High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detection

Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC(50) value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases.     

PAK'n it in: identification of a selective PAK inhibitor

p21-activated kinases are important signaling kinases for which no specific chemical inhibitors are known. In this issue of Chemistry & Biology, Deacon et al. target allosteric transitions undergone during PAK activation to identify a selective inhibitor (Deacon et al., 2008).     

Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.     

A small molecule discrimination map of the antibiotic resistance kinome

Kinase-mediated resistance to antibiotics is a significant clinical challenge. These enzymes share a common protein fold characteristic of Ser/Thr/Tyr protein kinases. We screened 14 antibiotic resistance kinases against 80 chemically diverse protein kinase inhibitors to map resistance kinase chemical space. The screens identified molecules with both broad and narrow inhibition profiles, proving that protein kinase inhibitors offer privileged chemical matter with the potential to block antibiotic resistance. One example is the flavonol quercetin, which inhibited a number of resistance kinases in vitro and in vivo. This activity was rationalized by determination of the crystal structure of the aminoglycoside kinase APH(2″)-IVa in complex with quercetin and its antibiotic substrate kanamycin. Our data demonstrate that protein kinase inhibitors offer chemical scaffolds that can block antibiotic resistance, providing leads for co-drug design.     

Downfalls of Chemical Probes Acting at the Kinase ATP-Site: CK2 as a Case Study

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase’s biology, with wide-reaching implications for drug development.     

Discovery of Potent and Exquisitely Selective Inhibitors of Kinase CK1 with Tunable Isoform Selectivity

Casein kinases 1 (CK1) are key signaling molecules that have emerged recently as attractive therapeutic targets in particular for the treatment of hematological malignancies. Herein, we report the identification of a new class of potent and highly selective inhibitors of CK1α, δ and ϵ. Based on their optimal in vitro and in vivo profiles and their exclusive selectivity, MU1250, MU1500 and MU1742 were selected as quality chemical probes for those CK1 isoforms. At proper concentrations, MU1250 and MU1500 allow for specific targeting of CK1δ or dual inhibition of CK1δ/ϵ in cells. The compound MU1742 also efficiently inhibits CK1α and, to our knowledge, represents the first potent and highly selective inhibitor of this enzyme. In addition, we demonstrate that the central 1H-pyrrolo[2,3-b]pyridine-imidazole pharmacophore can be used as the basis of highly selective inhibitors of other therapeutically relevant protein kinases, e.g. p38α, as exemplified by the compound MU1299.     

Globally Approved EGFR Inhibitors: Insights into Their Syntheses,Target Kinases,Biological Activities,Receptor Interactions,and Metabolism

Targeting the EGFR with small-molecule inhibitors is a confirmed valid strategy in cancer therapy. Since the FDA approval of the first EGFR-TKI, erlotinib, great efforts have been devoted to the discovery of new potent inhibitors. Until now, fourteen EGFR small-molecule inhibitors have been globally approved for the treatment of different types of cancers. Although these drugs showed high efficacy in cancer therapy, EGFR mutations have emerged as a big challenge for these drugs. In this review, we focus on the EGFR small-molecule inhibitors that have been approved for clinical uses in cancer therapy. These drugs are classified based on their chemical structures, target kinases, and pharmacological uses. The synthetic routes of these drugs are also discussed. The crystal structures of these drugs with their target kinases are also summarized and their bonding modes and interactions are visualized. Based on their binding interactions with the EGFR, these drugs are also classified into reversible and irreversible inhibitors. The cytotoxicity of these drugs against different types of cancer cell lines is also summarized. In addition, the proposed metabolic pathways and metabolites of the fourteen drugs are discussed, with a primary focus on the active and reactive metabolites. Taken together, this review highlights the syntheses, target kinases, crystal structures, binding interactions, cytotoxicity, and metabolism of the fourteen globally approved EGFR inhibitors. These data should greatly help in the design of new EGFR inhibitors.     

Resorcylic acid lactones: A pluripotent scaffold with therapeutic potential

The recent discoveries of potent HSP90 and MAP kinase inhibitors amongst the resorcylic acid lactones (RALs) have revived interest in this family of natural products. Both HSP90 and MAP kinase inhibition hold tremendous therapeutic potential, particularly in the treatment of cancer. Our synthetic efforts towards the RALs and, in particular, selective inhibitors of HSP90 and kinases are reviewed.     

