Imatinib: a selective tyrosine kinase inhibitor.

The understanding of the pathophysiology of a large number of cancer types provides a strategy to target cancer cells with minimal effect on normal cells. Protein phosphorylation and dephosphorylation play a pivotal role in intracellular signaling; to regulate signal transduction pathways, there are approximately 700 protein kinases and 100 protein phosphatases encoded within the human genome. In cancer, as well as in other proliferative diseases, unregulated cell proliferation, differentiation and survival frequently results from abnormal protein phosphorylation. Although it is often possible to identify a single kinase that plays a pivotal role in a given disease, the development of drugs based upon protein kinase inhibition has been hampered by unacceptable side effects resulting from a lack of target selectivity. With the growing understanding of the molecular biology of protein tyrosine kinases and the use of structural information, the design of potential drugs directed towards the bind adenosine triphosphate (ATP)-binding site of a single target has become possible. These advances have transferred emphasis away from the identification of potent kinase inhibitors and more towards issues of target selectivity, cellular efficacy, therapeutic effectiveness and tolerability. In this paper, the relationship between molecular biology and drug discovery methods, as utilized for the identification of anticancer drugs, will be illustrated.

Total synthesis and biological evaluation of the nakijiquinones.

The Her-2/Neu receptor tyrosine kinase is vastly overexpressed in about 30% of primary breast, ovary, and gastric carcinomas. The nakijiquinones are the only naturally occurring inhibitors of this important oncogene, and structural analogues of the nakijiquinones may display inhibitory properties toward other receptor tyrosine kinases involved in cell signaling and proliferation. Here, we describe the first enantioselective synthesis of the nakijiquinones. Key elements of the synthesis are (i) the reductive alkylation of a Wieland-Miescher-type enone with a tetramethoxyaryl bromide, (ii) the oxidative conversion of the aryl ring into a p-quinoid system, (iii) the regioselective saponification of one of the two vinylogous esters incorporated therein, and (iv) the selective introduction of different amino acids via nucleophilic conversion of the remaining vinylogous ester into the corresponding vinylogous amide. The correct stereochemistry and substitution patterns are completed by conversion of two keto groups into a methyl group and an endocyclic olefin via olefination/reduction and olefination/isomerization sequences, respectively. This synthesis route also gave access to analogues of nakijiquinone C with inverted configuration at C-2 or with an exocyclic instead of an endocyclic double bond. Investigation of the kinase-inhibiting properties of the synthesized derivatives revealed that the C-2 epimer 30 of nakijiquinone C is a potent and selective inhibitor of the KDR receptor, a receptor tyrosine kinase involved in tumor angiogenesis. Molecular modeling studies based on the crystal structure of KDR and a model of the ATP binding site built from a crystal structure of FGF-R revealed an insight into the structural basis for the difference in activity between the natural product nakijiquinone C and the C-2 epimer 30.

Tyrosine kinase inhibitors: from rational design to clinical trials.

Protein kinases play a crucial role in signal transduction as well as in cellular proliferation, differentiation, and various regulatory mechanisms. The inhibition of growth related kinases, especially tyrosine kinases, might provide new therapies for diseases such as cancer. The progress made in the crystallization of protein kinases has confirmed that the ATP-binding domain of tyrosine kinases is an attractive target for drug design. Three successful examples of drug design at Novartis using a tyrosine kinase as a molecular target are described. PKI166, a pyrrolo[2,3,-d]pyrimidine derivative, is a dual inhibitor of both the EGFR and the ErbB2 kinases. The compound entered clinical trials in 1999, based on its favorable preclinical profile: potent inhibition of EGF-mediated signalling in cells, in vivo antitumor activity in several EGFR overexpressing xenograft tumor models in nude mice, long-lasting inhibition of EGF-stimulated EGFR autophosphorylation in tumor tissue, good oral bioavailability in animals, and no prohibitive in vitro and in vivo toxicity findings. The anilino-phthalazine derivative PTK787/ZK222584 (Phase I, co-developed by Schering AG, Berlin) is a potent and selective inhibitor of both the KDR and Flt-1 kinases with interesting anti-angiogenic and pharmacokinetic properties (orally bioavailable). STI571 (Glivec, Gleevec), a phenylamino-pyrimidine derivative, is a potent inhibitor of the Abl tyrosine kinase, which is present in 95% of patients with chronic myelogenous leukemia (CML). The compound specifically inhibits proliferation of v-Abl and Bcr-Abl expressing cells (including cells from CML patients) and shows anti-tumor activity as a single agent in animal models at well-tolerated doses. Pharmacologically relevant concentrations are achieved in the plasma of animals (oral administration). Promising data from phase I and II clinical trials in CML patients (98% haematological response rate in Phase I) support the fact that the STI571 represents a new treatment modality for CML. In addition, potent inhibition of the PDGFR and c-Kit tyrosine kinases also indicates its possible clinical use in solid tumors.

