results in an impaired intrinsic GTPase activity; (b) reduces the activation energy of the GTPase enzymatic activity of said GTP-binding protein by introduction of at least one functional group into said active and/or activates the nucleophilic water thereby increasing the GTPase reaction rate; and (c) is of synthetic or semisynthetic origin. In connection with the present invention, the term "normal or mutated GTP- binding protein" is intended to mean that said compounds will bind to (i) a normal GTP-binding protein, (ii) a mutated GTP-binding protein, or (iii) to both (i) and (ii). Surprisingly, it has been found in accordance with the present invention that the introduction of a functional group into the active site of a GTP-binding protein wherein the active site mediates GTPase activity and wherein said functional group preferably is an arginine finger or is based on or derived from an arginine finger or mimics the action of an arginine finger or is an aromatic amine is sufficient to reduce the activation energy of the GTPase enzymatic activity or to activate the nucleophilic water and consequenty increases the GTPase reaction rate. Although previous findings showed that a mutation destroyed the ability of GAP to activate GTP hydrolysis or that the mutations of a particular arginine other than the one mentioned in the present invention would destroy the ability of GAP to stabilize the transistion state, all of these effects could have been caused by indirect effects of these mutations of the integrity of the 3D structure. Only the present invention shows conclusively that another arginine than the one mentioned earlier participates directly in catalysis. In addition, the present invention for the first time identifies a chemical compound useful to trigger catalysis.This finding has enormous implications in the medical field. On the basis of the finding of the present invention, it is now possible to devise other compounds that fulfill the same function as the natural arginine finger with the unmutated GTP-binding protein. For example, such compounds would be required to trigger the GTPase reaction in cases where the mutation has occurred in the active site of the GTP binding protein, said mutation barring the access of the arginine finger into the active site due to steric hinderances. Mutations of this kind are known for Ras proteins, for example, in amino acid positions 12 and 61 of Ras proteins. Additionally and most importantly, compounds may be devised on the basis of the the present invention that activate or increase the GTPase activity of a GTP-binding protein with an active site which, under physiological conditions, is not activated by an arginine finger. Such compounds may be devised, for example, on the basis of the knowledge that other phosphoryl transfer enzymes supply positive charges into the active site of such GTPases and therefore stabilize the transition state. Accordingly, the invention also comprises compounds that comprise amines, amino groups or guanidino groups or three-dimensional structures that are based on that groups and may be derived therefrom, for example by peptidomimetics. The compound of the invention should preferably be of small size in order to allow a convenient synthetic or semisynthetic production thereof. For pharmaceutical applications, the compounds should preferably, but not necessarily, be of small size. Other factors such as hydrophilicity also contribute to the pharmaceutical applicability. All these factors are known to the person skilled in the art, who is, on the basis of the teachings of the present invention, in a position to devise compounds with pharmaceutically advantageous properties that may be most conveniently administered to patients in need thereof. The compound of the invention may also be prepared by chemical synthesis out of chemical modification of peptides. Phage display or combinatorial library techniques may also be used for developing and for selecting appropriate compounds or precursores thereof such as peptidic precursors. The compound of the invention may also be prepared by combining two or more of the above recited methods or by combining one or more of these methods with other methods. Another application of the compound of the invention relates to cases, where the cellular GAPs are either absent or non-activating. In these cases, the active centre of the GTPase may be unimpaired and not distorted by mutation. The compound of the invention thus serves the purpose to replace the GAP and interact with the "wildtype" GTPase to trigger its normal function. A pharmaceutical application relating to this embodiment is neurofibromatosis (for more detailed information, see below). Again, the compound must be small enough (and lipophilic enough) to be able to enter the cell. The compound of the invention is, on the basis of its biochemical features, capable of being introduced into, for example, cancerous cells with an impaired function of a GTP binding protein and there fulfill the desired pharmaceutical function, namely activate or increase the GTPase activity of said GTP-binding protein. Furthermore, the compound of the invention preferably is, at least when applied for therapeutic purposes, not be of a purely proteinaceous nature in order to avoid an immediate degradation in the human or animal body before the desired pharmaceutical activity can take place. The compound of the invention may be of synthetic origin. In an alternative embodiment, the compound of the invention is of semisynthetic origin. When used in the kit of the invention, the compound may also be of peptidic origin. The compound of the invention may be devised starting from the natural three- dimensional peptide portion comprising the arginine finger and synthesizing a corresponding three-dimensional structure by chemical means, for example, by peptidomimetics. Alternatively, the compound of the invention may comprise peptidic parts such as the arginine finger and otherwise consist of portions that have been devised by, for example, peptidomimetics. Other embodiments of the compound of the invention can be devised by the person skilled in the art using known techniques where it has to be kept in mind, however, that a rapid degradation corresponding to a therapeutical ineffectiveness is to be avoided. In a preferred embodiment of the compound of the invention, said mutated GTP- binding protein is a mutated Ras-related protein. This embodiment of the invention is preferred since it is known in the art that many tumor formations in humans are related to mutated Ras-related proteins. Particularly preferred is that said mutated Ras-related protein is a mutated Ras- protein. It is further particularly preferred that said Ras protein is K-Ras, H-Ras or N-Ras. Also particularly preferred is that the mutated Ras protein bears a mutation in amino acid position 12 and/or in amino acid position 61. These mutations are known in the art to be involved in the formation of tumors in humans. Accordingly, the compound of the invention has the advantage over natural and thus prior art compounds such as GAPs to trigger the GTPase enzymatic activity of these proteins. This is because, in contrast to prior art compounds, the compound of the invention fits into the active site by, for example, a key and lock mechanism and thus is not barred from activating the GTPase enzymatic activity due to the changed conformity of the GTPase active site. Thus, with the teachings of the present invention, it is not only possible to confirm on a structural level the hypothesis that GAP actively participates in the chemical cleavage step of the GTPase reaction via an arginine finger (Arg789), that appears to be the major contributor to the enzymatic reaction by showing the arrangement of the active site residues in the catalytically competent conformation of the complex. In addition, it demonstrates that GAP stabilizes the position of Gln61 , the most crucial residue in Ras mediated GTP hydrolysis, which is very mobile in isolated Ras. Most importantly the structure also allows to formulate a unifying concept why mutations in both Gly12 and Gln61 activate the transforming potential of Ras: Both are in van der Waals distance from each other and from GAP in the transition state mimic. In a further preferred embodiment of the compound of the invention, said functional group is an arginine finger or a derivative thereof or a compound that mimics the activity of an arginine finger. This embodiment of the invention is particularly useful for activating or increasing the GTPase activity of heterotrimeric G proteins as well as of RAS-related proteins known to interact, under normal physiological conditions, with the arginine fingers of GAPs. Rho and Rac proteins are believed to be involved in tumor formation because guanine nucleotide exchange factors of Rac and Rho are frequently found as oncogenes. Therefore, compounds that would inactivate Rac/Rho by activating the GTPase reaction might be useful for the prophylaxis or treatment of such tumors, since all RhoGAP's have been shown to have a conserved arginine residue. It is additionally preferred in accordance with the present invention that said functional group comprised in said compound is or comprises an aromatic amine. It is particularly preferred that said aromatic amine is a 4-amino-benzophenone- derivative. In accordance with one of these preferred embodiments of the present invention, it has been shown that GTPase activity of oncogenic, GTPase-deficient Ras mutants is preferentially stimulated by a unique GTP analogue which carries an aromatic amino group which is shown to be essential for catalysis. The analog works with all the oncogenic mutants tested with a maximal stimulation of 900- 1000-fold. Structural analysis shows that the aromatic amino group is positioned in the active site to activate the nucleophilic water. Biochemical studies indicate that the mechanism of hydrolysis is likely to be different from normal GTP hydrolysis, with proton transfer, most likely to the aromatic amine, becoming the rate-limiting step of the GTPase reaction. One of the preferred compounds comprising a preferred embodiment of the compound of the invention, namely DABP-GTP is not hydrolysed with a rate comparable to that of the GAP-catalysed reaction which in the 5-20 s "1 range. However, it has been estimated that a 30-50 fold stimulation of the GTPase reaction of oncogenic mutants should be sufficient to serve as an anti-Ras