Forecasting roles of combinatorial chemistry in the age of genomically derived drug discovery targets

Genomics has caused an explosion in the number of potential therapeutic targets with varying degrees of validated pathophysiology. Among the first applications of combinatorial chemistry in genomics-driven drug discovery is the search for surrogate ligands or substrates. In the event that no surrogate is found for molecular assays, more exotic functional screens in whole cells or model organisms are used. Protein-protein interaction mapping by yeast and mammalian two-hybrid systems dominates empirical functional genomics, and this will lead to a bias for screening projects targeting this type of interaction. Drug discovery for protein-protein interactions has a poor track record, and this will challenge prevailing views on the design of combinatorial libraries. Genomics based on structural homology will yield many putative kinases, receptors, enzymes, transporter proteins, ion channels and GPCRs. Most of these projects will require new surrogate agonists, ligands or substrates, and then pharmaceutically useful agonists or antagonists will need to be found. Again, combinatorial chemistry might be essential to these studies. Given the need to screen hundreds of targets at great risk of irrelevance to pathophysiology, combined with the challenge of finding surrogate or natural ligands for these new targets, there is an urgent need for efficiency. Different groups are addressing these concerns by developing biologically-driven combinatorial libraries in order to achieve a higher density of bioactivity. Early efforts in this regard will be described.     

An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase

Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation and identified the inhibitor IPA-3. Remarkably, preactivated Pak1 is resistant to IPA-3. IPA-3 also inhibits activation of related Pak isoforms regulated by autoinhibition, but not more distantly related Paks, nor >200 other kinases tested. Pak1 inhibition by IPA-3 in live cells supports a critical role for Pak in PDGF-stimulated Erk activation. These studies illustrate an alternative strategy for kinase inhibition and introduce a highly selective, cell-permeable chemical inhibitor of Pak.     

Structurally sophisticated octahedral metal complexes as highly selective protein kinase inhibitors

The generation of synthetic compounds with exclusive target specificity is an extraordinary challenge of molecular recognition and demands novel design strategies, in particular for large and homologous protein families such as protein kinases with more than 500 members. Simple organic molecules often do not reach the necessary sophistication to fulfill this task. Here, we present six carefully tailored, stable metal-containing compounds in which unique and defined molecular geometries with natural-product-like structural complexity are constructed around octahedral ruthenium(II) or iridium(III) metal centers. Each of the six reported metal compounds displays high selectivity for an individual protein kinase, namely GSK3α, PAK1, PIM1, DAPK1, MLCK, and FLT4. Although being conventional ATP-competitive inhibitors, the combination of the unusual globular shape and rigidity characteristics, of these compounds facilitates the design of highly selective protein kinase inhibitors. Unique structural features of the octahedral coordination geometry allow novel interactions with the glycine-rich loop, which contribute significantly to binding potencies and selectivities. The sensitive correlation between metal coordination sphere and inhibition properties suggests that in this design, the metal is located at a "hot spot" within the ATP binding pocket, not too close to the hinge region where globular space is unavailable, and at the same time not too far out toward the solvent where the octahedral coordination sphere would not have a significant impact on potency and selectivity. This study thus demonstrates that inert (stable) octahedral metal complexes are sophisticated structural scaffolds for the design of highly selective chemical probes.     

7-Aminoalkoxy-Quinazolines from Epigenetic Focused Libraries Are Potent and Selective Inhibitors of DNA Methyltransferase 1

Inhibitors of epigenetic writers such as DNA methyltransferases (DNMTs) are attractive compounds for epigenetic drug and probe discovery. To advance epigenetic probes and drug discovery, chemical companies are developing focused libraries for epigenetic targets. Based on a knowledge-based approach, herein we report the identification of two quinazoline-based derivatives identified in focused libraries with sub-micromolar inhibition of DNMT1 (30 and 81 nM), more potent than S-adenosylhomocysteine. Also, both compounds had a low micromolar affinity of DNMT3A and did not inhibit DNMT3B. The enzymatic inhibitory activity of DNMT1 and DNMT3A was rationalized with molecular modeling. The quinazolines reported in this work are known to have low cell toxicity and be potent inhibitors of the epigenetic target G9a. Therefore, the quinazoline-based compounds presented are attractive not only as novel potent inhibitors of DNMTs but also as dual and selective epigenetic agents targeting two families of epigenetic writers.     

