Abstract:
Posttranslational lipidation is an essential modification for membrane-bound proteins being involved in intracellular signaling pathways. Ras proteins, belonging to the Ras (rat-adeno-sarcoma) superfamily as some of such are known to play a decisive role in the signal transduction cascade resulting in cell differentiation and proliferation. Ras is lacking a transmembrane domain and it can only function when bound near the growth factor receptor at the cell membrane. Therefore it requires farnesylation to increase its hydrophobicity and ability to anchor to the plasma membrane. This posttranslational modification is catalyzed by the enzyme farnesyltransferase (FTase) which transfers a farnesyl (C15) residue to the carboxy-terminal rest of the Ras protein. Scientists nowadays assume that the cause of approximately 30% of all human tumors is mutated Ras, mainly being involved in myeloid malignancies and carcinomas of e.g. the colon, breast, pancreas, lung and thyroid. Point mutations in the genes lead to the inability of Ras to regulate itself and Ras as a molecular switch remains constantly activated. A selective blocking of FTase by farnesyltransferase inhibitors (FTIs) is viewed as a promising target for therapeutic cancer treatment: Preventing the prenylation of Ras hinders it from attachment with the plasma membrane and thus interrupts the oncogenic Ras function.
Signaling pathway:
A novel and hopeful starting point in the development of new therapies to treat malign illnesses is the investigation and modification of processes, which control the proliferation and differentiation of cells. A promising target is the Ras signal transduction pathway: The membrane-bound Ras protein acts as molecular switch, allowing to transfer a signal coming from the outside of the cell (e.g. a growth factor) into the nucleus (see Fig. 1). The first step in this cascade is the dimerisation of the monomeric receptor tyrosine kinase by stimulation with an extracellular ligand. Grb2 as the first adaptor protein recognizes a binding site enabling in turn a second adaptor molecule called Sos ("Son of sevenless") to attach. Sos subsequently stimulates the inactive GDP-carrying Ras to exchange GDP for GTP and thus becomes active. Thereafter the active Ras can forward the signal to other downstream effectors, e.g. Raf which is the first protein kinase in the MAP-kinase signaling cascade [1][2][3], which finally results in the transcription of different genes. [4][5][6]
Figure 1: Signaling pathway
Ras and cancer:
Approximately 30% of all human tumors arise from a mutated Ras oncogene [7][8][9], particularly point mutations in the corresponding Ras genes are found in over 90% of human pancreatic carcinomas [10] and 50% of human colon cancers [11]. As a biochemical consequence of those mutations the Ras protein loses its GTPase activity (an intrinsic enzyme, hydrolyzing GTP to GDP) and therefore permanently stays in the GTP-bound active state sending constantly signals into the nucleus. This in turn results in uncontrolled cell division being characteristic for cancer diseases.
Posttranslational modifications of Ras:
The crucial point for normal as well as mutated Ras proteins is the necessity to be bound to the cell membrane to ensure signal transduction. The anchorage to the cell membrane is achieved by a series of posttranslational modifications, in which the enzyme farnesyltransferase (FTase) first transfers the lipophilic farnesyl residue of farnesyl pyrophosphate (FPP) to the mercapto function of a cystein. This amino acid is a component of the so called "CaaX-box" located at the C-terminus of Ras, whereat "C" stands for cystein, "a" for an aliphatic amino acid and "X" is methionine, serine, alanine, or glutamine. [12] The mode of binding in the active site of the FTase was investigated with the help of X-ray crystal structures of crystallized complexes from FTase, FPP and a CaaX mimetic [13][14]: A central zinc ion promotes the deprotonation of the cystein to its thiolate which in turn substitutes the pyrophosphate group of FPP generating a C-S linkage and thus connecting the farnesyl residue to the Ras protein. In the next steps the "aaX" part is cleaved off by a specific endoprotease and the free S-farnesylcystein at the C-terminus is finally methylated with the help of a methyltransferase enabling the Ras protein to bind to the cell membrane. [15]
Farnesyltransferase inhibitors (FTIs)
Different classes and strategies to inhibit the enzyme farnesyltransferase have been designed which are listed below.
Inhibitors based on the CaaX motif try to simulate the C-terminal tetrapeptide and compete with Ras to be farnesylated. Their development was initiated by just varying some amino acids (e.g. 1, IC50 = 25 nM in vitro, [16]), which in vivo suffered from membrane permeability and proteolysis. Combinatorial chemistry and high throughput screening eventually led to highly efficient inhibitors, where the rational concept is hardly recognizable (e.g. 2, IC50 = 0.7 nM, [17]).
Farnesyl pyrophosphate analogues (e.g. 3, IC50 = 75 nM, [18]) are in general less attractive due to the possibility to compete with FPP as a substrate also in other enzymatic processes such as squalene synthase.
Bisubstrate inhibitors are based on the theory that a complex consisting of the enzyme and both substrates (FPP and CaaX-box) is formed prior to the catalysis. As a consequence they combine both strategies mentioned above: They incorporate a farnesyl and a CaaX mimetic (see for example 4, MIC = 0.1 mM, [19]) and display enhanced selectivity and activity. Other inhibitors can be found in the pool of natural products, e.g. pepticinnamin E (5, IC50 = 42 mM, [20][21][22]).
Recent studies deal with metal chelating FTIs: The inhibitor design is based on the combination of a Zn-chelating core with a substituent, recognizing the aromatic residues which are situated around the zinc ion in the active site of the FTase (e.g. 6, IC50 = 1.9 mM, [23]).
Beside the aforementioned classes, FTIs from natural product sources (e.g. 7, IC50 = 5 mM, [24]) and from compound libraries (e.g. 8, IC50 = 40 nM, [25]) are also known.
Figure 2: Few examples of potent FTIs
Conclusion:
Ras as one of the most frequent oncogenes found in human cancers emerged as a valuable target for anti-cancer therapy. One approach to decrease the activity of Ras-proteins is to avert its anchorage to the plasma membrane with the help of farnesyltransferase inhibitors (FTIs). Those substances prevent the enzyme farnesyltransferase (FTase) from adhering a lipophilic farnesyl residue to the C-terminus of Ras, and hence to impede Ras to bind to the cytosolic side of the membrane. Apart from a number of lead structures, showing potent inhibitory activities, have been investigated, also at least three FTIs are currently undergoing clinical evaluation [26][27][28] (Zarnestra® (9) [29][30], Sarasar® (10) [31] and BMS-214662 (2) [32]) which demonstrates in a striking way the potential of this strategy to treat malign illnesses.
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