drug. The reason is that P12Ras is the only non-oncogenic Gly12 mutant (Seeburg, P. H., Colby, W. W., Capon, D. J., Goedel, D. V. and Levinson, A. D. (1984) Nature 312, 71 ). Its intrinsic GTPase activity is 2-3 fold higher than that of wild type but it is not stimulated by GAP (Schweins, T., Geyer, M., Kalbitzer, H.R., Wittinghofer, A. and Warshel, A. (1996) Biochemistry 35, 14225; H. Gutfreund, Kinetics of the Life Sciences, Cambridge University Press 1995) (this invention). Considering that most oncogenic mutants of Ras have a 10-20 fold reduction of its intrinsic GTPase, it might be sufficient to stimulate their GTPase reaction up to the level of the Pro12 mutant. The results presented here, with stimulations of 12 to several hundred fold suggest that this will be possible. In a further preferred embodiment of the compound of the invention, said compound has at least one of the following properties: (a) an aromatic amino group with a proper pK a positioned such that it can accept protons from the nucleophilic water; (b) shielding the water from the bulk water such that its nucleophilicity is increased; and (c) a guanidinium side chain positioned such that it stabilizes the transition state. Methods of selecting or devising such a compound can be carried out by the person skilled in the art on the basis of the teachings of this invention as well as on his general common knowledge. Further preferred in accordance with the present invention is a compound which is a compound derived from a peptide comprising the arginine finger referred to above by peptidomimetics. Peptidomimetics has, in pharmaceutical applications, obtained a growing importance in recent years. The main reason therefor is that peptide analogs obtained by chemical synthesis out of chemical modification of peptides enjoy a much greater resistance to proteolytic degradation and thus allow an oral formulation of pharmaceutical compositions. An example of the development by peptidomimetics is the development of an anti-Ras drug directed against membrane targeting of the protein. Here a tetrapeptide from the CaaX motif was found to be a good inhibitor of the enzyme protein-famesyltransferase and was modified such that its potency in vitro and its bioavailability in vivo was dramatically improved (60). In an additional preferred embodiment of the compound of the invention, the increase of the GTPase reaction rate is at least 20-fold as compared to the oncogenic GTP binding protein. It has been shown that a particular mutation of Ras, where glycine 12 has been mutated to proline has a 2-3 fold increase in the GTPase reaction rate and is not oncogenic. Since oncogenic versions of Ras have on average about a tenfold lower intrinsic GTPase reaction rate, a 20-30 fold increase as compared to the oncogenic Ras should block the oncogenic properties of these mutants. Although a higher increase level would, at least in some cases, also be desirable, the preferred level of a 20-fold increase is considered advantageous to trigger the transition from an oncogenic to a non-oncogenic state. Preferably, the increase is 20 to 30-fold. Further preferred is an increase of at least 100-fold. The present invention further relates to a pharmaceutical composition comprising the compound of the invention and a pharmaceutically acceptable carrier and/or diluent. The pharmaceutical composition of the invention may comprise one or a variety of compounds of the invention. It may be administered to a patient in need thereof at a suitable dose. The dose regimen will be determined by the attending physician considering the condition of the patient, the severity of the disease and other clinical factors. Generally, the regimen as a regular administration as well the dose administered will depend on the type of GTP-binding protein and the nature if any of the mutation of the GTP-binding protein. Administration of the suitable compositions may be effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular or other routes. Most preferred is the oral route of administration. Examples of suitable pharmaceutical carriers are well known in the art and include buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions and others. Compositions comprising such carriers can be formulated by well known conventional methods. The invention also relates to a method of treating a patient suffering from an oncogenic activation of the Ras gene comprising administering an effective amount of the pharmaceutical composition of the invention. Further, the invention relates to a method of treating a patient suffering from neurofibromatosis type I or an oncogenic activation associated with neurofibromatosis type I comprising administering an effective amount of the pharmaceutical composition of the invention. The molecular basis for neurofibromatosis type I has been elaborated on above: According to the current model neurofibromatosis is caused by an abnormal regulation of Ras. More particularly, said disease the pleiotropic symptoms of which include rare malignancies and universal benign tumors is associated with a defunct rasGAP termed neurofibromin. A more detailed recent review on neurofibromatosis type I can be found in (58). The successful treatment of neurofibromatosis type I with the pharmaceutical composition of the invention is one example wherein the compound of the invention interacts with an unmutated counterpart, in this case a Ras protein, to substitute for an - in healthy persons - interacting and activating partner that is rendered non-functional in the disease state. In a preferred embodiment of the method of the invention, said oncogenic activation has resulted in a tumor. It is to be understood that said oncogenic activation can have resulted in more than one tumor and indeed in different types of tumors which can develop concomitantly or successively. Most preferably, said tumor is a tumor of neural crest derived tissue, for example, neurofibrosarcoma, glioma, astrocytoma, pheochromocytoma, embryonal rhabdomyosarcoma, or is colon cancer, pancreatic tumor, lung or breast cancer, or leukemia. Additionally, the invention relates to a method of treating a patient suffering from a tumor caused by a malfunction of a G protein comprising administering an effective amount of the pharmaceutical composition of the invention. The present invention also relates to the use of the compound of the invention for the treatment of any one of the above recited malfunctions or diseases and it relates to the use of the compound of the invention for the preparation of a pharmaceutical composition for the treatment of any one of the above recited malfunctions or diseases. Additionally, the present invention relates to a kit comprising the compound of the invention or a peptide analogue thereof. The kit of the invention may be used for the generation of novel compounds of the invention specifically interacting with wildtype or mutated GTP-binding proteins. Said compounds may be devised according to the special scientific or therapeutic interests involved. The kit of the invention may further be used in any of the methods of the invention described hereinbelow, for example, in the development of a so-called lead compound. The invention relates also to a method of devising a compound of the invention comprising: (a) screening an at least partially randomized peptide library for peptides that bind with a sufficient activity to the surface of a GTP-binding protein to allow a contact with the active site thereof and reduces the activation and energy of the GTPase enzymatic activity of said GTP-binding protein by introduction of at least one functional group into said active site; and optionally (b) repeating step (a) one or more times. The compound will be found by screening a library of compounds, the design of which is based on the structure of the Ras:RasGAP complex such that it contains reactive groups, preferably amines, amidines or guanidines which are able to bind to these phosphate groups of GTP and neutralize and stabilize its charges. These compounds are, for example, screened against a GTP-binding protein such as a Ras protein loaded with radioactivity [γ-32P]-labeled GTP by incubating both for various times in an appropriate buffer, filtrating the solution through a nitrocellulose filter and determining the radioactivity remaining bound to Ras. The libraries may be 1 ) peptide libraries which have defined and degenerate positions in the sequence, 2) phage display libraries where a similar series of peptides is constrained in its conformation by insertion into a surface loop of a phage protein, 3) a combinatorial library whose main features are the combination of positive charges and hydrophobic groups which mimic the GTP-binding protein (e.g. Ras)-(Ras)GAP interface in the active site. The first screens would be made for compounds that are marginally active and are able to increase the GTPase rate 2-3 fold. In further rounds of screening, the conditions of the assay would be modified such that higher activities are needed to produce a detectable readout of release of the radioactive label. The compound identified after step (b) may be further characterized for its dose response curve and by NMR, whether it binds to the active site of Ras or other GTP-binding proteins. It may be directly used as an active ingredient in the pharmaceutical composition of the invention. Alternatively, it may be further improved with regard to, e.g. its therapeutic features by one of the methods described hereinbelow. The compound will be improved in its bioavailability properties such as the maximum plasma concentration and plasma elimination half-life. In a preferred embodiment, said method further comprises: (c) improving the capacity of said compound obtained in step (a) or (b) to reduce the activation energy of the GTPase enzymatic activity by peptidomimetics, and/or by involving phage displays techniques and/or combinatorial library techniques. All those techniques and uses thereof are known to the person skilled in the art or are immediately apparent in the light of the teachings of the present invention. Particularly preferred is a method, wherein said GTP-binding protein used in step (a) and/or (b) is a wildtype GTP-binding protein and said GTP-binding protein in step (b) and/or step (c) is a mutated GTP-binding protein. Using this embodiment of the invention, a compound can be designed which binds to wildtype and/or mutated G protein. The method of the invention thus allows