As outlined above, small molecule kinase inhibitor targeting the conserved ATP-binding site can be quite unselective. Determining the full target spectrum of a kinase inhibitor still remains a challenge33, 68. Nevertheless, this information is crucial to understand the molecular mechanisms for tumor response as well as potential toxicities upon drug treatment. Various methods have been developed over the years to address this issue. Traditional methods employ in vitro screening panels that contain large numbers of recombinant kinases. Here, kinase inhibitors are tested for inhibition of activity by measuring the transfer of a radioactively labeled phosphate group of ATP to a substrate. This has successfully been performed with smaller numbers of up to 65 inhibitors against 80 kinases69-71. Anastassiadis et al. expanded it to 178 kinase inhibitors against 300 kinases72 and recently, 183 inhibitors were tested against mutant kinase variants applying the same technology73. Other groups use DNA-tagged recombinant kinases to assess binding of free inhibitors in competition with an immobilized ligand. Binding affinity is then determined by a qPCR readout74-76. Nowadays, new compounds are often evaluated in recombinant assays against selected kinases in the intended target’s family.
Recombinant kinase assays are quite powerful and provide good insight into the target spectrum of a drug, but do not take all molecular characteristics of endogenous target proteins like posttranslational modifications, cofactors, or interaction partners in the cell into account. Most drugs target proteins, which are part of complex networks and pathways in a cellular environment and may change depending on their physiological or functional context. Therefore, it seems natural to investigate the effect of a drug on the whole proteome. Proteomics has developed several methods during the last years to evaluate the target spectrum of a drug of interest77.
1.3.1 Target deconvolution on sub-proteome level
Chemical proteomic technologies often employ protein affinity chromatography approaches with immobilized small molecules, like kinase inhibitors, followed by protein identification via mass spectrometry78. Classical methods directly immobilize the compound of interest to beads.
Therefore, the molecule often needs to be modified to facilitate covalent linking to chromatography resin. Simultaneously, protein binding capabilities should not be affected. Inhibitor-protein binding sites might be deduced from available co-crystal structures or inferred from structure-activity relationship data78-80. First efforts in chemical proteomics at the beginning of the millennium identified RPS6KA3 as target of bisindolylmaleimide81 and investigated targets of CDK inhibitors82, 83. Back then, target identification was biased towards highly abundant cellular kinases, but could be improved by altering biochemical conditions during enrichment and elution79, 84. By using excess of free inhibitor, the targets of the compound are competed from the beads, whereas background binding stays the same. This approach reduces false positive identifications (Figure 5a).
Cravatt and colleagues developed the so-called activity-based protein profiling (ABPP) approach.
Here, the compound is modified as reactive probe, which covalently attaches to the active site of a target protein. The covalent interaction occurs with suitable amino acids inside or in close proximity of the catalytic/reactive site. These molecules feature chemical moieties like biotin for streptavidin enrichment, or can be linked to fluorescent tags for monitoring proteins in vitro or fluorescent gels (Figure 5b). This technology is often applied for serine hydrolases but has also been used to study HDACs and kinases85-87. The technology can also be used for competitive profiling of an inhibitor of choice. The active site of the enzyme is then blocked and cannot be assessed by the probe88.
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Figure 5: Chemical Proteomic strategies. a) Affinity-based profiling: the compound of interest or a functional derivative is immobilized. Subsequent incubation with cell or tissue lysate enriches for target proteins, which are then identified by mass spectrometry. Competition with free inhibitor prevents target proteins from binding. b) Activity-based profiling: a reactive probe binds the active site of the enzyme, followed by affinity enrichment and MS analysis. c) Strategies for affinity pulldowns. d) Influence of affinity and time on residual binding (adapted from 77, 89, 90.
Various ways exist to immobilize the compound on a solid support like agarose beads or magnetic particles. Besides the use of a direct linker, the compound can be modified with an alkyne handle and then be ‘clicked’ to an azide bearing support. Trifunctional probes not only reversibly interact with the target but are also equipped with a reactive group that stabilizes this interaction after photo activation, for instance. A common sorting function (e.g. biotin or alkyne handle) then allows affinity enrichment of the drug target complex (Figure 5c)77, 89.
In each of these methods, the compound of interest needs to be chemically modified to a functionalized analog, to be immobilized on beads or to covalently bind to the target. This might hamper target identification as the compound doesn’t reflect the true inhibitor anymore and its synthesis is a time consuming step.
