Nancy Gerits1 and Ugo Moens2
Faculty of Medicine, Department of Microbiology and Virology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, Norway. Email-addresses : nancy@sigtrans.org1 and ugom@fagmed.uit.no2
Phosphate transferring enzyme inhibitors.
Enzymes are specialized proteins that facilitate biochemical reactions without being affected by them. They exist in all living organisms and consist of several different classes. One class of enzymes are the protein kinases, which transfer a γ-phosphoryl group of ATP donor molecules to acceptor hydroxyl groups of serine, threonine and tyrosine residues in proteins. Many protein kinases are components of signal transduction pathways that regulate cellular processes such as division, differentiation, migration and survival. Their perturbed function often contributes to the development of cancer and other diseases. Therefore, molecules that can inhibit the activity of protein kinases, protein kinase inhibitors (PKI), can be used both in investigating the function of a specific kinase in a particular signalling pathway, as well as in preventing the aberrant action of protein kinases in pathophysiological conditions.
After their synthesis (translation), most proteins go through a maturation process, called post-translational modification that affects their activity. One common post-translational modification of proteins is phosphorylation. Two functional classes of enzymes mediate this reversible process: protein kinases add phosphate groups to hydroxyl groups of serine, threonine and tyrosine in their substrate, while protein phosphatases remove phosphate groups. The phosphate-linking reaction requires the presence of three specific sites within the protein kinase: an ATP-binding site, a domain catalysing the transfer of the phosphate group, and a substrate-binding site that recruits the phosphoaccepting target protein. In order to prevent the activity of a particular kinase, PKI are often directed against one or several of these three sites. The diversity of mechanisms of action of PKI are outlined here and summarized in Figure 1. Examples of PKI that possess promising therapeutic potentials and that are currently being successfully applied in clinical trials or as therapy against diseases (e.g. cancer, inflammation, autoimmune diseases, diabetes, cardiovascular disease and viral infections) are given in section 4.
Besides cytoplasmic protein kinases, membrane receptors can exert protein kinase activity. These so-called receptor tyrosine kinases (RTK) contain a ligand-binding extracellular domain, a transmembrane motif, and an intracellular catalytic domain with specificity for tyrosine residues. Upon ligand binding and subsequent receptor oligomerization, the tyrosine residues of the intracellular domain become phosphorylated by the intrinsic tyrosine kinase activity of the receptor. The phosphotyrosine residues function as docking sites for other proteins that will transmit the signal received by the RTK.
One strategy to interfere with RTK activity is to prevent ligand binding to its cognate receptor. This may be achieved by antagonists that block the accessibility of the receptor for its natural ligand or by antibodies that associate with the ligand or the extracellular part of the receptor, thereby avoiding ligand-receptor interaction. To increase the therapeutic potential of antibodies against RTK, toxic proteins are conjugated to the antibodies. These fusion proteins simultaneously block aberrant signalling from the receptor and destroy the target cells. However, a potential immune response against the toxin complicates the clinical application of such inhibitors. Monoclonal antibodies exist that bind both a specific RTK and an immunologic effector cell. Molecules that efficiently prevent receptor oligomerization, and subsequently abrogate RTK activity and downstream signalling have been designed as well. However, such PKI have not yet entered clinical trials.
The RTK activity phosphorylates tyrosine residues within the intracellular domain of the receptor. These phosphorylated residues function as docking sites for proteins that will convey the signal to downstream signal transduction components. PKI can be developed that bind these phosphorylated docking sites in order to abrogate inappropriate downstream signalling.
Another strategy to inhibit the expression of a specific protein kinase exists in preventing translation of its transcripts. One mode to accomplish this relies on the use of ribozymes, which are modified RNA molecules that can cut other RNA molecules. Ribozymes consist of a central catalytic domain with RNA degrading activity, flanked by RNA sequences that are complementary to the target mRNA. In this way, a catalytic domain with no particular specificity can be made to cut up the mRNA encoding a specific protein kinase. Another way to thwart translation is by degradation of target RNA by RNA interference. Synthetic RNA sequences that are complementary to target protein kinase mRNA will form dsRNA structures with their target. These dsRNA molecules will be recognized and digested by specific RNA degrading enzyme complexes.
Most of the protein kinase inhibitors currently in clinical trials are small molecules that compete for the ATP-binding site. They prevent the phosphate donor ATP to bind to the protein kinase, and hence the target protein will not become phosphorylated and the perturbed signalling can be terminated.