the gradual improvement of the binding and activation capacity of the compound of the invention towards mutated G proteins. Starting from a known interaction, namely the interaction of wildtype GTP-binding protein with a compound comprising an arginine finger or an functional equivalent thereof, the person skilled in the art can, for example, aided by, e.g., peptidomimetics or phage display or combinatorial library techniques or combinations thereof, generate over several rounds of screening and selection, a compound that has the desired GTPase activating properties towards wildtype and mutant GTPase and can successfully be employed for therapeutic purposes. In a further particularly preferred embodiment of the method of the invention, said at least partially randomized peptide library is presented for screening by phage display. This embodiment of the invention is particularly preferred because of the high- throughput screening that has become possible using phage display screening techniques. These techniques are well known in the art and need not be described here in greater detail. For a review, it is referred to (59). Further, the invention relates to a method of devising a lead compound for the development of a compound of the invention, comprising screening an at least partially randomized peptide library for peptides that bind with a sufficient activity to the surface of a GTP-binding protein to allow a contact with the active site thereof and reduce the activation energy of the GTPase enzymatic activity of said GTP-binding protein by introduction of at least one functional group into said active site. The development of a lead compound is particularly useful in cases where different mutations leading to different conformations of active centres to be influenced require the development of a variety of compounds each of which is particularly useful to influence, for example activate a specific mutant having a unique conformation. Since a mutation of the Ras proteins is, according to the current knowledge, involved in about 25-30% of all human tumors, the GTP-binding protein involved in the method of the invention is a Ras-related protein and preferably a Ras- protein. These results presented in accordance with the invention show for the first time that oncogenic Ras mutants can be chemically inactivated. Therefore, this invention forms the path to design or screen for chemical entities that incorporate the relevant chemistry found to be necessary for the hydrolysis of GTP and at the same time bind with high enough affinity into the active site of Ras. The data provided in accordance with the invention suggest that the incorporation into the compound of the features recited herein below would be desirable to achieve efficient catalyis of GTP on oncogenic Ras; accordingly the invention also relates to a method for selecting for a compound that activates efficient catalyis of GTP on oncogenic Ras having at least one of the following properties: i. an aromatic amino group with a proper pK a positioned such that it can accept protons from the nucleophilic water; ii. shielding the water from the bulk water such that its nucleophilicity is increased; and iii. a guanidinium side chain positioned such that it stabilizes the transition state. With the structures of the transition state complex of Ras and GAP available (Scheffzek, K. et al. (1997) Science 277, 333) and the structures of DABP-GTP bound to Ras shown here it will be possible to design appropiate scaffolds as the basis to apply combinatorial chemistry in order to find compounds specifically homing in into the active site of Ras. The figures show: Fig.1 Segment of the 2 F 0 -F c electron density map (Ras in blue, GAP-334 in red, waters in light blue, contoured at 1.2σ ) covering the active site region in the complex. Fig. 2 The complex between GAP-334 and Ras. A. Ribbon representation of the complex model drawn with 'Molscript' (53) and 'Raster3D' (54) according to the assignment of secondary structure elements obtained with DSSP (55). The extra and catalytic domains of GAP-334 are shown in green and red, Ras in yellow, GDP and AIF 3 as ball and stick models. B. Schematic drawing with selected interactions included. Polar interactions between individual residues of GAP-334 and Ras are shown as red lines for interactions of side chains, as red arrows for contacts from side chain to main chain atoms where the arrow head marks the residue contributing the main chain group. Yellow lines indicate van der Waals or hydrophobic interactions. Of 8 water molecules found so far in the interface region only 4 are included for simplicity. Residues belonging to switch I or switch II are denoted in elliptical or hexagonal boxes, respectively. Interaction between Lysδδ and T791 is indicated by a dashed arrow, since the electron density in this region is presently not of sufficient quality to unambiguously define the contact. Fig. 3 Sequence alignment of portions of Ras (A) and of the catalytic (red) domain of RasGAPs (B) from the indicated organisms relevant for complex formation, together with secondary structure assignment for GAP-334 according to the program DSSP (55). Residues participating in catalysis are marked as closed circles, selected residues involved in Ras-RasGAP interaction (+) are indicated, also residues that have been found mutated in neurofibromin from patients with type I neurofibromatosis and solid tumors (closed triangles). Fig. 4 A. Hydrophobic and polar interaction of the effector region of Ras with GAP-334. Residues 32-39 of Ras with side chain as ball and stick models are seen to contact the hydrophobic and negatively charged surface of GAP-334 in the vicinity of the 'catalytic track', as calculated by GRASP (56). For orientation the same area of the complex is shown as ribbon model (colors corresponding to Fig. 2A, water molecules in light orange). B. Interaction of helix α7 c and variable loop including the 'KE'-motif of GAP-334 with part of the effector region in Ras. C. Close approach of the finger loop of GAP to loops L1 (P-loop) and L4 of Ras. In position 12 of Ras Ala has been introduced (white side chain) into the model to show that even small substitutions would be within van der Waals distance (gray sphere) to Gln61 (NH 2 ) (blue sphere) and Arg789 (CO, red sphere), whereas Gly13 is more remote from GAP. Fig. 5 Structural (A) and schematic (B) view of the active site, with the important elements of catalysis. C. Superposition of the active site of G iα1 «GDP«AIF 4 " (9) (green) and the Ras«GDP«AIF 3 «GAP-334 (orange/red) complexes (aligned via the G domains). Note that Arg178 of G iα1 , derived from the same molecule and Arg789, supplied in trans by GAP-334, are basically in the same position, as are Gln61 and AIF 3 from Ras and Gln204 and AIF 4 " from G iα1 , respectively. Fig. 6 Chemical structure of the GTP-phosphonoamidate derivatives. DABP and MABP, di- and monoaminobenzophenone; PDA and PMA, Phenylenedi- and - monoamine. Fig. 7 HPLC-analysis of the hydrolyis reaction of wildtype and Q61A Ras loaded with either GTP or DABP-GTP, respectively, a. Single turnover hydrolysis of 150 μM wildtype (full symbols) or mutant Q61A mutant Ras (open symbols) with 140 μM of either GTP (triangle) or DABP-GTP (circle), in standard buffer at 30°C. Aliquots of the reaction mixture at the indicated time points are analyzed by HPLC as described in Materials and Methods, b. HPLC analysis of time points of DABP- GTP hydrolysis by wild type Ras. Elution times of standards GDP (3.7 min), DABP-GTP (5.7 min) and DABP-Pi (7.3 min) are indicated. Fig. 8 Real time fluorescence kinetic of the GTP, DABP- and PDA-GTP hydrolysis by wild type W32Ras (A) and W32V12Ras mutant (B). Time course of tryptophan fluorescence emission at 350 nm of 1 μM GTP-bound Ras mutants (red) has been followed in standard buffer at 30°C. The hydrolysis of the GTP-analogs is measured by rapid-mixing of 1.1 μM nucleotide free Ras-mutants with 1 μM DABP-GTP (green) or PDA-GTP (blue) using stopped-flow apparatus and tryptophan fluorescence in standard buffer at 30°C. For calculation of the rate constant the data were fitted to a single exponential. Fig. 9 Three-dimensional structure of the active site of Ras(G12P) and Ras(G12V) bound to the GTP analogs DABP-GppNHp. Fig. 10 Kinetic Isotope effect of the GTP hydrolysis reaction. Ras(Y32W) was incubated with either GTP (A) or DABP-GTP (B) in either H 2 O or D 2 O as indicated. The reaction was monitored by the fluorescence increase as shown in Fig. 8. The data were analyzed by single exponentials. Fig. 11 Metal ion effect of the reaction. Ras(Y32W) loaded with either GTP (A) or DABP- GTP (B) was incubated in standard buffer in the presence of 10 mM of either Mg 2+ or Mn 2+ and the hydrolysis reaction analyzed as before. Examples The examples which follow are set forth to aid in understanding the invention but are not intended to, and should not construed to, limit its scope in any way. The examples do not include detailed descriptions for conventional methods employed in the construction of vectors, the insertion of genes encoding polypeptides into such vectors or the introduction of the resulting plasmids into hosts. The examples also do not include detailed description for conventional methods employed for assaying the polypeptides produced by such host vector systems. Such methods are well known to those of ordinary skill in the art and are described in numerous publications. Wildtype and mutant H-Ras proteins were prepared from Eschericchia coli using the ptac-expression system as described (Tucker, J., Sczakiel, G., Feuerstein, J., John, J., Goody, R.S. and Wittinghofer, A. (1986) EMBO J. 5, 1351 ). The nucleotide-free and GTB-bound form of Ras proteins were prepared as described (John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A. and Goody, R.S. (1990) Biochemistry 29, 6058). The phosphonoamidate derivatives of GTP and GppNHp were synthesized as described (Zor, T., Bar-Yaacov, M., Elgavish, S., Shaanan, B. and Selinger Z. (1997) Eur. J. Biochem. 