2-Anilino-4-aryl-8H-purine derivatives as inhibitors of PDK1 (original) (raw)
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Journal of Biological Chemistry, 2010
The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K m values were determined as 5.6 M for ATP and 40 nM for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1؋ and 10؋ K m concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1. . The abbreviations used are: MAPK, mitogen-activated protein kinase; PDK1, phosphoinositide-dependent protein kinase-1; uHTS, ultrahigh throughput screening; PIF, PDK1-interacting fragment; HM, hydrophobic motif; TR-FRET, time-resolved fluorescence resonance energy transfer; PH, pleckstrin homology; PRK, protein kinase C-related kinase; DMSO, dimethyl sulfoxide; DTT, dithiothreitol.
Chemistry & Biology, 2012
The PIF-pocket of AGC protein kinases participates in the physiologic mechanism of regulation by acting as a docking site for substrates and as a switch for the transduction of the conformational changes needed for activation or inhibition. We describe the effects of compounds that bind to the PIF-pocket of PDK1. In vitro, PS210 is a potent activator of PDK1, and the crystal structure of the PDK1-ATP-PS210 complex shows that PS210 stimulates the closure of the kinase domain. However, in cells, the prodrug of PS210 (PS423) acts as a substrate-selective inhibitor of PDK1, inhibiting the phosphorylation and activation of S6K, which requires docking to the PIF-pocket, but not affecting PKB/Akt. This work describes a tool to study the dynamics of PDK1 activity and a potential approach for drug discovery.
Journal of Biological Chemistry, 2012
Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.
Acta Crystallographica Section A Foundations and Advances, 2021
Phosphoinositide-dependent protein kinase 1 (PDK1) is a master AGC kinase of the PI3K signalling pathway that phosphorylates at least other 23 AGC kinases, being PKB/Akt the most relevant substrate for growth and cell survival, and therefore a potential drug target for cancer treatment. Over the years, our laboratory used a chemical and structural biology approach to study and characterize in detail the allosteric regulation of the catalytic domain of PDK1. We developed small compounds that bind to a regulatory site we termed the PIF-pocket and activate PDK1, mimicking the mechanism of activation of AGC kinases by phosphorylation. Using an integrative approach between biochemistry, crystallography and molecular dynamics, we showed how PS653, a small compound that binds to the active ATP-Binding site, displaces through a reverse allosteric mechanism the in vitro interaction between the PIF-pocket and PIFtide, which is a peptide derived from the hydrophobic motif of a PDK1 substrate. Thus, we not only demonstrated an allosteric regulation from a regulatory site to the active site, but also showed experimentally the existence of the reverse process [1]. This bidirectional allosteric mechanism of regulation between both pockets can therefore be modulated by small molecules that bind to their specific orthosteric site and either enhance or inhibit interactions at the allosteric site. Taking this into consideration, it is not surprising that while the pharmaceutical industry has been developing compounds that bind at the ATP-binding site of kinases, they unwillingly developed drugs that affect protein-protein interactions [2]. Moreover, we now provide further evidence of the bidirectional system using hydrogen/deuterium exchange (HDX) experiments and present a rather complete model for a kinase that can be modulated bidirectionally with small compounds. This concept of bidirectional allostery in kinases can be exploited to produce drugs that enhance or disrupt the formation of multi-protein complexes. Could this mechanism be already in use physiologically? We found out that adenosine binds at the ATP-binding site and allosterically enhances the interaction between PIFtide and PDK1, which demonstrates that bidirectional allostery is a phenomenum that can also be modulated by metabolites. But interestingly, adenine, AMP, ADP, or ATP do not produce this effect. The findings open the possibility that the physiological regulation of the kinase complexes may be modulated by metabolites and implies that the metabolic state of cells could be linked to cell signalling.
Fragment based discovery of a novel and selective PI3 kinase inhibitor
Bioorganic & Medicinal Chemistry Letters, 2011
We report the use of fragment screening and fragment based drug design to develop a PI3c kinase fragment hit into a lead. Initial fragment hits were discovered by high concentration biochemical screening, followed by a round of virtual screening to identify additional ligand efficient fragments. These were developed into potent and ligand efficient lead compounds using structure guided fragment growing and merging strategies. This led to a potent, selective, and cell permeable PI3c kinase inhibitor with good metabolic stability that was useful as a preclinical tool compound.
Journal of Biomolecular Screening, 2009
The PI3K/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis. 3-Phosphoinositidedependent protein kinase 1 (PDK1) is a Ser/Thr protein kinase, which catalyzes the phosphorylation of a conserved residue in the activation loop of a number of AGC kinases, including proto-oncogenes Akt, p70S6K, and RSK kinases. To find new small-molecule inhibitors of this important regulator kinase, the authors have developed PDK1-specific high-throughput enzymatic assays in time-resolved fluorescence resonance energy transfer (TR-FRET) and AlphaScreen ® formats, monitoring phosphorylation of a biotinylated peptide substrate derived from the activation loop of Akt. Development of homogeneous assays enabled screening of a focused kinase library of ~21,500 compounds in 1536-well TR-FRET format in duplicate. Upon validation of hits in an alternative 384-well AlphaScreen ® assay, several classes of structurally diverse PDK1 inhibitors, including tetracyclics, tricyclics, azaindoles, indazoles, and indenylpyrazoles, were identified, thus confirming the utility and sensitivity of the developed assays. Further testing in PC3 prostate cancer cells confirmed that representatives of the tetracyclic series showed intracellular modulation of the PDK1 activity, as evident from decreased phosphorylation levels of AKT, RSK, and S6-ribosomal protein.