Coupling of the antennapedia third helix to a potent antagonist of the p53/hdm2 protein-protein interaction.

The use of cell-membrane translocating sequences for intracellular delivery of peptides can be a powerful approach to validate drug discovery targets in cellular settings. To accomplish this, a protocol has been implemented to couple the antennapedia third helix (residues 43-58) to a potent antagonist of the p53/hdm2 protein-protein interaction without affecting its in vitro inhibitory activity.

A new structural class of selective and non-covalent inhibitors of the chymotrypsin-like activity of the 20S proteasome.

We describe the identification and in vitro characterization of a series of 2-aminobenzylstatine derivatives that inhibit non-covalently the chymotrypsin-like activity of the 20S proteasome. Our initial SAR data demonstrate that the 2-aminobenzylstatine core structure can effectively serve as the basis for designing potent, selective and non-covalent inhibitors of the chymotrypsin-like activity of the 20S proteasome.

Modeling of the binding mode of a non-covalent inhibitor of the 20S proteasome. Application to structure-based analogue design.

The 2-aminobenzvlstatine derivative I is a 20S proteasome inhibitor of a novel chemical type identified by high throughput screening. The compound specifically inhibits the chymotrypsin-like catalytic activity of the human proteasome with an IC50 value in the micromolar range. Using the crystal structure of the yeast proteasome, we modeled the structure of the human proteasome in complex with 1. As one of the first applications of the model in our oncology programme targeting the proteasome, we designed an analogue of the inhibitor having enhanced stacking/hydrophobic interactions with the enzyme. One order of magnitude in inhibitory potency was gained.

Identification of cylin-dependent kinase 1 inhibitors of a new chemical type by structure-based design and database searching.

We have selected cyclin-dependent kinase 1 (CDK1), an enzyme participating in the regulation of the cell cycle, as a target in our efforts to discover new antitumor agents. By exploiting available structural information, we designed an ATP-site directed ligand scaffold that allowed us to identify 4-(3-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylamino)-benzenesulfonamide as a new potent inhibitor of CDK1 in a subsequent database search. The synthesis and testing of some analogues confirmed the interest of this class of compounds as novel CDK1 inhibitors.

Convergent synthesis of potent peptide inhibitors of the Grb2-SH2 domain by palladium catalyzed coupling of a terminal alkyne.

A new strategy was developed to prepare in a very efficient and convergent manner C-terminal modified tripeptides with high affinities for the Grb2-SH2 domain. Using Pd(PPh3)2Cl2 as catalyst, selected naphthyl iodides and triflates were coupled to Ac-Pmp(t-Bu)2-Ac6c-Asn-NH(prop-2-ynyl). The resulting alkyne derivatives were hydrogenated and deprotected to afford potent Grb2-SH2 inhibitors.

Selective in vivo and in vitro effects of a small molecule inhibitor of cyclin-dependent kinase 4.