249, 330). Example 1 : Structure determination Crystals were obtained from solutions of Ras«GDP and GAP-334 mixed with aluminum fluoride (12). The structure was determined by the molecular replacement method using the coordinates of GAP-334 (11 ) and Ras (13) as search models. Both rotation and translation functions, calculated with the program X-PLOR (14) showed unique peaks for the GAP-334 and Ras models. The electron density map based on the phases derived from the GAP-model after rigid body refinement showed clear density for the Ras molecule, that was subsequently adjusted manually in the map. Rigid body and powell minimization resulted in an electron density map that showed a strong peak for the bound nucleotide which was incorporated as GDP into the complex model. After rebuilding of residues 46 to 54 and 61 to 66 in Ras and of residues 830 to 839, and 947 to 952 in GAP-334 another round of positional refinement was carried out, yielding an R-factor of 32% (R free =37%). Local errors were corrected in subsequent rounds of alternate model building and positional- and B-factor refinement along with simulated annealing and a bulk solvent correction included (programs 'O' (15) and X-PLOR3.8 (14)). Water molecules were added in F 0 -F c maps using the 'water pick' option of X-PLOR. A maximum of electron density in the position usually occupied by the g-phosphate present in all maps during refinement was interpreted as aluminum fluoride. From the outline in F 0 -F c maps it appeared to be a trigonal AIF 3 moiety rather than a tetragonal AIF 4 " as observed in the structures of activated α-subunits of the heterotrimeric G-proteins G iα1 and G tα (9). A summary of refinement statistics is given in Table 1. Residues 981 to 990 are currently ill defined in the electron density and were modeled stereochemically. The present model comprises residues 718 to 1037 of GAP- 334, 1-166 of H-Ras, GDP, AIF 3 , Mg 2+ , and 35 water molecules. A representative part of the electron density map is shown in Fig. 1. Example 2: Complex model of the GAP-334/Ras A ribbon representation of the complex model is shown in Fig. 2A along with a schematic drawing in Fig. 2B. The overall structures of GAP-334 and Ras are very similar to those of the isolated molecules. In Ras, residues of the L4-region (switch II, s. below) become ordered and the loop connecting b2 with b3 is somewhat shifted in the complex. In GAP-334 the extra domain (GAP ex ) appears to be slightly displaced towards the central domain (GAP C ), and part of the loop connecting α7 c with α8 c has moved to come into contact with Ras. In addition residues 834 to 839 that have been modeled stereochemically in unligated GAP- 334 mostly showed reasonable electron density and were modeled accordingly. The rmsd-values for superposition of corresponding Ca atoms are 0.9 A for GAP- 334 and 0.54 A for Ras using free GAP-334 (11 ) and Ras » GppNHp (13) for the comparison. The orientation of the two proteins in the complex structure is similar to that of our earlier docking approach of the Ras-RasGAP interaction (Fig. 4 in ref. 11 ) which was based on a number of biochemical and genetic data. Activated Ras binds into the shallow groove of the central-catalytic domain GAP C of GAP- 334, with the tip of its triangular shape most deeply penetrating it. 3145 A 2 of solvent-accessible surface area become buried in the interface due to complex formation which is much larger when compared with other protein-protein interactions (16). Consistent with numerous mutational studies, residues of the phosphate-binding (P-) loop, switch regions I, II and possibly α3 on Ras (Fig. 3A, 2B) are involved in the protein-protein interaction (17-19). The switch regions of Ras have been defined as the regions which change their structure upon transition from the GTP- to the GDP-bound state (20). On the GAP side, the catalytic track consisting of helices α6 c and α7 c , loops L1 c and L6 C are involved (Fig. 3B, 2B). We call L1 c the 'finger loop' because it points into the active site and L6 C the 'variable loop' because with the present alignment it is not strictly conserved (see below). In contrast to the complex of the Ras related Rap1A»GppNHp with RafRBD (an effector of Ras) which shows many strong polar hydrogen bonds and salt bridges between the two proteins (21 ), the interface of the Ras«RasGAP complex consists of weak van der Waals and a number of polar interactions involving also several water molecules. As the affinity between GAP-334 and Ras«GTP (5 μM) decreases strongly with increasing salt concentration, the contribution of polar interactions appears to be predominant for the affinity of GAP-334 for Ras. The catalytic activity appears to be also dependent on non-polar interactions, since it increases with increasing salt concentration, at least in the case of neurofibromin and most likely also for GAP- 334 (22). The structure suggests that the extra domain of GAP-334, GAP ex is dispensable for Ras-RasGAP interaction as seen in the current model, consistent with studies on neurofibromin, which showed a 230 residue fragment corresponding to the catalytic domain of GAP-334 (red in Fig. 2A) to be sufficient for GTPase activation (23). Accordingly, we will focus on the catalytic domain. Contact area 1 : determinants of binding in Switch I Switch I comprises residues 30-38 and overlaps with the Ras effector region (24). It interacts predominantly with the central helices α6 c and α7 c , which we have called the 'catalytic track', and with the 'variable loop'. A hydrophobic patch is formed by the invariant Leu902 and Leu910 on α6 c and by Pro34, Ile36 and Tyr32 (Fig. 2B, 4A). The phenyl ring of Tyr32 points into a small cavity between 6 c and the finger loop and is situated close to the aliphatic portion of Arg894. Like most other residues from the effector region Tyr32 adopts a conformation close to that observed in crystals of isolated Ras*GppNHp (13) but different from that observed with RaplA in the complex with Raf(RBD) (21 ). The whole network of hydrophobic interactions in which also Tyr64 is involved (see below) is presumably the target of inhibitory lipids such as phosphatidic acid and arachidonic acid (25, 26) the latter of which has been shown to inhibit the Ras- RasGAP interaction in a partly competitive mode with respect to Ras*GTP (27). The hydrophobic patch might also explain the finding that the oncogenic mutation Q61→L in Ras, although unable to hydrolyze GTP efficiently, has a higher affinity to either GAP-334 or neurofibromin (26, 28-30) and that apparently small perturbations such as the mutation L9O2— l have drastic consequences for catalysis (29, 30). Apart from the hydrophobic amino acids mentioned above, switch I contains 5 acidic residues, Asp30, Glu31 , Asp33, Glu37, Asp38, creating a negatively charged surface patch which has been shown to be used for the interaction with effectors (21 ) and is also involved in the interaction with GAP-334 (Fig. 2B, 4A, B). Lys949, located on the tip of a conspicuously exposed part of loop L6 C , points into the highly negatively charged surface patch. While the side chains of Asp30, Glu31 , Glu37 are not directly involved in the interaction, Asp33 and Asp38 are oriented towards Lys949, with only Asp33 making a direct and Asp38/Thr35 water-mediated polar contacts. From mutational studies one would have expected more direct interactions with GAP-334, although the magnitude of the effects of the mutations on either the affinity and/or k rat are controversial (17, 26), most likely due to the chosen experimental conditions. Since the affinity of GAP-334 for Ras is rather low (K D = 5 μM), slight perturbations of the interaction are scored as completely negative. If the interaction is investigated with neurofibromin with its higher affinity for Ras, mutations of Asp38 result in a tenfold decrease in affinity (26) without affecting k^ under saturating conditions (31 ). Diverging from our earlier assumption that the invariant residues Lys935, Gln938, Asn942, located one helical turn apart on helix α7 c , would form direct contacts with Ras, we find only Asn942 to do so. It forms a hydrogen bond with Asp33 and its own orientation is stabilized by the carbonamide group of Gln938 (Fig. 2B, 4B). Lys935 forms an intramolecular salt bridge with Glu950 in loop L6 C , whose carboxylate group is also contacting the main chain amide group of S39 (Fig. 4B). Lys1423 in neurofibromin, (Lys935 in p120GAP), has been found mutated to Glu or Gin in neurofibromas and solid tumors (32), and it has been argued that this residue is important for catalysis, since the GAP activity was dramatically reduced, with conflicting results on its affinity for Ras (32, 33). Assuming a similar role of the corresponding residues in p120GAP and considering the structural finding of the intramolecular salt bridge between Lys935 and Glu950, it is unlikely that Lys935 is directly involved in catalysis or binding. One would postulate instead that most of its mutations destabilize the protein, consistent with the observation that the mutation K1423-»M in neurofibromin renders the protein thermosensitive (34). Two consecutive residues in loop L6 C , Lys949 and Glu950 appear to be of crucial importance for complex formation with Ras. In the model they form a bifurcated clamp with helix α7 c and the effector loop (Fig. 4B). They have so far not been recognized as being important and in former sequence alignments the 'KE'-motif was only moderately conserved. Considering its crucial involvement in the interface of the complex we are proposing a structure-based alignment (Fig. 3B) where the 'KE'-motif is situated in a loop of variable length (hence 'variable loop') with almost complete conservation of this motif and other residues. In this context it is worth mentioning that point mutations in this loop have consistently been identified as mutants with increased affinity for Ras (33, 35) or as intragenic suppressors of Lys1423 mutations in genetic screens of neurofibromin. In one of these, Phe1434 close to the 'KE' motif has been mutated to Leu, while in the other Lys1436 of the 'KE'-motif itself was replaced by Arg (35). Judging from the way Lys949 (corresponding to Ly1436 in neurofibromin) points into the negatively charged patch one might well imagine an arginine being able to make more direct interactions than lysine. Contact area 2: stabilization of Switch II Switch II comprises the loop L4 together with helix α2 in Ras. In contrast to the structures of isolated Ras where L4 was found to be highly mobile (13, 20, 36), this region is very well defined in the complex with GAP-334, due to a number of contacts. Residues 61 to 63 are now arranged in a short 3 10 helix preceding α2. Interestingly the L4/α2 region adopts a similar conformation as found for elongation factor Tu in its triphosphate form (37). Tyr64 participates in the formation of the hydrophobic interface and in addition forms a polar