Selective blockade of cancer cell proliferation and anchorage-independent growth by Plk1 activity–dependent suicidal inhibition of its polo-box domain

Polo-like kinase 1 (Plk1) plays a critical role in proper M-phase progression and cell proliferation. Plk1 is overexpressed in a broad spectrum of human cancers and is considered an attractive anticancer drug target. Although a large number of inhibitors targeting the catalytic domain of Plk1 have been developed, these inhibitors commonly exhibit a substantial level of cross-reactivity with other structurally related kinases, thus narrowing their applicable dose for patient treatment. Plk1 contains a C-terminal polo-box domain (PBD) that is essentially required for interacting with its binding targets. However, largely due to the lack of both specific and membrane-permeable inhibitors, whether PBD serves as an alternative target for the development of anticancer therapeutics has not been rigorously examined. Here, we used an intracellularly expressed 29-mer-long PBIP1-derived peptide (i.e., PBIPtide), which can be converted into a “suicidal” PBD inhibitor via Plk1-dependent self-priming and binding. Using this highly specific and potent system, we showed that Plk1 PBD inhibition alone is sufficient for inducing mitotic arrest and apoptotic cell death in cancer cells but not in normal cells, and that cancer cell–selective killing can occur regardless of the presence or absence of oncogenic RAS mutation. Intriguingly, PBD inhibition also effectively prevented anchorage-independent growth of malignant cancer cells. Thus, targeting PBD represents an appealing strategy for anti-Plk1 inhibitor development. Additionally, PBD inhibition–induced cancer cell–selective killing may not simply stem from activated RAS alone but, rather, from multiple altered biochemical and physiological mechanisms, which may have collectively contributed to Plk1 addiction in cancer cells.     

Genetic and pharmacological inhibition of PDK1 in cancer cells: characterization of a selective allosteric kinase inhibitor

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.     

Selective Targeting of CTNNB1-, KRAS- or MYC-Driven Cell Growth by Combinations of Existing Drugs

The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for β-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in Wnt-pathway-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes.     

NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors.     

Quantitative chemical proteomics reveals new potential drug targets in head and neck cancer

Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. To identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant intercell line differences for 42 kinases (p < 0.05), and loss of function experiments using siRNA in high and low expressing cell lines identified kinases including EGFR, NEK9, LYN, JAK1, WEE1, and EPHA2 involved in cell survival and proliferation. EGFR inhibition by the small molecule inhibitors lapatinib, gefitinib, and erlotinib as well as siRNA led to strong reduction of viability in high but not low expressing lines, confirming EGFR as a drug target in 10-20% of HNSCC cell lines. Similarly, high, but not low EPHA2-expressing cells showed strongly reduced viability concomitant with down-regulation of AKT and ERK signaling following EPHA2 siRNA treatment or EPHA1-Fc ligand exposure, suggesting that EPHA2 is a novel drug target in HNSCC. This notion is underscored by immunohistochemical analyses showing that high EPHA2 expression is detected in a subset of HNSCC tissues and is associated with poor prognosis. Given that the approved pan-SRC family kinase inhibitor dasatinib is also a very potent inhibitor of EPHA2, our findings may lead to new therapeutic options for HNSCC patients. Importantly, the strategy employed in this study is generic and therefore also of more general utility for the identification of novel drug targets and molecular pathway markers in tumors. This may ultimately lead to a more rational approach to individualized cancer diagnosis and therapy.     

Tyrosine kinases. New target of anticancer therapy

Recently, clinical studies of new drugs development to target specific forms of cancer were reported. Herceptin, a monoclonal antibody against the Her2/neu receptor tyrosine kinase, prolonged the survival of women with Her2/neu positive metastatic breast cancer. STI571, a small molecule inhibitor of the BCR/ABL, c-Kit and platelet derived growth factor receptor tyrosine kinase, produced pronounced clinical responses in patients with BCR/ABL positive chronic myeloid leukemia and c-Kit positive gastrointestial stromal tumors. In order to consider the use of the inhibitor of tyrosine kinases activity as anticancer drug, their mechanisms of the oncogenic activation and their impact on tumor transformation should be studied. The treatment with tyrosine kinase inhibitors such as STI571 or herceptin was a spectacular clinical success which stimulated research on the structure and function of both kinases and their inhibitors.     

Identification of direct target engagement biomarkers for kinase-targeted therapeutics

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3'-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1(Ser410) and p-PDK1(Thr513) are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1(Ser241)), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.     

