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.

Structural basis for the high affinity of amino-aromatic SH2 phosphopeptide ligands.

An anthranyl moiety placed at the N terminus of a phosphotyrosine peptide potentiates the inhibitory effect of this small peptide on the binding of the Grb2 SH2 domain to the EGF receptor. Using molecular modeling procedures based on the Lck SH2 domain structure, this observation was rationalized in terms of a suitably favorable pi-pi stacking interaction between the anthranyl moiety and the arginine alphaA2 (ArgalphaA2) residue side-chain of Grb2 SH2. The crystal structure of the Grb2 SH2 domain in complex with the inhibitor 2-Abz-EpYINQ-NH2 (IC50 26 nM) has been solved in two different crystal forms at 2.1 and 1.8 A resolution. This structure confirms the modeling based on the Lck SH2 domain. The ArgalphaA2 residue is conserved in most SH2 domains. Thus, as expected, the anthranyl group also confers high affinity to small peptide ligands of other SH2 domains such as Lck-, PLC-gamma-amino-terminal and p85 amino-terminal SH2 domains as demonstrated by structure affinity relationships (SAR) data. These potent peptides with an amino-terminal surrogate group and the structure of Grb2 SH2 domain in complex with one such peptide represent good starting points for the design and optimization of new inhibitors of many SH2 domains.

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.

Discovery of 3-aminobenzyloxycarbonyl as an N-terminal group conferring high affinity to the minimal phosphopeptide sequence recognized by the Grb2-SH2 domain.

The observation that anthranilic acid as N-terminal group produces a dramatic increase of the binding affinity of the phosphopeptide sequence Glu-pTyr-Ile-Asn for the Grb2-SH2 domain was rationalized by molecular modeling. The model, which invokes a stacking interaction between the N-terminal group and the SH2 domain residue Arg alpha A2, was subsequently used to design the 3-aminobenzyloxycarbonyl N-terminal group. The latter confers high affinity (IC50 = 65 nM in an ELISA assay) to the minimal sequence pTyr-Ile-Asn recognized by the Grb2-SH2 domain.

Structure-based design and synthesis of high affinity tripeptide ligands of the Grb2-SH2 domain.

The X-ray structure of the Grb2-SH2 domain in complex with a specific phosphopeptide ligand has revealed the existence of an extended hydrophobic area adjacent to the primary binding site of the ligand on the SH2 domain. This has been exploited to design hydrophobic C-terminal groups that improve the binding affinity of the minimal sequence pTyr-Ile-Asn recognized by the Grb2-SH2 domain. The most significant increase in affinity (25-fold compared to that of the reference peptide having a nonsubstituted carboxamide C-terminus) was obtained with a 3-naphthalen-1-yl-propyl group which was predicted to have the largest contact area with the SH2 domain hydrophobic region. This modification combined with replacement of the minimal sequence isoleucine residue by 1-aminocyclohexane carboxylic acid to stabilize the beta-turn conformation required for recognition by the Grb2-SH2 domain resulted in the high affinity (47 nM in an ELISA assay) and selective phosphopeptide Ac-pTyr-Ac6c-Asn-NH(3-naphthalen-1-yl-propyl).

DNA binding properties of the marine sponge pigment fascaplysin.

Association of fascaplysin with double-stranded calf thymus DNA was investigated by means of isothermal titration calorimetry, absorption spectroscopy, and circular dichroism. The UV spectroscopic data could be well interpreted in terms of a two-site model for the binding of fascaplysin to DNA revealing affinity constants of K1 = 2.5 x 10(6) M(-1) and K2 = 7.5 x 10(4) M(-1) (base pairs of DNA). Based on the typical change observed in the absorption and circular dichroism spectra, intercalation of fascaplysin is regarded as the major binding mode. The calorimetric titration curves showed an exothermic reaction which was exhausted at a 2:1 base pair/drug; ratio. This finding is in agreement with an intercalation model comprising nearest neighbor exclusion. In addition, significantly weaker non-intercalative DNA interactions can be observed at high drug concentration. By comparison of all these data with the binding behavior of known intercalating agents, it is concluded that fascaplysin intercalates into DNA.

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.

The substrate specificity of tocopherol cyclase.

The substrate specificity of the enzyme tocopherol cyclase from the blue-green algae Anabaena variabilis (Cyanobacteria) was investigated with 11 substrate analogues revealing the significance of three major recognition sites: (i) the OH group at C(1) of the hydroquinone, (ii) the (E) configuration of the double bond, and (iii) the length of the lipophilic side chain. Experiments with two affinity matrices suggest that substrates approach the enzyme's active site with the hydrophobic tail.