contact with the main chain carbonyl group of Leu902 from the FLR signature motif, consistent with the observation that it can be mutated to Phe but not to Giu without affecting Ras-RasGAP interaction (17). Interactions between Glu62 and Arg749 or between Glu63 and Arg903 appear to contribute to the stabilization of switch II. However, this contribution is obviously not critical as Glu62/Glu63 can be replaced by Ala without significant consequences for successful Ras-RasGAP interaction. In contrast, Gln61 is extremely critical since it is essential for GTP hydrolysis: its mutation to any other amino acid (except Glu) blocks Ras-mediated GTP hydrolysis (18, 19, 26, 38, 39) and leads to tumor formation. In the structure of G a proteins in the GDP*AIF 4 " conformation the Gln61 homologue has been found to stabilize the transition state of the GTPase reaction (9) and a similar role has been postulated for Gln61 of Ras (40). In the complex with GAP-334, it points towards the phosphate chain of the nucleotide and is stabilized in its orientation by a hydrogen bond with the main chain carbonyl group of the invariant Arg789. Looking at the B-factor distribution (not shown) we find a strong minimum for the L4-region, suggesting that binding of GAP to Ras results in stabilization of switch Contact area 3: structural basis for oncoαenicitv of Glv12 mutants Gly12 of the P-loop is critical for oncogenic activation of Ras since any mutation of this residue (except G12→P) activates the oncogenic potential of Ras (41 ). Ras(G12P), although insensitive to GAP, is not transforming presumably because its intrinsic rate of GTP hydrolysis is slightly increased (6, 22, 42). No consistent explanation has so far been put forward to explain the defect in both the intrinsic and GAP-accelerated GTP hydrolysis of all these mutants, although some cause more obvious rearrangement of the active site (28, 43). In the structure of the complex Gly12 can be seen to contact the loop L1 c region of GAP-334. The main chains are approaching each other, the closest encounter being a van der Waals contact between the C a atom of Gly12 and the main chain CO of Arg789 (Fig. 4C). This mode of interaction imposes severe constraints on the space that may be filled by amino acids in positions 12 in Ras and provides an obvious explanation for the block in GAP-accelerated GTP hydrolysis. Although the interaction with GAP has not been investigated for all Gly12 mutants it is clear from the structure that even replacement with alanine would be highly unfavorable, leading to steric clashes with the main chain of Arg789 and with the side chain of Gln61 , which is consistent with experimental findings (31 ). Gly12 mutants of Ras have been shown to bind to GAP with similar affinity as compared to wildtype (39) without subsequent GTP hydrolysis. This suggests that Arg789 is not close to Gly12 in the Ras»GTP»GAP ground state as compared to the transition state mimic observed here or that it is pushed aside in such unproductive complexes. The structure also shows that the steric requirements are less strict for position 13 and maybe therefore Gly13 is less likely a target for oncogenic transformation in Ras. It has indeed been observed, that some side chains in position 13 do and others do not interfere with Ras-RasGAP interaction, consistent with their transforming potential (39). The active site GDP is bound in a mode similar to that observed in isolated Ras*GDP or Ras«GppNHp complexes with Mg 2+ present in the expected position (13, 20). The 3'-OH group of the ribose which is exposed in isolated Ras is hydrogen bonded to the hydroxyl group of Thr785 in the 'finger loop'. Aluminum fluoride, modeled as AIF 3 , appears to be in a position/orientation where the fluoride ligands contact Mg 2+ ,Thr35 (NH), Lys16 (NH 3 + ), Gln61 (NH2), and most importantly the positively charged guanidinium group of Arg789 (Fig. 5A, B). This residue from the 'finger loop', which is somewhat flexible in unligated GAP-334, points into the active site and is well defined in the structure of the complex. It is additonally stabilized by interaction with Thr 785 (CO) (Fig. 5A) and communicates with Lys949 of the 'KE'- motif via two water molecules one of which interacts with the main chain carbonyl group of Glu31. Most importantly, the main chain CO of Arg789 forms a hydrogen bond with the amide group of the Gln61 side chain. As in uncomplexed GAP-334, the conformation of the 'finger loop' appears to be stabilized by the interaction of Arg903 with the main chain carbonyl oxygens of Phe788, Arg789 and Ala790. In the structures of activated G lα and G tα and of myosin, the putative transition state mimic was identified as GDP/ADP«AIF 4 " , respectively (9, 44). This discrepancy to our Ras*RasGAP structure is not understood at the present moment, but is not without precedent, since in the transition state mimic of the NDP kinase structure, aluminum fluoride was also identified as AIF 3 (45) and this reflects the ability of aluminum fluoride to form trigonal-bipyramidal complexes (46). A water molecule could be modeled in a position apical to the AIF 3 plane and within hydrogen bonding distance to the carbonyl group of the Gln61 side chain. This water molecule basically matches the site of Wat175 found in the GTP- conformation of Ras (13) and presumably represents the attacking nucleophile in the GTPase reaction, or the hydroxyl group derived from it. Although the location of atoms in the g-phosphate site was identified from electron density maps obtained without including an AIF 3 model during refinement, further crystallographic analysis at increased resolution will be necessary to establish the exact geometry of the interaction pattern in this region. The structural view of the Ras»GAP-334 complex along with mutational analyses (31 ) identifies Arg789 as the proposed 'arginine finger' which stabilizes charge on the g-phosphate and is most crucial for catalysis. In contrast, Arg903 is not critical for the reaction itself but rather appears to stabilize the position of the 'arginine finger'. This is surprising considering the fact that Arg903, which is in the most highly conserved FLR fingerprint regions of RasGAPs was found to be important for the reaction, depending on the mutation investigated. Its conservative replacement by Lys results in a comparatively slight reduction of GAP activity with only a minor effect on binding to it (29, 30, 31 ). This rather suggests an indirect role of Arg903 in catalysis by stabilizing an essential amino acid, an interpretation which is in excellent agreement with the 3D-structure of GAP-334 (11 ) and especially of its complex with Ras presented in this work. The mechanism During phosphoryl transfer reactions there is a development of negative charge: it is on the g-phosphate if the reaction is associative and goes through a pentavalent phosphorous intermediate. It is on the leaving group, which in the hydrolysis of GTP is the b,g-bridging oxygen atom, if the mechanism is dissociative and shows a metaphosphate-like transition state (7). Proteins such as GAP could potentially stabilize the transition state by compensating these developing charges and thus catalyze the chemical reaction. It has been demonstrated that the intrinsic GTPase reaction of Ras proceeds via substrate- assisted catalysis where the substrate GTP itself acts as the general (or specific) base (6). Although this is favorable only in the case of an associative mechanism it is not universally accepted that the Ras-GTPase proceeds by such a mechanism, mostly because in solution hydrolysis of phosphoanhydrides (such as ATP/GTP) or phosphate monoesters proceeds via a dissociative mechanism and from the finding that the main chain amide of Gly13 in Ras is presumed to be hydrogen-bonded to the b,g-bridging oxygen (7). A number of suggestions have been put forward to explain the role of GAP in the GTPase of Ras. It has originally been questioned whether the actual chemical step of the phosphoanhydride cleavage is catalyzed by GAP or whether Ras is in principle a fast GTPase enzyme and GAP catalyzes a rate-limiting isomerization reaction without actual participation in the reaction (8). Given the recent findings that stoichiometric amounts of GAP are necessary to stabilize the transition state (10) it could be argued that the principal role of GAP would be to contact the b,g- bridging oxygen or, if the mechanism is associative, one of the g-phosphate oxygens. Now, with the structure of the Ras*RasGAP complex at hand we can extend our view of the catalysis to three dimensions and attempt to define the mechanism as well as the transition state of the phosphoryl transfer reaction. The structural model suggests a polar interaction between the charged guanidinium group of Arg789 and a fluoride ligand of aluminum in the g- phosphate binding site of Ras (Fig. 5A, B). Since Al-F and P-O bond lengths are very similar (46), and since an AIF 3 trigonal base mimics the trigonal base of the transferred phosphoryl group, we can assume that the observed geometry in the active site is a close mimic of the transition state (or intermediate) of the GTPase reaction. We can identify one axial ligand of the pentavalent aluminum as the oxygen of the GDP leaving group, the second being derived from the attacking nucleophilic water. Based on the arguments put forward earlier (7) that residues which neutralize charges on the g-phosphate would act anticatalytically, the arrangement of active site residues presently seen would support a mostly associative mechanism in the GTPase reaction with a pentavalent bipyramidal transition (intermediate) state. Based on the structure it is likely that the interaction of arginine with phosphate residues in the active site contributes significantly to the catalysis of the GTPase reaction. The actual quantitative evaluation of this contribution has been tested by mutational analysis which shows it to be the major factor of the 10 5 fold stimulation of the Ras GTPase by GAPs (31 ). Viewing the complex structure it is unlikely that other residues of GAP are directly involved in the catalytic activity. Apart from Arg789 no additional residue from GAP-334 was identified in the active site region, i.e. one that could function as a general base and thus increase the nucleophilicity of the attacking water. However, another major factor for the acceleration of the Ras-GTPase by GAP appears to arise from the stabilization of the switch II region. It has been