BACKGROUND: Cyclin-dependent kinase 4 (Cdk4) represents a prime target for the treatment of cancer because most human cancers are characterized by overexpression of its activating partner cyclin D1, loss of the natural Cdk4-specific inhibitor p16, or mutation(s) in Cdk4's catalytic subunit. All of these can cause deregulated cell growth, resulting in tumor formation. We sought to identify a small molecule that could inhibit the kinase activity of Cdk4 in vitro and to then ascertain the effects of that inhibitor on cell growth and tumor volume in vivo. METHODS: A triaminopyrimidine derivative, CINK4 (a chemical inhibitor of Cdk4), was identified by screening for compounds that could inhibit Cdk4 enzyme activity in vitro. Kinase assays were performed on diverse human Cdks and on other kinases that were expressed in and purified from insect cells to determine the specificity of CINK4. Cell cycle effects of CINK4 on tumor and normal cells were studied by flow cytometry, and changes in phosphorylation of the retinoblastoma protein (pRb), a substrate of Cdk4, were determined by western blotting. The effect of the inhibitor on tumor growth in vivo was studied by use of tumors established through xenografts of HCT116 colon carcinoma cells in mice. Statistical tests were two-sided. RESULTS: CINK4 specifically inhibited Cdk4/cyclin D1 in vitro. It caused growth arrest in tumor cells and in normal cells and prevented pRb phosphorylation. CINK4 treatment resulted in statistically significantly (P: =.031) smaller mean tumor volumes in a mouse xenograft model. CONCLUSIONS: Like p16, the natural inhibitor of Cdk4, CINK4 inhibits Cdk4 activity in vitro and slows tumor growth in vivo. The specificity of CINK4 for Cdk4 raises the possibility that this small molecule or one with a similar structure could have therapeutic value.

Structure-based design of compounds inhibiting Grb2-SH2 mediated protein-protein interactions in signal transduction pathways.

Receptor protein tyrosine kinases are usually activated upon binding their growth factors, or other suitable ligands, to their extracellular domains. These activated receptors initiate cytoplasmic signalling cascades which, when aberrant, can result in different disease states, such as oncogenic transformation. Many receptor protein tyrosine kinases use Src homology 2 domains (SH2) to couple growth factor activation with intracellular signalling pathways to mediate cell control and other biological events. The characterization of the components involved in these signal transduction pathways has resulted in the identification of new attractive targets for therapeutic intervention. Such is the case for the protein-protein interactions involving the SH2 domain of growth factor receptor bound protein 2 (Grb2). Agents that specifically disrupt Grb2-SH2 binding interactions involved in aberrant signalling could potentially shut down these oncogenic pathways and thus block human malignancies. This paper reviews the structural characteristics of the Grb2-SH2 domain and the approaches which have been used to identify antagonists of the Grb2-SH2 domain. Examples have been selected from our own research to illustrate how the unique structural features of the ligand-bound Grb2-SH2 have been exploited to design potent and selective Grb2-SH2 antagonists.

Structure-based design and synthesis of phosphinate isosteres of phosphotyrosine for incorporation in Grb2-SH2 domain inhibitors. Part 1.

Based on X-ray crystal structure information, mono charged phosphinate isosteres of phosphotyrosine have been designed and incorporated in a short inhibitory peptide sequence of the Grb2-SH2 domain. The resulting compounds, by exploiting additional interactions, inhibit binding to the Grb2-SH2 domain as potently as the corresponding doubly charged (phosphonomethyl)phenylalanine analogue.

Structure-based design and synthesis of phosphinate isosteres of phosphotyrosine for incorporation in Grb2-SH2 domain inhibitors. Part 2.

A series of novel phosphinates, derived from 4-phosphonomethylphenylalanine, are described as isosteres of phosphotyrosine. Benzyl (or alkyl) phosphinomethylphenylalanine derivatives were prepared by alkylation of an amino acid P-H phosphinate.

Inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin, a marine natural product.

Small chemical molecules that interfere with biological proteins could be useful for gaining insight into the complex biochemical processes in mammalian cells. Cdk4 is a key protein whose activity is required not only for emergence of cells from quiescence but also at the G1/S transition in the cell cycle and which is misregulated in 60-70% of human cancers. We set out to identify chemical inhibitors of Cdk4 and discovered that, in vitro, fascaplysin specifically inhibited Cdk4. Molecular modelling based on the crystal structure of Cdk2 suggests that fascaplysin inhibits Cdk4 by binding to the ATP pocket of the kinase. Treatment of tumour (p16(-), pRb(+)) and normal (p16(+), pRb(+)) cell lines with fascaplysin caused G1 arrest and prevented pRb phosphorylation at sites implicated as being specific for Cdk4 kinase. Fascaplysin will therefore prove to be a useful tool in studying the consequence of Cdk4 inhibition, especially in cells containing inactivated p16.