Novel Cdk inhibitors restore TGF-beta sensitivity in cdk4 overexpressing epithelial cells.

Transforming growth factor-beta (TGF-beta) is a potent mitogen that effects a wide variety of cells by blocking cell growth. TGF-beta acts by interacting with components of cell cycle machinery to cause G1 arrest and in mink lung epithelial cells (Mv1Lu) it does so by inhibiting Cdk4 synthesis. Overexpression of Cdk4 in these cells (B7) renders them resistant to the effects of TGF-beta. Here we report that two novel Cdk inhibitors (pyridopyrimidines) that not only inhibit Cdk4 and Cdk2 in an in vitro kinase assay but also, in the absence of TGF-beta, block growth of Mv1Lu cells in G1 more efficiently than their B7 (overexpressing Cdk4) counterparts. Interestingly, these inhibitors restored sensitivity of B7 cells towards TGF-beta. This may have implications for the treatment of tumors that have lost TGF-beta responsiveness due to deregulated cellular growth in vivo. These Cdk inhibitors could therefore be used in conjunction with TGF-beta to understand the mechanism of growth arrest in normal versus tumour cells.

Aspects of the molecular structure and dynamics of neuropeptide Y.

Human neuropeptide Y (hNPY) and the Q34-->P34 mutant (P34-hNPY) have been characterized by CD spectroscopy. hNPY self-associates in aqueous solution with a dimerization constant in the micromolar range. The self-association correlates with an increase in secondary-structure content which was studied as a function of concentration, temperature and pH. The effects of temperature were measured in water (5-84 degrees C) and in ethanediol/water (2 : 1) (-90 degrees to +90 degrees C). A single-residue mutation, Q34-->P34, affects the pH, thermal and self-association properties of NPY. The CD results are correlated with photochemically induced dynamic nuclear polarization NMR experiments which show that the tyrosines at the interface between two monomer units present limited accessibility to a photoreactive dye. An equilibrium state is described, involving a PP-fold monomer form and a handshake dimer form, that accommodates the physicochemical properties of NPY.

Dual specificity of Src homology 2 domains for phosphotyrosine peptide ligands.

SH2 domains mediate protein-protein interactions and are involved in a wide range of intracellular signaling events. SH2 domains are 100-amino acid stretches of protein that bind to other proteins containing phosphotyrosine residues. A current major research goal is formulation of the structural principles which govern peptide-binding specificity in SH2 domains. Several structures (both X-ray and NMR) of SH2 domains have now been determined. Short peptide fragments on the carboxyl-terminal side of the phosphotyrosine residue carry the sequence specific information for SH2 recognition. The bound peptides are held in an extended conformation. However, for the GRB2 SH2 domain, the peptide adopts a beta-turn as the motif for recognition [Rahuel, J., et al. (1996) Nat. Struct. Biol. 3, 586-589]. Our SAR data and molecular modeling studies suggest that many SH2 domains, such as the SH2 domains of Lck, Src, and p85, can interact with high affinity with short peptide sequences at least in two ways which are sequence-dependent. The peptide forms either an extended chain across the D-strand of SH2 domains with anchors at pY and pY+3 or, as in the case of GRB2 SH2, a beta-turn with anchors at pY and pY+2. Due to a bulky tryptophan in its EF1 loop, GRB2 SH2 cannot bind peptide conformations such as the extended chain and thus has a unique specificity.

Structure-based design of peptidomimetic ligands of the Grb2-SH2 domain.

We have designed and synthesized a (3-aminomethyl-phenyl)-urea scaffold to mimic the X+1-Asn part of the minimal phosphopeptide sequence, Ac-pTyr-X+1-Asn-NH2, recognized by the Grb2-SH2 domain. The resulting compounds show the same degree of affinity as their peptide counterparts for the Grb2-SH2 domain. This is the first example reported to date of ligands of the Grb2-SH2 domain with substantially reduced peptidic character.

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.

Highly potent inhibitors of the Grb2-SH2 domain.

Highly potent inhibitors of the Grb2-SH2 domain have been synthesized. They share the common sequence: Ac-Pmp-Ac6c-Asn-NH-(3-indolyl-propyl). Different substituents at the 3-indolyl-propylamine C-terminal group were explored to further improve the activity. This is the first example of inhibitors of SH2 domains with sub-nanomolar affinity reported to date.

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.