shown both by the crystallographic analysis of Ras in the di- and triphosphate form as well as by nuclear magnetic resonance spectroscopy that the region around loop L4 and a2 is very mobile with respect to the rest of the molecule (13, 20, 36). Loop L4 contains residue Gln61 which is invariant in heterotrimeric G a as well as in Ras-like GTP-binding proteins except Rap. The block of GTP hydrolysis in Gln61 mutants, even in the presence of GAPs underscores the importance of this residue for catalysis. This has been supported by experiments with G a proteins which showed that the mutation of the corresponding Gin results in oncogenic activation (47). The structures of G tα and G iα complexed with AIF 4 ~ have shown that Gin stabilizes the transition state of the GTPase reaction by making hydrogen bonds to both the fluoride of AIF 4 " and to the axial ligand, i.e. the nucleophile, of the pentavalent transition state (9). In the Ras«GAP-334 structure, Gln61 also contacts both one of the fluoride ions and the axial ligand derived from the nucleophilic water molecule. This supports an earlier notion that Gln61 stabilizes the transition of the Ras GTPase reaction (40) and that GAP stabilizes the conformation of the region of Ras containing Gln61 (6). The most remarkable feature of the active site structure in relation to the oncogenic potential of Ras is that both Gly12 and Gln61 are in van der Waals distance to each other in the model of the transition state mimic. Fig. 4C shows that the replacement of Gly12 with a methyl group to make it Ala, let alone by larger side chains would sterically interfere with the position of Gln61 required for catalysis, and with the main chain carbonyl group of Arg789. This would at last give a unifying concept how mutations in either of the two positions activate the oncogenic potential of Ras by blockage of the GAP-catalyzed reaction GTPase. If we assume that the intrinsic GTPase reaction has a similar transition state geometry with only Arg789 missing and Gln61 being very flexible, the structure would also give an explanation why most oncogenic mutations show a decrease in the intrinsic GTPase reaction rate as well. Example 3: Comparison of Ras G a proteins Compared to Ras and other small GTP-binding proteins, the a subunits of heterotimeric G proteins contain an extra helical domain as an insertion into the G-domain. It has originally been postulated (1 ) and proven later that this extra domain is responsible for the 100-fold higher GTPase rate of G a proteins as compared to the Ras-like proteins (48). The helical domain contains an invariant arginine at the C-terminus which is critical for the GTPase reaction since its ADP- ribosylation by cholera toxin reduces the GTPase of G sa and its mutation in G sα or G lα renders the proteins oncogenic (47, 49). The structure of the G a » GDP » AIF 4 " complex of both transducin and G lα1 has shown that this arginine contacts the fluorides of AIF 4 " as well as the b,g-bridging oxygen and is thus involved in stabilization of the transition state (9). When we superimpose the G domains of the Ras«GAP-334 structure and of G lα1 , we find the helical domain of G lα to cover a different region of the G domain with respect to the catalytic domain of GAP. We also see that the transition state mimics for the two GTPases, the intrinsic of G lα1 and the GAP-catalyzed reaction by Ras, look very similar (Fig. 5C). Thus, in both structures, the guanindinium groups of the respective arginines are in a similar position, although they point into the active site from different directions and have different positions for their main chain atoms. The significant difference is that in G a the 'arginine finger', located in the linker connecting the helical and the G domain, is supplied in cis i.e. from the same molecule, while in the Ras- RasGAP system it is supplied in trans. The superimposition of the two active sites also brings the corresponding glutamine residues into close proximity, confirming a similar role for these conserved residues in the GTPase reaction. The rate of the GTPase reaction of G a subunits is 2-5 min "1 and thus 100 fold higher than the intrinsic rate of Ras (0.03 min "1 ), but GAP accelerates the GTPase reaction of Ras 10 5 -fold such that it is now 500-1000 times faster than that of G a proteins. Comparing the two active sites (Fig. 5C) it is not obvious why the GAP catalyzed reaction is much faster than that of G a proteins, considering that the same two most relevant residues Gin and Arg are found in similar positions. Recently GAPs for G a proteins, called RGS (for Regulator of G protein Signaling) have been described which increase the GTPase reaction rate and which have no sequence homology to RasGAPs or other GAPs for Ras-like proteins (50). Although the maximal rate of GTP hydrolysis has not been reported it is estimated to be in the same order of magnitude as the maximally stimulated GTPase on Ras. Some RGS proteins bind with higher affinity to the GDP»AIF 4 " complex of G α proteins than to the GTPγS bound state which indicates that they act by stabilizing the transition state (51 ). The structure of a complex of RGS4 with G iα1 »GDP«AIF 4 " has now been solved by X-ray crystallography (52). It shows that RGS is a purely helical protein which contacts the switch residues of G iα1 . The interaction between G iα1 and RGS is thus similar to the Ras-RasGAP interaction. However, RGS does not appear to contribute catalytic residues to the active site except a single asparagine that could possibly help orient a water molecule for nucleophilic attack. Example 4: Determination of a chemical compound that stimulates the GTPase reaction of oncogenic mutants of Ras A chemical agent was selected that might stimulate the GTPase reaction of oncogenic mutants of Ras and might overcome the steric and catalytic constraints of the GAP-catalysed reaction. To this end a unique GTP analog, 3,4- diaminobenzophenone-phosphonoamidate of GTP (DABP-GTP, Fig. 6) was used, which has recently been shown to rescue the signaling properties of a GTPase-deficient (Q227L) G s α mutant (Zor, T., Bar-Yaacov, M., Elgavish, S., Shaanan, B. and Selinger Z. (1997) Eur. J. Biochem. 249, 330). It was argued that this was due to substrate-assisted catalysis, in which the mutant inactive enzyme lacking a catalytically important amino acid, is activated by a substrate supplying one or more functional groups (Zor, T., Bar-Yaacov, M., Elgavish, S., Shaanan, B. and Selinger Z. (1997) Eur. J. Biochem. 249, 330). In order to study the mechanism of this effect and to explore its possible pharmacological relevance the interaction of the Ras oncoproteins with DABP-GTP and other GTP analogs was investigated . GTP or DABP-GTP were bound to excess nucleotide-free wild type Ras and the single turnover hydrolysis at 30°C was analysed by HPLC. These GTPase reaction rates of Ras-proteins have been determined using different methods, (i) HPLC-experiments have been performed to measure the GTP and DABP-GTP hydrolysis as described (Schweins, T., Geyer, M., Scheffzek, K., Warshel, A., Kalbitzer, H.R., and Wittinghofer, A. (1995) Nature Str. Biol. 2, 36). Aliquots from a 140μM Ras.GTP solution or a mixture of 150μM nucleotide free Ras and 140μM DABP-GTP were taken at different time intervals and the reaction was terminated by freezing in liquid nitrogen. The samples were applied to a RP-18 HPLC column (250x4 mm) and isocratic acetonitril elution (7.5% for GTP analysis, 25% for DABP-GTP analysis) was performed at a flow rate of 1.8 ml/min. The HPLC solution contained 10 mM tetrabutylammonium bromide, 100 mM KPi buffer pH 6.5, and 2 mM NaNβ. GTP hydrolysis by different Ras-proteins have been measured on an LS50B Perkin-Elmer spectrofluorometer using 1 μM of the respective Ras.GTP using the tryptophan fluorescence of Y32W-mutant (excitation 295 nm, emission 350 nm). The measurements for DABP- or PDA- GTP hydrolysis have been performed in a Applied Photophysics SX16MV stopped-flow apparatus by rapidly mixing of 1 μM nucleotide free Ras-proteins and 1 μM of the respective nucleotides, respectively, using the tryptophan fluorescence of Y32W-mutant using an excitation wavelength of 295 nm and a cut-off-filter (320 nm) in front of the emission monochromator. All the reactions were followed at 30°C in 30 mM TrisHCI pH 7.5, 3 mM DTE and 5 mM MgCI 2 (standard buffer), unless indicated otherwise. Exponential fits to the data were done using the program Grafit (Erithacus software). Wild-Type Ras slowly hydrolysed GTP at a rate of 0.027 min "1 , which is comparable to previous measurements (Fig. 7a) (Ahmadian, M. R., Stege, P., Scheffzek, K. and Wittinghofer A. (1997) Nature Structural Biology 4, 686; Schweins, T., Geyer, M., Scheffzek, K., Warshel, A., Kalbitzer, H.R., and Wittinghofer, A. (1995) Nature Str. Biol. 2, 36). Ras-bound DABP-GTP is turned over with a much faster rate of 0.18 min "1 , 6.6 fold faster than that of GTP (Fig. 7a). The HPLC profile of the DABP- GTP hydrolysis reaction shows that the products of the reaction are indeed GDP and DABP-Pj (Fig. 7b), which means that water attacks the γ-phosphate group and that the anhydride bond between the β- and γ-phosphates is broken, as in normal GTP hydrolysis. Gln61 has been shown to be crucial for the GAP- mediated GTP hydrolysis since it stabilises the transition state by a double hydrogen bond involving both the transferred phosphate and the attacking nucleophile (Scheffzek, K. et al. (1997) Science 277, 333; Ahmadian, M. R., Stege, P., Scheffzek, K. and Wittinghofer A. (1997) Nature Structural Biology 4, 686). It is assumed that Gln61 plays a similar role for the transition state of the intrinsic reaction, partly because Gln61 mutants have a strongly reduced intrinsic GTPase. The crystal structure of Ras complexed with the hydrolysis resistant caged-GTP, which is an ester of GTP, is consistent with this suggestion. It revealed that the cage group (Schlichting, I. et al. (1990); Scheidig, A. J. et al. (1995) J. Mol Biol 253, 132), which is very similar in structure and position to the modification on DABP-GTP, significantly restricts the possible conformers of Gin 61 , which is highly mobile both in the crystal (Pai, E.F. et al. Nature (1989) 341 , 209; Ito, Y. et al. (1997) Biochemistry 36, 9109) and in solution. The Q61A mutant hydrolyses GTP very slowly (0.03 h "1 ), its GTPase reaction is not stimulated by GAP and it is expected to be oncogenic (Der, C.J., Finkel, T. and Cooper, G.M. (1986) Cell 44, 167). However, hydrolysis with the GTP analog DABP-GTP is strikingly faster (370-fold), the rate being identical to that of the -1 wild-type (0.18 min ; Fig. 7a). Previous experiments have shown that γ- phosphate esters of GTP are hydrolysis resistant, and it has been argued that this is due to the displacement of the catalytic glutamine by the bulky modification on the substrate (Zor, T., Bar-Yaacov, M., Elgavish, S., Shaanan, B. and Selinger Z. (1997) Eur. J. Biochem. 