The rationale behind substrate-mimicking molecules lies in their ability to compete with the genuine substrate for binding to the protein kinase. They occupy the binding site for the natural substrate, and therefore terminate the signal transduction event that contributes to the pathogenic state of the cell. Thymidylate synthase is an enzyme that is frequently overexpressed in tumours. The substrate-mimicking compound ThymectacinTM (a phosphoramidate derivative of brivudin) inhibits this enzyme and has successfully entered clinical evaluation against colon cancer. Although not a PKI, this example illustrates the therapeutic potential of substrate-mimicking compounds for PKI. Indeed, substrate-binding site compounds have been synthesized that efficiently ablate protein kinase activity in cell cultures, but none of them have been submitted to clinical trials so far.
Bisubstrate analogue inhibitors form a special group of protein kinase inhibitors that mimic both the phosphate donor (ATP) and the acceptor components (serine-, threonine-, and tyrosine-containing peptides). They can provide enhanced specificity for protein kinases as they block both the ATP- and the substrate-binding site of their target. Bisubstrate analogues that effectively inhibit the insulin receptor tyrosine kinase activity in vitro have been designed, and may thus comprise a new class of therapeutically useful agents.
Another way to restrain protein kinases is by altering their stability. Heat shock proteins, like e.g. heat shock protein 90 (Hsp90), function as molecular chaperones by binding to various cellular proteins, including protein kinases, thereby regulating the folding, stability and function of their substrates. Thus, chaperone-based inhibitors may prevent the associated chaperone to maintain the activated conformation state of the protein kinase and help to quench the oncogenic activity of this kinase in tumour cells. The inhibition of Hsp90 results in the proteasomal degradation of the client protein and in cell death of the targeted cancer cells.
An alternative strategy to inhibit a protein kinase relies on different conformations that active and inactive protein kinase can acquire. Antagonists for a protein kinase can be selected that exclusively bind to the inactive form of the kinase, so as to sequester the molecule in a state that cannot participate in signal transduction.
Antibodies that bind the ligand have been successfully used as inhibitors of RTK. Bevacizumab (Avastin) is a recombinant humanized monoclonal antibody that binds the vascular endothelial growth factor (VEGF), and thereby prevents activation of the VEGF receptor and the assembly of new blood vessels (angiogenesis), which ultimately leads to tumour growth regression. Bevacizumab seems beneficial in several clinical trials as an anti-angiogenic strategy in cancer patients and has been approved by the Food and Drug Administration as first-line treatment for metastatic colorectal cancer (Table 1). Bevacizumab has low toxicity, but patients possessed an increased risk of encountering thromboembolic events. Ranibizumab (Lucentis, another anti-VEGF monoclonal antibody) is used to treat macular degeneration, a medical condition leading to blindness. HerceptinTM (Trastuzumab) is a monoclonal antibody directed against the epidermal growth factor receptor 2 (EGFR2, HER-2, Neu, ErbB-2), which is overexpressed in ~25% of invasive human breast cancer and is associated with an aggressive tumour phenotype and reduced survival rate. Herceptin is now administered to treat metastatic breast cancer and tested in clinical trials with osteosarcoma and endometrium cancer patients. Most patients do not experience side effects, although some cases of temporary dizziness, fever or chill, headache, skin rash, nausea and shortness of breath have been reported. Despite its initial beneficial response, most patients develop resistance within one year. Combined therapy of Herceptin and conventional chemotherapy resulted in a synergistic effect on tumour regression and improved survival rates in patients. Pertuzumabisa is a monoclonal antibody that blocks dimerization of HER-2 with other EGFR receptors and has entered phase III clinical trials with breast cancer and other solid tumour patients. MDX-447, a bispecific antibody that binds EGFR and immunologic effector cells, is studied in phase II studies on patients with squamous cell carcinoma of the head and neck. Currently, a phase III trial with MDX-210 (inhibits EGFR2) in combination with monocyte-derived activated killer cell technology against ovarian cancer is under way. To increase the therapeutic potentials of antibodies against RTK, toxic proteins are fused to the antibodies. For example, anti-HER-2 antibodies linked to the fungal toxin maytansine DM-1 are being tested in preclinical studies. Peptides that occupy the receptor are also a strategy to prevent the natural ligand from binding to its receptor. The platelet-derived growth factor (PDGF) receptor is overexpressed in many carcinomas and some cancer patients have elevated serum levels of PDGF compared to healthy individuals. GFB-111 or its second-generation derivative GFB-204 bind the PDGF receptor and block PDGF-induced receptor autophosphorylation and downstream signalling. The GFB compounds possessed antiangiogenic and anticancer activity against human tumour xenografts in mice and showed no signs of gross toxicity. However, these compounds still await to enter clinical trials. DAB389EGF is a fusion of a specific peptide sequence of EGF and diphtheria toxin. This fusion molecule efficiently binds the EGFR that is overexpressed on tumour cells. DAB389EGF was cytotoxic for tumour cell cultures and caused tumour regression in animal models, but no clinical trials have been reported.
