Selectivity Profile Of Inhibitors Research Articles

STA‐55, an Easily Accessible, Broad‐Spectrum, Activity‐Based Aldehyde Dehydrogenase Probe

Aldehyde dehydrogenases (ALDHs) convert aldehydes into carboxylic acids and are often upregulated in cancer. They have been linked to therapy resistance and are therefore potential therapeutic targets. However, only a few selective and potent inhibitors are currently available for this group of enzymes. Competitive activity-based protein profiling (ABPP) would aid the development and validation of new selective inhibitors. Herein, a broad-spectrum activity-based probe that reports on several ALDHs is presented. This probe was used in a competitive ABPP protocol against three ALDH inhibitors in lung cancer cells to determine their selectivity profiles and establish their target engagement.

Computational analysis of kinase inhibitor selectivity using structural knowledge.

Kinases play a significant role in diverse disease signaling pathways and understanding kinase inhibitor selectivity, the tendency of drugs to bind to off-targets, remains a top priority for kinase inhibitor design and clinical safety assessment. Traditional approaches for kinase selectivity analysis using biochemical activity and binding assays are useful but can be costly and are often limited by the kinases that are available. On the other hand, current computational kinase selectivity prediction methods are computational intensive and can rarely achieve sufficient accuracy for large-scale kinome wide inhibitor selectivity profiling. Here, we present a KinomeFEATURE database for kinase binding site similarity search by comparing protein microenvironments characterized using diverse physiochemical descriptors. Initial selectivity prediction of 15 known kinase inhibitors achieved an >90% accuracy and demonstrated improved performance in comparison to commonly used kinase inhibitor selectivity prediction methods. Additional kinase ATP binding site similarity assessment (120 binding sites) identified 55 kinases with significant promiscuity and revealed unexpected inhibitor cross-activities between PKR and FGFR2 kinases. Kinome-wide selectivity profiling of 11 kinase drug candidates predicted novel as well as experimentally validated off-targets and suggested structural mechanisms of kinase cross-activities. Our study demonstrated potential utilities of our approach for large-scale kinase inhibitor selectivity profiling that could contribute to kinase drug development and safety assessment. The KinomeFEATURE database and the associated scripts for performing kinase pocket similarity search can be downloaded from the Stanford SimTK website (https://simtk.org/projects/kdb). Supplementary data are available at Bioinformatics online.

227 - Development of high-throughput screening assay to identify inhibitors of diacylglycerol kinases (DGKs): Utilization of DGK assay panel for the selectivity profiling of inhibitors

227 - Development of high-throughput screening assay to identify inhibitors of diacylglycerol kinases (DGKs): Utilization of DGK assay panel for the selectivity profiling of inhibitors

Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling

In addition to target efficacy, drug safety is a major requirement during the drug discovery process and is influenced by target specificity. Therefore, it is imperative that every new drug candidate be profiled against various liability panels that include protein kinases. Here, an effective methodology to streamline kinase inhibitor profiling is described. An accessible standardized profiling system for 112 protein kinases covering all branches of the kinome was developed. This approach consists of creating different sets of kinases and their corresponding substrates in multi-tube strips. The kinase stocks are pre-standardized for optimal kinase activity and used for inhibitor profiling using a bioluminescent ADP detection assay. We show that these strips can routinely generate inhibitor selectivity profiles for small or broad kinase family panels. Lipid kinases were also assembled in strip format and profiled together with protein kinases. We identified two specific PI3K inhibitors that have off-target effects on CK2 that were not reported before and would have been missed if compounds were not profiled against lipid and protein kinases simultaneously. To validate the accuracy of the data generated by this method, we confirmed that the inhibition potencies observed are consistent with published values produced by more complex technologies such as radioactivity assays.

New Affinity Probe Targeting VEGF Receptors for Kinase Inhibitor Selectivity Profiling by Chemical Proteomics

Solid tumors are dependent for growth on nutrients and the supply of oxygen, which they often acquire via neoangiogenesis. Vascular endothelial growth factors and the corresponding receptors (VEGFRs) play central roles in this process, and consequently, the blockade of this pathway is one therapeutic strategy for cancer treatment. A number of small molecules inhibiting VEGFR inhibitors have been developed for clinical use, and a comprehensive view of target selectivity is important to assess the therapeutic as well as risk potential of a drug molecule. Recent advances in mass spectrometry-based chemical proteomics allow analyses of drug-target interactions under close-to-physiological conditions, and in this study, we report on the design, synthesis, and application of a small molecule affinity probe as a tool for the selectivity profiling of VEGFR and other kinase inhibitors. The probe is capable of binding >132 protein kinases, including angiokinases such as VEGFRs, PDGFRs, and c-KIT from lysates of cancer cell lines or human placenta tissue. Combining the new probe with Kinobeads in competitive binding assays, we were able to identify nanomolar off-targets of the VEGFR/PDGFR inhibitors pazopanib and axitinib. Because of its broad binding spectrum, the developed chemical tool can be generically used for the discovery of kinase inhibitor targets, which may contribute to a more comprehensive understanding of the mechanisms of action of such drugs.