249, 330). DABP-GTP has a bulky aromatic group linked by an amide bond to the γ-phosphate and yet has an increased GTPase reaction rate. Thus in contrast to the normal GTPase reaction, the DABP-GTPase reaction does apparently not require Gln61. Example 5: Determination of the binding mode of the GTP analogue DABP-GTP It could be argued that the GTP analogue is bound in a different way than normal GTP. To get an idea about its binding mode the binding affinity of the modified nucleotide to Ras was determined. To do this and to monitor the GTPase reaction in real-time, the Y32W mutant of Ras (W32Ras) was employed which has a single tryptophan residue and shows a significant increase in tryptophan fluorescence upon nucleotide binding and hydrolysis of bound GTP (Scheidig, A. J. et al. (1995) J. Mol Biol 253, 132). Since the binding of DABP-GTP induces a fluorescence change also, both the binding reaction and the single-turnover hydrolysis of GTP and DABP-GTP on single, double or triple W32Ras mutants could thus be followed in real time by tryptophan fluorescence. In order to measure the binding of modified nucleotides to W32Ras, DABP- Gpp(NH)p, a double-modified GTP analog was used. Nucleotide binding experiments have been performed as described (Schmidt, G. et al. (1996) Oncogene 12, 87). As with GTP, the β, γ imido-derivative of DABP-GTP proved to be hydrolysis resistant which argues in favor of the assumption that the basic chemical step of bond cleavage is principally unaltered between GTP and DABP- GTP. Whereas the association rate of DABP-Gpp(NH)p to W32Ras, determined by stopped-flow experiments, was found to be identical to that found for Gpp(NH)p, the dissociation of DABP-Gpp(NH)p from W32Ras was 22.5-fold faster than that of Gpp(NH)p (Table 2). Therefore, the calculated K D (k off /k on ) of DABP-Gpp(NH)p is 5.4 nM, which means that the overall affinity of the analogue is 22-fold lower as compared to GppNHp, still high enough to ensure complete saturation of Ras under all experimental conditions and to assume that the basic binding mode is similar to GTP. Using W32Ras, the GTPase reaction rates could be followed by monitoring the increase of fluorescence after binding of the nucleotides. A single exponential fit to the fluorescence increase of W32Ras yielded rate constants of 0.039 min "1 and 0.079 min "1 for GTP and for DABP-GTP, respectively, as shown in Fig. 8a. The difference in hydrolysis rates between the two nucleotides is only 2-fold. However, control experiments with HPLC are in excellent agreement with the fluorimetric data, thus showing that the fluorescence amplitude indeed monitors the GTPase reaction and that the Y32W mutation has only a small effect on the properties of Ras (see below). We thus use W323Ras as quasi-wildtype and introduce a number of additional mutations to monitor their effect on the DABP-GTPase. As shown above, the oncogenic Q61 L mutant (in the Y32W background) shows a 30-fold rate enhancement with DABP-GTP to 0.11 min "1 , 1.6-fold slower than the Q61A mutant measured by HPLC (in the wild type background). Example 6: Assessment of Stimulation of DABP-GTP bound to W 32 Ras by Ras GAP For the reaction of Gα proteins with both GTP and DABP-GTP, the presence of the intrinsic arginine is necessary for an unperturbed hydrolysis rate (Zor, T., Bar- Yaacov, M., Elgavish, S., Shaanan, B. and Selinger Z. (1997) Eur. J. Biochem. 249, 330). In the case of Ras, the catalytic arginine is supplied in trans into the active site in order to stabilise the transition state and thereby increase the catalytic rate (Scheffzek, K. et al. (1997) Science 277, 333; Ahmadian, M. R., Stege, P., Scheffzek, K. and Wittinghofer A. (1997) Nature Structural Biology 4, 686). Therefore, it was tested whether the hydrolysis of the DABP-GTP bound to W32Ras is also stimulated by a specific RasGAP. Whereas, GTP-bound W32Ras could normally be stimulated by RasGAP, this activity is abolished when DABP- GTP is used instead of GTP. These findings argue for and are supported by the structure of the Ras « GDPΑIF 3 'GAP334 complex (Scheffzek, K. et al. (1997) Science 277, 333) that the insensitivity of the Ras.DABP-GTP complex to the GTPase-activating protein probably results from a steric interference of the bulky modification with the catalytic arginine finger of GAP. Example 7: Stimulation of mutant Ras by DABP-GTP Mutations of glycine 12 of Ras are the most common oncogenic mutations in human tumours (Bos, J.L. (1989) Cancer Res. 49, 4682; Seeburg, P. H., Colby, W. W., Capon, D. J., Goedel, D. V. and Levinson, A. D. (1984) Nature 312, 71 ). Therefore, it was of major importance to test whether the inability of these mutants to hydrolyse GTP could be bypassed by DABP-GTP. The intrinsic hydrolysis of GTP of Ras is decreased 12-fold by the mutation G12V (0.0024 min "1 at 37°C) and GAP can not further catalyse the reaction (Thrahy, E.M. and McCormick, F. (1987) Science 238, 542; Gideon, G. et al. (1992) Mol. Cell. Biol. 12, 2050). Most remarkably, this mutant rapidly hydrolysed DABP-GTP. The estimated hydrolysis half life is less than one min at 30°C, too high to be accurately measured by the HPLC method. Therefore, several Gly12 mutations were introduced into W32Ras and the GTPase and the DABP-GTPase reaction was measured as described above. The rate constant of DABP-GTP hydrolysis on V12Ras is dramatically faster than the corresponding GTP hydrolysis which is too slow to be accurately measurable (Fig. 8b, Table 3). In general, the GTPase reactions of all the Gly12-mutants tested (Table 3) are dramatically increased up to 750-fold, using DABP-GTP instead of GTP such that the rate constants of the DABP-GTPase are 5 to 34-fold higher than the GTPase of wild type Ras (0.028 min "1 ) and also higher than the DABP-GTPase of wildtype Ras or W32Ras. This shows that the analogue selectively increases the hydrolysis reaction of oncogenic G12 mutants, and this increase is more pronounced by the more bulky group isopropyl than the methyl side chain, if the Val12 is compared to Ala12. The increase is not however simply an increase in hydrophobicity as a large increase is even observed for the G12D mutation with its acidic carboxylate side chain The rescue of the catalytic properties is not only independent of the presence of Gln61 , but is actually more efficient in the absence of Gln61 as shown by the experiment employing the double mutants V12A61 Ras and V12L61 Ras which show an almost thousandfold rate enhancement as compared to the GTP- hydrolysis (Table 3). The structure of DABP-GTP (Fig. 6) shows two possible functional groups that might be involved in stimulating catalysis when bound to normal and oncogenic Ras. It has been suggested for Gα that the bulky aromatic group on DABP-GTP pushes aside the catalytic glutamine and that the aromatic amino group has a role in the reaction (Zor, T., Bar-Yaacov, M., Elgavish, S., Shaanan, B. and Selinger Z. (1997) Eur. J. Biochem. 249, 330). In support of the crucial role of the amino group, MABP-GTP without the ortho-amino group shows almost no accelerating effect on the GTPase reaction of Ras. To test whether other substituents of DABP-GTP are contributing to catalysis, the hydrolysis rate of PDA-GTP (Fig. 6) was tested. It binds to Ras (Table 2) and stimulates the GTPase of oncogenic mutants but not of wild type W32Ras (Fig. 8; Table 3). The stimulatory effects are somewhat smaller but the degree of susceptibility of various mutants is similar except for the Q61 L and the G12D mutants. On conclusion, it appears that the benzoyl group has some effect on the phosphoryl transfer reaction, but that the most important contribution comes from the aromatic amine group. Example 8: Determination of the three-dimensional structure of Ras bound to DABP-GppNHp In order to get a detailed view of the active site and clues to the possible mechanism of hydrolysis we solved the three-dimensional structure of Ras bound to DABP-GppNHp, the non-hydrolyzable analogue of DABP-GTP. For technical reasons the structure of the G12P and G12V mutants were solved. Protein » nucleotide complexes were crystallized and the structure was solved by molecular replacement using the RasGppNHp model (Pai, E.F. et al. Nature (1989) 341 , 209). The overall structures of the Ras mutants complexed with DABP-GppNH are very similar to those of the corresponding mutants containing GTP or GppNHp. A number of differences in the active site as compared to wildtype Ras (Pai, E.F. et al. Nature (1989) 341 , 209) are likely to arise from the presence of either the mutation or the GTP modification (Fig. 9). The effector region is disordered and residues 30-37 could not be traced reliably within the electron densitiy maps of the mutants. Unlike in other Ras triphosphate structures, the Mg 2+ ion is not coordinated as in all other triphosphate structures. The L4 region could not be built unambiguously but appears to be more flexible in the VaM 2 mutant in G12P. The DABP moiety is involved in hydrophobic interactions with Val12/Pro12 and Gly 13 and also with the DABP from the neighboring molecule (Fig. 9). It is an orientation that brings the o-amino group close to a position where it might contact a water molecule and could thus in principle activate the nucleophile. Candidate water molecules were found in the Pro 12 but not in the Val 12 structure. The close proximity of the DABP moiety to position 12 might explain the different rate of DABP-GTP hydrolysis of different Gly 12 mutants. Following an earlier suggestion (Zor, T., Bar-Yaacov, M., Elgavish, S., Shaanan, B. and Selinger Z. (1997) Eur. J. Biochem. 249, 330) it appeared plausible that the aromatic amino group of DABP-GTP would, at least partially substitute for the loss of function of the carbonamide side chain (Zor, T., Bar-Yaacov, M., Elgavish, S., Shaanan, B. and Selinger Z. (1997) Eur. J. Biochem. 