Preventing docking of signalling molecules to the phosphotyrosine motifs in the intracellular domain of the activated RTK may form another mode to interrupt aberrant signalling. CGP78850 can impede the usual SH2-phosphopeptide interactions upon activation of the EGFR, thereby blocking signalling downstream of this receptor. However, CGP78850 has not yet been tested on patients with inappropriate EGFR activity.
The anti-sense oligonucleotide LErafAON against the serine/threonine kinase c-Raf has been tested in phase I clinical trials. The anti-sense oligonucleotides ISIS-5132, which also inhibits c-Raf, and ISIS-3521, which inhibits PKC, went through different phase clinical trials with solid tumour patients. Unfortunately, no objective responses occurred with these PKI. GEM-231, an oligonucleotide targeting the RI? subunit of protein kinase A is currently undergoing phase I/II clinical trials alone or in combination with traditional therapy for the treatment of solid cancers.
Angiozyme is a ribozyme that specifically recognizes the mRNA for FLT-1, one of the most important vascular endothelial growth factor receptors involved in angiogenesis. Angiozyme, which has gone through phase I and II clinical trials, showed biological activity in metastatic breast cancer, although it could not be used as monotherapy. Angiozyme is now being investigated in combination therapy for metastatic colorectal cancer. Herzyme is also a ribozyme in phase I clinical trial for the treatment of HER2-overexpressing breast cancer.
Imatinib (STI-571 or Gleevec) was one of the first PKI developed to treat Philadelphia chromosome positive leukaemia, and gastrointestinal stromal tumours (GIST). This small compound occupies the ATP-binding site and prevents access of donor ATP to the kinase and subsequent phosphorylation of the substrate. Although the inhibitor was well tolerated in clinical trials and showed high bioavailability and mild side effects, resistance to the drug quickly arose. This resistance lies in functional inactivation of the compound, loss of BCR-ABL kinase target, mutations in the target protein kinase. Second generation derivatives that circumvent resistance are being developed and tested.
Erlotinib (TarcevaTM) competes with ATP in the HER1/EGFR ATP-binding pocket. It is used in the clinic in locally advanced or metastatic non-small cell lung cancer after failure of at least one chemotherapy regime.
Destabilization of an abnormally functioning protein kinase by interrupting the association of the kinase with its chaperone may be beneficial in pathogenic conditions. Encouraging in vitro results with the histone deacetylase inhibitor LBH589 and an analogue of geldanamycin (17-allyl-amino-demethoxy geldanamycin or 17-AAG) demonstrated that both compounds disrupt the chaperone association of Hsp90 with its targets proteins BCR-ABL and mutant FLT-3, resulting in ubiquitination and proteasomal degradation of both proteins. Currently, both 17-AAG and LBH589 are in Phase I and II clinical trials as treatment for a variety of solid tumours.
KC706 stabilizes the inactive conformation of the mitogen-activated protein kinase p38?, a protein kinase involved in inflammatory reactions and cardiovascular functions. KC706 therefore holds the potential to treat conditions such as rheumatoid arthritis, psoriasis, inflammatory bowel disease and cardiovascular disease. This compound is currently being tested in phase II clinical trials with patients suffering from rheumatoid arthritis.
|
Table 1: Examples of PKI currently investigated in clinical trials or administered in the clinic. Abbreviation : MAb, monoclonal antibody. |
||||
|
drug |
type |
target |
disease |
clinical status |
|
Bevacizumab (Avastin) |
monoclonal antibody against ligand |
VEGFR |
cancer |
clinic |
|
Herceptin, (Trastuzumab) |
monoclonal antibody against receptor |
EGFR2; ErbB-2/neu/HER-2 |
cancer |
clinic |
|
ISIS-5132
Angiozyme (RPI4610) |
antisense RNA
ribozyme |
c-Raf
Flt-1 |
cancer
cancer |
phase II
phase II |
|
Gleevec (Imatinib, STI571) |
ATP-analogue
|
BCR-ABL
|
cancer
|
clinic
|
|
LBH589, 17-AAG |
chaperone inhibitor |
Hsp90/BCR-ABL |
cancer |
phase I |
|
KC706 |
Inhibitor enzyme conformation |
MAP kinase p38 |
inflammatory diseases |
phase II |
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Receptor tyrosine kinase
cell surface receptor with an extracellular ligand-binding region and an intracellular domain with kinase activity that catalyses autophosphorylation of the receptor at tyrosine residues upon ligand binding.
Protein kinase inhibitors
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