In ‘binding mode centric’ profiling approaches, the immobilized probe or a mixture of probes is constructed in a way that a particular protein target class (kinases) can be enriched from a lysate77. This requires a conserved and druggable binding site in the target class. Besides enriching subproteomes for closer and more complete coverage, this approach can also be used for selectivity profiling of a compound of interest. The compound of interest competes with the affinity matrix for the active site of the enzymes present in the lysate. If the protein is a target of the particular inhibitor, its binding site is blocked for enrichment by the beads. Bead-bound proteins are eluted and can subsequently be analyzed by immunodetection (if looking for known targets) or mass spectrometry (unbiased target identification). Target proteins should then show reduced amounts of signal intensity. Competition with a range of inhibitor concentrations leads to a dose dependent decrease of target proteins enabling the determination of affinity values (EC50) of each protein in the lysate towards the free drug. This approach combines the identification of off-targets and allows ranking of targets according to their affinity. In 2007, Bantscheff and coworkers introduced the concept of Kinobeads91 that represent such an affinity matrix for kinases. In the original version,
11 seven unselective small molecule inhibitors were immobilized on Sepharose beads and mixed, leading to the identification of novel targets for the small molecule inhibitors Imatinib, Dasatinib and Bosutinib. Médard et al. optimized these Kinobeads to an improved version, featuring only five immobilized inhibitors with greater kinase coverage by using a mixture of four different cancer cell lines92. Other groups employed similar strategies to profile kinases in breast cancer and leukemia93, 94.
Kinases and nucleotide binding proteins can also be enriched using modified ATP or ADP. In the KiNativ technology, biotinylated acyl phosphates of ATP and ADP react irreversibly with conserved lysine residues in the ATP-binding site, thus, labeling the protein with biotin. Upon prior inhibitor treatment, the reactive ATP-probe cannot bind anymore. After digestion and streptavidin enrichment, mass spectrometry readout allows for identification and quantification of target proteins95, 96. The use of only one peptide per protein (containing the active site labeled residue) for identification and quantification reduces sample complexity but can lead to less accurate measurements. Compared to the Kinobeads technology, these ATP-probes generally enrich more nucleotide binding proteins97.
These so-called chemical proteomic methods are powerful, as they investigate the target proteins and the inhibitors close to physiological conditions; the proteins are at endogenous expression levels, contain their natural modification status and can be investigated in a cell line or tissue lysate of interest. Combined with mass spectrometry, not only kinases are investigated as targets but the inhibitor is also profiled against various other proteins binding to ATP-like molecules. These approaches identify direct targets of bioactive molecules. Furthermore, selective targeting of protein complexes is possible, providing insight into regulatory mechanisms of protein-protein interactions98.
Using either of such assays, many novel and sometimes quite surprising protein-drug interactions have been identified in recent years. To name a few, FLT3 or MAP4K4 appear to be frequently hit by kinase inhibitors72, DDR1 was discovered as a new target for Imatinib and other BCR-ABL inhibitors91, 99 and Pazopanib as well as Ponatinib were identified as inhibitors of cellular necroptosis100. Interestingly, enantiomers of kinase inhibitors can also have different targets. Huber and co-workers have found that the (S)-enantiomer of the approved MET/ALK inhibitor Crizotinib selectively inhibits the 7,8-dihydro-8-oxoguanine triphosphatase MTH1 while the actual (R)-enantiomer drug does not101.
1.3.2 Parameters influencing competition binding assays
The success of competition binding assays with reversible inhibitors is dependent on several biochemical factors: (i) the affinity of the target protein to the immobilized probe (Figure 5d); (ii) the concentration of the probe; (iii) the concentration of the target protein (e.g. kinase) or its expression/abundance in the tissue/cell line used; (iv) the concentration and affinity of the free compound; (v) the koff-rate of the enriched protein from the beads. The first three factors are different from experiment to experiment and vary between proteins, ligands, and lysates.
Diminishing the influence of (i)-(iii) in a competition experiment results in IC50 values that are close to ‘true’ dissociation constants (Kd).