Polyprenyl-hydroquinones and -furans from three marine sponges inhibit the cell cycle regulating phosphatase CDC25A

The CDC25 phosphatases regulate the cell division cycle by controlling the activity of cyclin-dependent kinases. While screening for inhibitors of phosphatases among natural products we repeatedly found that some polyprenyl-hydroquinones and polyprenyl-furans (furanoterpenoids) (furospongins, furospinosulins) were potent CDC25 phosphatase inhibitors. These compounds were extracted, isolated and identified independently from three sponge species (Spongia officinalis, Ircinia spinulosa, Ircinia muscarum), collected at different locations in the Mediterranean Sea. The compounds were inactive on the Ser/Thr phosphatase PP2C-alpha and on three kinases (CDK1, CDK5, GSK-3), suggesting that some potent and selective CDC25 phosphatase might be designed from these initial structures.     

A general framework for inhibitor resistance in protein kinases

Protein kinases control virtually every aspect of normal and pathological cell physiology and are considered ideal targets for drug discovery. Most kinase inhibitors target the ATP binding site and interact with residue of a hinge loop connecting the small and large lobes of the kinase scaffold. Resistance to kinase inhibitors emerges during clinical treatment or as a result of in vitro selection approaches. Mutations conferring resistance to ATP site inhibitors often affect residues that line the ATP binding site and therefore contribute to selective inhibitor binding. Here, we show that mutations at two specific positions in the hinge loop, distinct from the previously characterized "gatekeeper," have general adverse effects on inhibitor sensitivity in six distantly related kinases, usually without consequences on kinase activity. Our results uncover a unifying mechanism of inhibitor resistance of protein kinases that might have widespread significance for drug target validation and clinical practice.     

Rational inhibitor design and iterative screening in the identification of selective plasmodial cyclin dependent kinase inhibitors

New chemical classes of compounds must be introduced into the malaria drug development pipeline in an effort to develop new chemotherapy options for the fight against malaria. In this review we describe an iterative approach designed to identify potent inhibitors of a kinase family that collectively functions as key regulators of the cell cycle. Cyclin-dependent protein kinases (CDKs) are attractive drug targets in numerous diseases and, most recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. Our approach uses experimental and virtual screening methodologies to identify and refine chemical inhibitors and increase the success rate of discovering potent and selective inhibitors. The active pockets of the plasmodial CDKs are unique in terms of size, shape and amino acid composition compared with those of the mammalian orthologues. These differences exemplified through the use of screening assays, molecular modeling, and crystallography can be exploited for inhibitor design. To date, several classes of compounds including quinolines and oxindoles have been identified as selective inhibitors of the plasmodial CDK7 homologue, Pfmrk. From these initial studies and through the iterative rational drug design process, more potent, selective, and most importantly, chemically unique compound classes have been identified as effective inhibitors of the plasmodial CDKs and the malarial parasite.     

Evaluation of "credit card" libraries for inhibition of HIV-1 gp41 fusogenic core formation

Protein-protein interactions are of critical importance in biological systems, and small molecule modulators of such protein recognition and intervention processes are of particular interest. To investigate this area of research, we have synthesized small-molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motif, appended with a variety of chemical functions, which we have termed "credit-card" structures. From two of our "credit-card" libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation, and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors.     

Current literature highlights - June 2002

Selective Cdk4 Inhibitors: Cyclins and cyclin-dependent kinases (Cdks) play important roles in regulation of the cell cycle. In particular, D-type cyclins, which have been shown to be amplified or over-expressed in several tumour cells, associate with Cdk4/6 to activate their phosphorylation activity. Cyclin D-Cdk4/6 complexes phosphorylate the retinoblastoma protein (pRB) and regulate the cell cycle during G₁/S transition. Loss of function or deletion of p16^ink4a (endogenous Cdk4/6 specific inhibitor protein) frequently occurs in clinical cancer cells. Thus, selective Cdk4/6 inhibitors should be useful as a new class of cytostatic antitumour agents. In order to overcome the problem of selectivity for Cdk4/6 over the hundreds of homologous kinases in the superfamily, specific amino acid residues were identified around the ATP binding pocket of Cdk4 by comparing the amino acid sequences of 390 representative kinases. Subsequently, a chemical library was designed using this information about the locations of these amino acid residues (A novel approach for the development of selective Cdk4 inhibitors: Library design based on locations of Cdk4 specific amino acid residues, Honma, T. et. al., J. Med. Chem., 44, (2001), 4628-4640).     

Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by hymenialdisine, a marine sponge constituent

BACKGROUND: Over 2000 protein kinases regulate cellular functions. Screening for inhibitors of some of these kinases has already yielded some potent and selective compounds with promising potential for the treatment of human diseases. RESULTS: The marine sponge constituent hymenialdisine is a potent inhibitor of cyclin-dependent kinases, glycogen synthase kinase-3beta and casein kinase 1. Hymenialdisine competes with ATP for binding to these kinases. A CDK2-hymenialdisine complex crystal structure shows that three hydrogen bonds link hymenialdisine to the Glu81 and Leu83 residues of CDK2, as observed with other inhibitors. Hymenialdisine inhibits CDK5/p35 in vivo as demonstrated by the lack of phosphorylation/down-regulation of Pak1 kinase in E18 rat cortical neurons, and also inhibits GSK-3 in vivo as shown by the inhibition of MAP-1B phosphorylation. Hymenialdisine also blocks the in vivo phosphorylation of the microtubule-binding protein tau at sites that are hyperphosphorylated by GSK-3 and CDK5/p35 in Alzheimer's disease (cross-reacting with Alzheimer's-specific AT100 antibodies). CONCLUSIONS: The natural product hymenialdisine is a new kinase inhibitor with promising potential applications for treating neurodegenerative disorders.     

The chemical ecology of crucifers and their fungal pathogens: boosting plant defenses and inhibiting pathogen invasion

Fungal plant diseases can cause very substantial yield losses in crucifer crops such as rapeseed and canola, or vegetables such as cabbage and broccoli. To devise sustainable methods to prevent and deter crucifer pathogens, the chemical interaction between crucifers and their fungi is under intense investigation. Crucifers produce complex blends of secondary metabolites with diverse ecological roles that include protection against microbial pathogens and other pests. The secondary metabolites involved in crucifer defense, namely phytoalexins and phytoanticipins, and their metabolism by fungal pathogens indicate that some fungi produce different enzymes to detoxify these metabolites and that some fungal detoxifying enzymes are rather specific. Chemical synthesis and screening of phytoalexin analogue libraries using cultures of fungal pathogens, as well as protein extracts, have shown that such detoxification reactions can be inhibited and that some inhibitors are strongly antifungal. Overall results of current work show the feasibility of using selective inhibitors of fungal detoxifying enzymes, i.e., paldoxins, to protect plants by boosting their chemical defenses.     

Identification of Potent,Selective Protein Kinase C Inhibitors Based on a Phorbol Skeleton

The elucidation of specific functions of protein kinase C (PKC) subtypes in physiological processes is an important challenge for the future development of new drug targets. Subtype‐selective PKC agonists and antagonists are useful biological tools for this purpose. Most of the currently used PKC modulators elicit their activities through binding to the ATP binding site of PKC, which shares many features with other kinases. PKC modulators that target the PKC regulatory domain are considered to be advantageous in terms of selectivity, because the structure of the regulatory domain is intrinsic to each PKC subtype. In this paper, we describe the identification of new potent and conventional PKC‐selective inhibitors that target the regulatory domain. The inhibitors contain a phorbol skeleton, a naturally occurring potent and selective PKC regulatory domain binder, with a perfluorinated alkyl group and a polyether hydrophilic chain on a terephthaloyl aromatic ring at the C12 position. Both of these substituents are essential for the potent inhibitory activity. Specifically, the binding affinity between PKC and the phorbol ester analogues was improved by an electron‐deficient aromatic ring at C12. This finding cannot be explained by the previously proposed binding model and suggests a new binding mode between phorbol esters and PKC.