Simultaneous Protein Expression and Modification: An Efficient Approach for Production of Unphosphorylated and Biotinylated Receptor Tyrosine Kinases by Triple Infection in the Baculovirus Expression System

Protein kinases can adopt multiple protein conformations depending on their activation status. Recently, in drug discovery, a paradigm shift has been initiated, moving from inhibition of fully activated, phosphorylated kinases to targeting the inactive, unphosphorylated forms. For identification and characterization of putative inhibitors, also interacting with the latent kinase conformation outside of the kinase domain, highly purified and homogeneous protein preparations of unphosphorylated kinases are essential. The kinetic parameters of nonphosphorylated kinases cannot be assessed easily by standard kinase enzyme assays as a result of their intrinsic autophosphorylation activity. Kinetic binding rate constants of inhibitor-protein interactions can be measured by biophysical means upon protein immobilization on chips. Protein immobilization can be achieved under mild conditions by binding biotinylated proteins to streptavidin-coated chips, exploiting the strong and highly specific streptavidin–biotin interaction. In the work reported here, the cytoplasmic domains of insulin receptor and insulin-like growth factor-1 receptor fused to a biotin ligase recognition sequence were coexpressed individually with the phosphatase YopH and the biotin-protein ligase BirA upon triple infection in insect cells. Tandem affinity purification yielded pure cytoplasmic kinase domains as judged by gel electrophoresis and HPLC. Liquid chromatography-mass spectrometry analysis showed the absence of any protein phosphorylation. Coexpression of BirA led to quantitative and site-specific biotinylation of the kinases, which had no influence on the catalytic activity of the kinases, as demonstrated by the identical phosphorylation pattern upon autoactivation and by enzymatic assay. This coexpression approach should be applicable to other protein kinases as well and should greatly facilitate the production of protein kinases in their phosphorylated and unphosphorylated state suitable for enzymatic and biophysical studies.     

Target validation and biomarker identification in oncology : the example of aurora kinases

The strong link between gene expression of mitotic Aurora kinases and cancer has stimulated a very high interest in developing Aurora kinase inhibitors for cancer therapy. Validation of Aurora kinases as targets, and development of pharmacodynamic biomarkers for inhibitors of Aurora kinases, provides an example of how target validation can help the drug discovery process, and also of how to interpret results depending on the technology used. In this review, we outline the principal tools, concepts, and strategies of target and biomarker validation for Aurora kinases, with emphasis on validation results derived from RNA-interference experiments. These data were essential for the decision to enter the next steps in drug development and for the selection of the appropriate biomarkers for clinical trials.     

Steered Molecular Dynamics Simulations Reveal the Likelier Dissociation Pathway of Imatinib from Its Targeting Kinases c-Kit and Abl

Development of small molecular kinase inhibitors has recently been the central focus in drug discovery. And type II kinase inhibitors that target inactive conformation of kinases have attracted particular attention since their potency and selectivity are thought to be easier to achieve compared with their counterpart type I inhibitors that target active conformation of kinases. Although mechanisms underlying the interactions between type II inhibitors and their targeting kinases have been widely studied, there are still some challenging problems, for example, how type II inhibitors associate with or dissociate from their targeting kinases. In this investigation, steered molecular dynamics simulations have been carried out to explore the possible dissociation pathways of typical type II inhibitor imatinib from its targeting protein kinases c-Kit and Abl. The simulation results indicate that the most favorable pathway for imatinib dissociation corresponds to the ATP-channel rather than the relatively wider allosteric-pocket-channel, which is mainly due to the different van der Waals interaction that the ligand suffers during dissociation. Nevertheless, the direct reason comes from the fact that the residues composing the ATP-channel are more flexible than that forming the allosteric-pocket-channel. The present investigation suggests that a bulky hydrophobic head is unfavorable, but a large polar tail is allowed for a potent type II inhibitor. The information obtained here can be used to direct the discovery of type II kinase inhibitors.     