Characterization of a Chemical Affinity Probe Targeting Akt Kinases

Protein kinases are key regulators of cellular processes, and aberrant function is often associated with human disease. Consequently, kinases represent an important class of therapeutic targets and about 20 kinase inhibitors (KIs) are in clinical use today. Detailed knowledge about the selectivity of KIs is important for the correct interpretation of their pharmacological and systems biological effects. Chemical proteomic approaches for systematic kinase inhibitor selectivity profiling have emerged as important molecular tools in this regard, but the coverage of the human kinome is still incomplete. Here, we describe a new affinity probe targeting Akt and many other members of the AGC kinase family that considerably extends the scope of KI profiling by chemical proteomics. In combination with the previously published kinobeads, the synthesized probe was applied to selectivity profiling of the Akt inhibitors GSK690693 and GSK2141795 in human cancer cells. The results confirmed the inhibition of all Akt isoforms and of a number of known as well as CDC42BPB as a novel putative target for GSK690693. This work also established, for the first time, the kinase selectivity profile of the clinical phase I drug GSK2141795 and identified PRKG1 as a low nanomolar kinase target as well as the ATP-dependent 5'-3' DNA helicase ERCC2 as a potential new non-kinase off-target.

Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP

Most kinase inhibitor drugs target the binding site of the nucleotide cosubstrate ATP. The high intracellular concentration of ATP can strongly affect inhibitor potency and selectivity depending on the affinity of the target kinase for ATP. Here we used a defined chemoproteomics system based on competition-binding assays in cell extracts from Jurkat and SK-MEL-28 cells with immobilized ATP mimetics (kinobeads). This system enabled us to assess the affinities of more than 200 kinases for the cellular nucleotide cofactors ATP, ADP, and GTP and the effects of the divalent metal ions Mg(2+) and Mn(2+). The affinity values determined in this system were largely consistent across the two cell lines, indicating no major dependence on kinase expression levels. Kinase-ATP affinities range from low micromolar to millimolar, which has profound consequences for the prediction of cellular effects from inhibitor selectivity profiles. Only a small number of kinases including CK2, MEK, and BRAF exhibited affinity for GTP. This extensive and consistent data set of kinase-nucleotide affinities, determined for native enzymes under defined experimental conditions, will represent a useful resource for kinase drug discovery.

Structural origins of AGC protein kinase inhibitor selectivities: PKA as a drug discovery tool

The era of structure-based protein kinase inhibitor design began in the early 1990s with the determination of crystal structures of protein kinase A (PKA, or cyclic AMP-dependent kinase). Although many other protein kinases have since been extensively characterized, PKA remains a prototype for studies of protein kinase active conformations. It serves well as a model for the structural properties of AGC subfamily protein kinases, clarifying inhibitor selectivity profiles. Its reliable expression, constitutive activity, simple domain structure, and reproducible crystallizability have also made it a useful surrogate for the discovery of inhibitors of both established and emerging AGC kinase targets.

Abstract 5499: Kinase inhibitor selectivity profiling utilizing a versatile TR-FRET kinase binding assay platform

Abstract There is great potential for the utilization of selective kinase inhibitors in the treatment of many clinical indications, especially in oncology and autoimmune conditions. Kinase inhibitor selectivity profiling has become an important tool during lead development to understand a compound's selectivity, potential off-target effects and possible new indications for the compound. We have utilized the LanthaScreen® Eu Kinase Binding Assay platform to develop a simple, robust means for profiling compounds across the kinome utilizing pre-plated reagents and controls. The assay is based on competitive displacement of a tracer (a fluorophore conjugated to a kinase inhibitor scaffold) from specific kinases of interest. Binding of the tracer to a kinase is detected by addition of a europium-labeled anti-tag antibody. Binding of the tracer and antibody to a kinase results in a high degree of FRET, whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET. This platform is useful to explore activation-state binding selectivity, time-dependent binding, and binding to kinases for which substrates have not been identified. The assay detects both ATP-competitive as well as non-competitive, allosteric kinase inhibitors. We will present data demonstrating the utility of these reagents for kinase profiling efforts with a variety of kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5499.

High-Throughput Biochemical Kinase Selectivity Assays: Panel Development and Screening Applications

Kinases represent attractive targets for drug discovery. Eight small-molecule kinase inhibitors are currently marketed in the area of oncology, and numerous others are in clinical trials. Characterization of the selectivity profiles of these compounds is important to target appropriate patient populations and to reduce the potential of toxicity due to off-target effects. The authors describe the development, validation, and utilization of a biochemical kinase assay panel for the selectivity profiling of inhibitors. The panel was developed as 29 radiometric Flashplate assays, and then an initial 13 were transitioned to a nonradiometric Caliper mobility shift assay format. Generation of high-quality data from the panel is detailed along with a comparison of the assay formats. Both assay technologies were found to be suitable for panel screening, but mobility shift assays yielded higher data quality. The selectivity data generated here should be useful in computational modeling and help facilitate, in conjunction with sequence and structural information, the rational design of inhibitors with well-defined selectivity profiles.

In-cell Selectivity Profiling of Serine Protease Inhibitors by Activity-based Proteomics

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.

Classifying protein kinase structures guides use of ligand‐selectivity profiles to predict inactive conformations: Structure of lck/imatinib complex

We report a clustering of public human protein kinase structures based on the conformations of two structural elements, the activation segment and the C-helix, revealing three discrete clusters. One cluster includes kinases in catalytically active conformations. Each of the other clusters contains a distinct inactive conformation. Typically, kinases adopt at most one of the inactive conformations in available X-ray structures, implying that one of the conformations is preferred for many kinases. The classification is consistent with selectivity profiles of several well-characterized kinase inhibitors. We show further that inhibitor selectivity profiles guide kinase classification. For example, selective inhibition of lck among src-family kinases by imatinib (Gleevec) suggests that the relative stabilities of inactive conformations of lck are different from other src-family kinases. We report the X-ray structure of the lck/imatinib complex, confirming that the conformation adopted by lck is distinct from other structurally-characterized src-family kinases and instead resembles kinases abl1 and kit in complex with imatinib. Our classification creates new paths for designing small-molecule inhibitors.

Functional Interrogation of the Kinome Using Nucleotide Acyl Phosphates

The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.