𝐾𝑑 = 𝐾𝑑(𝑝𝑟𝑜𝑏𝑒)
𝐾𝑑(𝑝𝑟𝑜𝑏𝑒)+ [𝑝𝑟𝑜𝑏𝑒] 𝑥 𝐼𝐶50 (2)
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with [𝑝𝑟𝑜𝑏𝑒] ≪ 𝐾𝑑(𝑝𝑟𝑜𝑏𝑒)
𝐾𝑑= 𝐼𝐶50 (3)
Equation (3) shows that by using concentrations of the immobilized compounds ([probe]) below the affinity of a protein towards the immobilized probe (Kd(probe)), the binding constant Kd of protein and free compound are independent of Kd(probe)76, 102.
In competition experiments, depletion of a protein from the lysate is defined as the fraction of this protein bound to the immobilized probe. It influences the correct determination of an EC50 value and shifts it to higher values. Depletion can be avoided using nanomolar concentrations of immobilized compounds. Moreover, large amounts of lysate reduce the influence of individual protein expression levels. Immobilized compounds with low nanomolar to picomolar affinity towards their targets also result in depletion and underestimation of EC50s. If the depletion of protein is higher than 40%, competition experiments will not be possible anymore91. Contrary to assuming that there should be no depletion in the case of unselective Kinobead-probes, experimental evidence shows otherwise. To correct for this, Sharma et al. introduced a correction factor103 which was slightly modified for Kinobeads. It allows the correction of the obtained EC50
values by multiplication with the correction factor r to an apparent binding constant Kdapp. Therefore, two subsequent pulldowns of the vehicle treated lysate are performed. For each protein, a depletion factor can be calculated by determining the ratio (r) of the MS1 intensity in the second pulldown divided by the MS1 intensity in the first enrichment step97.
r =𝑖𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 2
with T being the total amount of a target and f being the fraction of captured target that remains constant over subsequent pulldowns.
Some studies characterized interactions with affinities of up to 40 mM104, but generally nanomolar potencies for free compounds are needed for target identification. Moreover, washing of the beads after enrichment and the time needed for the whole affinity purification process limit the recovery of certain proteins (Figure 5d). Improved sensitivity and accuracy of mass spectrometers also help the detection of low abundant proteins and more robust quantification leads to better determination of binding constants77.
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1.3.3 Target deconvolution on proteome wide level
The methods described above are still limited to sub-proteomes and rely on competition of the compound at the same site as the immobilized compounds. This can lead to an incomplete view on the target spectrum of a drug. Recent advances have enabled target deconvolution on a proteome-wide level. They make use of differences in biophysical properties upon drug binding. In drug affinity responsive target stability (DARTS) measurements, target identification is based on the idea that a protein has reduced protease digestion susceptibility upon drug binding105. Protein oxidation as a function of denaturation by hydrogen peroxide reduces thermodynamic stability as well and can be assessed by SPROX (stability of proteins from rates of oxidation)106. A protein’s thermal stability changes upon addition of a ligand and has been used extensively in drug discovery programs107. Here, purified proteins are heated with increasing temperatures in the presence or absence of a ligand, being it a small molecule, DNA or RNA molecule or even another protein. Upon heat denaturation, the protein unfolds and exposes its hydrophobic parts. This can be measured with the use of a fluorescent dye; the difference in melting is then dependent on the ligand’s affinity towards the protein108. Martinez-Molina et al. discovered that this principle can also be used in living cells or whole cell lysate without a dye and named it cellular thermal shift assay (CETSA). Upon drug-protein interaction, a target protein is stabilized and, thus, precipitates later compared to vehicle control treated samples. However, they only showed this for target proteins of interest, followed by western blot readout109. Combination of this method with multiplexed quantitative mass spectrometry allows investigation of protein thermal stability on a proteome wide level110. This allows the determination of melting temperatures for every protein identified in the sample.
Furthermore, target engagement can be assessed in a cellular context and enables unbiased target and off-target identification. A variant of the CETSA is the isothermal dose response (ITDR). Here, living cells or lysate are heated to the same temperature (the half-melting temperature) but are treated with increasing concentrations of inhibitor. The protein’s stability will increase with increasing compound concentrations enabling the determination of affinity values for half maximal stabilization109.
It has to be noted that most of the chemoproteomic approaches mentioned above measure binding and, therefore, only generate target hypotheses. Such identified targets then require further validation with purified proteins or cell culture models to investigate the influence on a desired phenotype77.