249, 330), which is involved in stabilisation of the transition state, but possibly also in increasing the nucleophilicity of water. The three-dimensional structure suggests that the amino group of DABP-GTP is in a position to act as a general base and thus activate the nucleophilic water. If indeed proton transfer is taking place and this step is rate- limiting it should be possible to measure a kinetic isotope effect of the DABP- GTPase reaction. Fig. 10a shows that the normal GTPase reaction of Ras has the same rate in either water or deuteriumoxide, in line with earlier observation (Schweins.T., Geyer, M., Kalbitzer, H.R., Wittinghofer, A. and Warshel, A. (1996) Biochemistry 35, 14225). The DABP-GTPase reaction of Ras shows a strong isotope effect, the reaction rate being reduced twofold in D 2 0 (Fig. 10b) It is even further reduced for the oncogenic mutant G12V, where a threefold reduction is found. Deuterium isotope effects for proton transfer step from water of up to 3 have been found before (H. Gutfreund, Kinetics of the Life Sciences, Cambridge University Press 1995). These results seem to indicate that the rate-limiting step of the DABP-GTPase reaction is the proton transfer from water to the aromatic amino group. The pKa of an aromatic amine such as aniline is 4.6, well in the range suitable to act as a general base in an aqueous environment. However it should be stressed that the pKa of PDA-GTP, which has lost its para-substituent as compared to DABP-GTP, should have a higher pKa and thus a higher rate of hydrolysis which is not found (Table 3). We can only deduce from this that other parameters are contributing to the observed effects. It was shown before that in the intrinsic GTPase reaction of Ras the pKa of the γ- phosphate itself determines the reaction rate but that the proton transfer is not rate-limiting. Thus, no isotope effect was observed (Schweins, T., Geyer, M., Scheffzek, K., Warshel, A., Kalbitzer, H.R., and Wittinghofer, A. (1995) Nature Str. Biol. 2, 36; Thrahy, E.M. and McCormick, F. (1987) Science 238, 542) and is indeed also found for Y32Ras (Fig. 10). For normal Ras and other GTP-binding proteins it was also shown that the replacement of Mg 2+ by Mn 2+ increases the reaction rate (Schweins.T., Scheffzek.K., Aβheuer, R. and Wittinghofer (1997) J. Mol. Biol. 266, 847). As a further indication of the different mechanism for the hydrolysis of the GTP-analog the DAB-GTPase in the presence of Mn 2+ was measured: No influence on the rate was observed (Fig. 11 ), whereas 'wildtype' shows the expected twofold difference in reaction rate. Table 1 : Summary of crystallographic analysis Data Collection: Resolution limit (A) 2.5 No. of reflections 34,938 No. of unique reflections 15,811 Completeness (%) 89 Molecular Replacement: Search model: GAP-334 (718-1042) H-Ras (1-166) Resolution range (A) 15-4 15-4 Θ 1 f Θ 2 , Θ 3 Rotation* 18.6, 36.8, 256 31.6, 89.8, 250.2 x, y, z Translation** 0.059, 0.0, 0.296 0.36, 0.0, 0.49 Refinement: Resolution (A) 30-2.5 No. of unique reflections 15,548 Rcryst (%) 23.3 Rfree (%) 32.3 R.m.s. bond (A) 0.007 R.m.s. angles (°) 1.27 + R symm = ∑ I ' h i - ' h I / Σ ' h i . where l hi is the scaled intensity of the rth symmetry- related observation of reflection h and l h is the mean value. * Eulerian angles Θ-,, Θ 2> Θ 3 , as defined in X-PLOR. **Fractional coordinates x, y, z as obtained by X-PLOR. § R yst = ∑ h I F oh - F ch I / ∑ h F oh , where F oh and F ch are the observed and calculated structure factor amplitudes for reflection h. $ value of R^ for 10% randomly chosen reflections not included in the refinement. 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Crystals were grown with the hanging drop method using 15-20% PEG 3350 in 100 mM HEPES pH8 with 20 mM ammonium sulfate and 100 mM guanidine hydrochloride as precipitant and seeding protocols for crystal improvement similar to (K. Scheffzek et al., 1997, Proteins: Struct. Funct. Genet. 27, 315-318, 1997). They belong to the monoclinic space group P2τ with unit cell dimensions a=71.9 A, b=41.1 A, c=89 A, α=90°. β=108.4°, γ=90° and contain one Ras»GAP-334, complex in the asymmetric unit. Only few crystals diffracted to resolutions higher than 3 A. A data set from one single crystal (cooled to 4°C) was collected with the rotation method using mirror focused X-rays from a rotating anode (Elliott GX18; 35 kV/50 mA) and a Siemens/Nicolett area detector for data recording. Space group determination, data processing, and scaling were done with the programs XDS (W. Kabsch, J. Appl. Crystallogr. 26, 795-800, 1993) and XSCALE (W. Kabsch), respectively. 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Claims: Claims 1. A compound characterized in that it: (a) binds with a sufficient affinity to the surface of a normal or mutated GTP-binding protein to allow a contact with the active site thereof, wherein the mutation results in an impaired intrinsic GTPase activity; (b) reduces the activation energy of the GTPase enzymatic activity of said GTP-binding protein by introduction of at least one functional group into said active site and/or activates the nucleophilic water thereby increasing the GTPase reaction rate; and (c) is of synthetic or semisynthetic origin. 2. The compound of claim 1 wherein said mutated GTP-binding protein is a mutated Ras-related protein. 3. The compound of claim 2 wherein said mutated Ras-related protein is a mutated Ras-protein. 4. The compound of claim 3, wherein said Ras protein is K-Ras, H-Ras or N-Ras. 5. The compound of claim 3 or 4 wherein said mutated Ras protein bears a mutation in amino acid position 12. 6. The compound of any one of claims 3 to 5 wherein said mutated Ras protein bears a mutation in amino acid position 61. 7. The compound of any one of claims 1 to 6, wherein said functional group is an arginine finger or a derivative thereof or a compound that mimics the activity of an arginine finger. 8. The compound of any one of claims 1 to 7 wherein said functional group is or comprises an aromatic amine. 9. The compound of claim 8 wherein said aromatic amine is a 3,4 diamino- benzophenone-phosphonoamidate. 10. The compound of any one of claims 1 to 9 having at least one of the following properties: (a) an aromatic amino group with a proper pK a positioned such that it can accept protons from the nucleophilic water; (b) shielding the water from the bulk water such that its nucleophilicity is increased; and (c) a guanidinium side chain positioned such that it stabilizes the transition state. 11. The compound of any one of claims 1 to 10 which is a compound derived from a peptide by peptidomimetics. 12. The compound of any one of claims 1 to 11 , wherein the increase of the GTPase reaction rate is at least 20-fold as compared to the oncogenic GTP binding protein. 13. A pharmaceutical composition comprising the compound of any one of claims 1 to 12 and a pharmaceutically acceptable carrier and/or diluent. 14. A method of treating a patient suffering from an oncogenic activation of the Ras gene comprising administering an effective amount of the pharmaceutical composition of claim 13. 15. A method of treating a patient suffering from neurofibromatosis type I or an oncogenic activation associated with neurofibromatosis type I comprising administering an effective amount of the pharmaceutical composition of claim 13. 16. The method of claim 14 or 15 wherein said oncogenic activation has resulted in a tumor. 17. The method of claim 16 wherein said tumor is a tumor of neural crest derived tissue, for example, neurofibrosarcoma, glioma, astrocytoma, pheochromocytoma or embryonal rhabdosarcoma or is a pancreatic tumor, leukemia, lung or breast cancer or colon cancer. 18. A method of treating a patient suffering from a tumor caused by malfunction of a G protein comprising administering an effective amount of the pharmaceutical composition of claim 13. 19. Kit comprising the compound of any one of claims 1 to 12 or a peptidic analogue thereof. 20. A method of devising a compound of any one of claims 1 to 12 comprising: (a) screening an at least partially randomized peptide library for peptides that bind with a sufficient activity to the surface of a GTP- binding protein to allow a contact with the active site thereof and reduces the activation and energy of the GTPase enzymatic activity of said GTP-binding protein by introduction of at least one functional group into said active site; and optionally (b) repeating step (a) one or more times. 21. The method of claim 20, further comprising: (c) improving the capacity of said compound obtained in step (a) or (b) to reduce the activation energy of the GTPase enzymatic activity by peptidomimetics, and/or by involving phage display techniques and/or combinatorial library techniques. 22. The method of claim 20 or 21 , wherein said GTP-binding protein used in step (a) and/or (b) is a wildtype GTP-binding protein and said GTP- binding protein in step (b) and/or step (c) is a mutated GTP-binding protein. 23. The method of any one of claims 20 to 22, wherein said at least partially randomized peptide library is presented for screening by phage display. 24. A method of devising a lead compound for the development of a compound of any one of claims 1 to 12, comprising screening an at least partially randomized peptide library for peptides that bind with a sufficient activity to the surface of a GTP-binding protein to allow a contact with the active site thereof and reduce the activation energy of the GTPase enzymatic activity of said GTP-binding protein by introduction of at least one functional group into said active site. 25. The method of any one of claims 20 to 24 wherein said GTP-binding protein is a Ras-related protein and preferably a Ras-protein. 26. A method for selecting for a compound that activates efficient catalyis of GTP on oncogenic Ras having at least one of the following properties: i. an aromatic amino group with a proper pK a positioned such that it can accept protons from the nucleophilic water; ii. shielding the water from the bulk water such that its nucleophilicity is increased; and iii. a guanidinium side chain positioned such that it stabilizes the transition state.

Inventors: AHMADIAN MOHAMMAD REZA, SCHEFFZEK KLAUS, SELINGER ZVI, WITTINGHOFER ALFRED, AHMADIAN, MOHAMMAD, REZA, SCHEFFZEK, KLAUS, SELINGER, ZVI, WITTINGHOFER, ALFRED
Applicants: AHMADIAN MOHAMMAD REZA, MAX PLANCK GESELLSCHAFT, SCHEFFZEK KLAUS, SELINGER ZVI, WITTINGHOFER ALFRED, AHMADIAN, MOHAMMAD, REZA, MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V., SCHEFFZEK, KLAUS, SELINGER, ZVI, WITTINGHOFER, ALFRED
Application year: 1998
Main classification: C07K14
IPC codes: A61K38, C07K14