Protein kinase biochemistry and drug discovery

Protein kinases are fascinating biological catalysts with a rapidly expanding knowledge base, a growing appreciation in cell regulatory control, and an ascendant role in successful therapeutic intervention. To better understand protein kinases, the molecular underpinnings of phosphoryl group transfer, protein phosphorylation, and inhibitor interactions are examined. This analysis begins with a survey of phosphate group and phosphoprotein properties which provide context to the evolutionary selection of phosphorylation as a central mechanism for biological regulation of most cellular processes. Next, the kinetic and catalytic mechanisms of protein kinases are examined with respect to model aqueous systems to define the elements of catalysis. A brief structural biology overview further delves into the molecular basis of catalysis and regulation of catalytic activity. Concomitant with a prominent role in normal physiology, protein kinases have important roles in the disease state. To facilitate effective kinase drug discovery, classic and emerging approaches for characterizing kinase inhibitors are evaluated including biochemical assay design, inhibitor mechanism of action analysis, and proper kinetic treatment of irreversible inhibitors. As the resulting protein kinase inhibitors can modulate intended and unintended targets, profiling methods are discussed which can illuminate a more complete range of an inhibitor's biological activities to enable more meaningful cellular studies and more effective clinical studies. Taken as a whole, a wealth of protein kinase biochemistry knowledge is available, yet it is clear that a substantial extent of our understanding in this field remains to be discovered which should yield many new opportunities for therapeutic intervention.     

Protein-tyrosine kinase and protein-serine/threonine kinase expression in human gastric cancer cell lines

Protein kinases play key roles in cellular functions. They are involved in many cellular functions including; signal transduction, cell cycle regulation, cell division, and cell differentiation. Alterations of protein kinase by gene amplification, mutation or viral factors often induce tumor formation and tumor progression toward malignancy. The identification and cloning of kinase genes can provide a better understanding of the mechanisms of tumorigenesis as well as diagnostic tools for tumor staging. In this study, we have used degenerated polymerase-chain-reaction primers according to the consensus catalytic domain motifs to amplify protein kinase genes (protein-tyrosine kinase, PTK, and protein-serine/threonine kinase, PSK) from human stomach cancer cells. Following amplification, the protein kinase molecules expressed in the gastric cancer cells were cloned into plasmid vectors for cloning and sequencing. Sequence analysis of polymerase-chain-reaction products resulted in the identification of 25 protein kinases, including two novel ones. Expression of several relevant PTK/PSK genes in gastric cancer cells and tissues was further substantiated by RT-PCR using gene-specific primers. The identification of protein kinases expressed or activated in the gastric cancer cells provide the framework to understand the oncogenic process of stomach cancer.     

Development of a cell-selective and intrinsically active multikinase inhibitor bioconjugate

Multikinase inhibitors are potent anticancer drugs that simultaneously intervene in multiple related signaling cascades, thus being capable of blocking salvage pathways that may play a role in the development of drug resistance. Multikinase inhibitors are increasingly evaluated for indications other than cancer, but long-term safety risks dictated by off-organ toxicities of these agents may prevent their safe and effective use. Here, we describe a new approach in which platinum coordination chemistry is applied for the development of a cell-selective multikinase inhibitor bioconjugate. The platinum(II) kinase inhibitor bioconjugate was designed to be active with the linker attached to the inhibitor and displayed improved activity by enhanced cell specificity as well as enhanced intracellular retention, thereby prolonging its pharmacological activity. In addition, the utilized platinum-based linkage technology potentiated the inhibitory activity of the multikinase inhibitor. These features in combination with carrier-mediated uptake in the target cells may revolutionize dosing regimens and safety profiles of (multi)kinase inhibitors.     

Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

Small-molecule protein kinase inhibitors are widely used to elucidate cellular signaling pathways and are promising therapeutic agents. Owing to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments that use these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we used functional assays to profile the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases. Quantitative analysis revealed complex and often unexpected interactions between protein kinases and kinase inhibitors, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can identify multitargeted inhibitors of specific, diverse kinases. The results have implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments involving kinase inhibitors.     

Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2

CK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2α catalytic subunit in the hinge/αD region, the p-loop and the β4β5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/αD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the β regulatory subunits.     

Targeting protein kinases benefits cancer immunotherapy

Small-molecule kinase inhibitors have been well established and successfully developed in the last decades for cancer target therapies. However, intrinsic or acquired drug resistance is becoming the major barrier for their clinical application. With the development of immunotherapies, in particular the discovery of immune checkpoint inhibitors (ICIs), the combination of ICIs with other therapies have recently been extensively explored, among which combination of ICIs with kinase inhibitors achieves promising clinical outcome in a plethora of cancer types. Here we comprehensively summarize the potent roles of protein kinases in modulating immune checkpoints both in tumor and immune cells, and reshaping tumor immune microenvironments by evoking innate immune response and neoantigen generation or presentation. Moreover, the clinical trial and approval of combined administration of kinase inhibitors with ICIs are collected, highlighting the precise strategies to benefit cancer immune therapies.