Structure-based design of potent CDK1 inhibitors derived from olomoucine.

Cyclin-dependent kinase 1 (CDK1), an enzyme participating in the regulation of the cell cycle, constitutes a possible target in the search for new antitumor agents. Starting from the purine derivative olomoucine and following a structure-based approach, potent inhibitors of this enzyme were rapidly identified. The molecular modeling aspects of this work are described.

New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis.

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values < 0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

A small synthetic peptide, which inhibits the p53-hdm2 interaction, stimulates the p53 pathway in tumour cell lines.

The hdm2 protein negatively regulates p53 tumour suppressor activity. Upon binding to p53, hdm2 stimulates p53 degradation and inhibits its transcriptional activity. Moreover, the hdm2 protein is overexpressed in various tumours inactivating p53. We report here that an octamer synthetic peptide derived from p53 inhibits the p53-hdm2 interaction in vitro. In cellular assays, this untagged peptide penetrates tumour cells and induces the accumulation of p53. The accumulation of p53 leads to its activation. Two gene products transcriptionally regulated by p53, p21Waf1/Cip1 and hdm2, are induced in the presence of the peptide. When used with tumour cells that overexpress hdm2, the peptide induces the death of these tumour cells by apoptosis. The mode of action of this peptide differs from that of DNA-damaging agents (e.g. cisplatin) in that it does not induce p53 phosphorylation on serine 15. This work validates with a low molecular mass molecule our current knowledge on the regulation of the p53 pathway by the hdm2 protein. It also shows that inhibitors of the p53-hdm2 interaction are very attractive candidates for the activation of the p53 pathway in tumours expressing wild-type p53.

Mapping the X(+1) binding site of the Grb2-SH2 domain with alpha,alpha-disubstituted cyclic alpha-amino acids.

A series of phosphopeptides containing alpha,alpha-disubstituted cyclic alpha-amino acids (Ac(n)c, 3 < or = n < or = 7; n refers to the number of carbons in the ring) at the X(+1) position of Ac-Tyr(PO3H2)-X(+1)-Asn-NH2 has been synthesised and their inhibitory activity as antagonists of the Grb2-SH2 domain has been determined in competitive binding assays. The SAR data obtained have been interpreted by using models constructed from the X-ray structure of the ligand-bound Grb2-SH2 domain. The used of alpha,alpha-disubstituted cyclic alpha-amino acids to map the binding pockets of proteins expands the classical alanine scan concept and takes advantage of the known conformational preferences of these amino acids.

Selective GRB2 SH2 inhibitors as anti-Ras therapy.

Given the key role of Ras in the mitogenic signaling by receptor tyrosine kinases, several targets upstream of Ras may prove to be excellent targets for drugs in the treatment of cancer caused by oncogenic tyrosine kinases. CGP78850 is a potent competitor of Grb2 SH2-phosphopeptide interactions. This inhibitor has been obtained by rational drug design and is specific toward the Grb2 SH2 vs. other SH2 domains and the PTB domain of SHC in vitro. Accordingly, CGP78850 blocks epidermal growth factor receptor (EGFR)-Grb2 and Shc-Grb2 interactions in living cells. It also inhibits the growth of cells transformed by receptor tyrosine kinases, which transmit a proliferative signal through Grb2 to Ras, but not cells transformed by oncogenic Raf or cells that contain activating Ras mutations. Moreover, our results demonstrate that, in cells overexpressing receptor tyrosine kinases, such as the EGFR, Grb2 SH2 inhibitors induce expression of the cell cycle inhibitors p21(Waf1/Cip1/CAP1) and p27(Kip1) and reverse transformation.