Abstract Cyclin-dependent kinases (CDKs) and their associated cyclins are central regulators of cell cycle progression and transcriptional control. Dysregulation of CDK/Cyclin complexes is a hallmark of many cancers, driving uncontrolled proliferation and tumor development. Consequently, these complexes have become critical targets in oncology, with CDK4/6 inhibitors already established as a cornerstone in the treatment of e.g. hormone receptor-positive breast cancer and under investigation for other malignancies. Early preclinical development of CDK inhibitors relies on biochemical screening assays to identify small molecules capable of inhibiting CDK/Cyclin activity. These assays, frequently based on kinase activity measurements, enable high-throughput evaluation of compound libraries and provide essential insights into inhibitor potency and selectivity. These approaches are mandatory for rational drug design, guiding the optimization of lead compounds toward improved efficacy and reduced off-target effects. Despite their obvious limitations in predicting cellular and in vivo responses, these early screening strategies are instrumental in shaping the current generation of CDK- and other kinase-targeted therapeutics. In subsequent development stages, cellular and in vivo model systems are employed to validate inhibitor activity and assess pharmacodynamics and toxicity. Many of these models are based on non-human mammalian species, such as murine or primate systems. These studies are essential for bridging the gap between biochemical screening and clinical application, ensuring that candidate molecules demonstrate efficacy and safety before entering human trials. However, despite the importance of such data, it is rarely assessed in early biochemical screening whether results from human and non-human assays are consistent. Such early biochemical evaluation of potential differences in the effects of drug candidates on the kinase target from different species could generate valuable insights to select the most relevant cellular or in-vivo model systems for advanced drug-development. Here, we present comparative biochemical data for late-stage development or already approved CDK inhibitors tested against CDK/Cyclin complexes from human, rat, mouse, dog and primate origin, focusing on CDK4/CycD1. Notably, differential inhibitory potency was observed for several compounds, including palbociclib, which showed an approximately ten-fold difference in CDK4/CycD1 inhibition between human and murine enzymes. Citation Format: Andreas Gericke, Frank Totzke, Constance Rademann, Carolin Heidemann-Dinger, Daniel Mueller. Comparative biochemical evaluation of small-molecule inhibitor efficacy on CDK/Cyclin complexes across diverse mammalian species [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1902.

Protein kinases are essential for the regulation of many biological processes, e.g. proliferation, differentiation, migration, and apoptosis. Deregulated kinase activity is observed in tumor cells and altered activity of specific kinases is essential for development and progression of cancer and other diseases. Consequently, regulation of protein kinase activity became a target for therapeutic intervention. Intensive research resulted in the development of many small molecule kinase inhibitors (SMI) being clinically approved as targeted therapies. Functional biochemical in-vitro assays are essential requirements to assess SMIs effects on target kinase activity after validation of specific kinases being molecular drivers of a pathological condition. Only limited information is gained by monitoring the interaction of kinase and compound in pure binding assays, but more conclusive data can be generated by in-vitro kinase activity assays. However, the relevance of results from both approaches depends on how well the assay setup responds to the mode-of-action of the compounds. Recombinant proteins and generic substrates are well established in development of protein kinase assays due to cost and technical feasibility considerations. Especially some generic substrates are highly artificial e.g. the commonly used Tyrosine-kinase substrate Poly(Glu/Tyr)4:1 and may be considered as phosphate group acceptors only. However, such generic substrates have successfully been used for development of numerous clinically approved SMIs. Other kinases however are not compatible with generic substrates and can only be established using less artificial substrates or even require their respective physiological in-vivo substrate for in-vitro activity. These kinases include several members of the RAF and MAPK family. Functional in-vitro assay using a generic substrate are rarely replaced by physiological substrate due to more complex assay conditions and increased cost for drug screening. We compared biochemical in-vitro kinase activity assays for the LIM kinases, LIMK1 and LIMK2, using either generic substrates or the published physiological substrates, CFL1 and CFL2. We further evaluated these results by comparing them in different assays, using radiometric and luminescent read-out technologies. Several published LIMK inhibitors displayed significant differences in the relative potency when tested with generic or physiological substrates. Generic substrates for biochemical in-vitro kinase activity assays in preclinical drug development have been well established and successfully applied in the past. However, our data indicate, that the choice of a more physiological substrate should be considered carefully, as it might significantly increase the relevance and therefore value of the results obtained from early-stage compound screening. Robert Torka, Andreas Gericke, Frank Totzke, Carolin Heidemann-Dinger, Constance Rademann, Thomas Weber, Daniel Mueller. Apparent potency of small molecule inhibitors is influenced by the choice of substrate in LIMK biochemical in-vitro kinase activity assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6966.

Abstract Essential biological signaling pathways affecting e.g., proliferation, differentiation, migration, and apoptosis are regulated by protein kinases. Deregulation of protein kinases is observed in many tumor cells and frequently the development and progression of human cancers and other diseases is found to be causally connected to altered activity of specific protein kinases. Therefore, protein kinases have become a prime molecular target for therapeutic intervention. Multiple small molecule inhibitors targeting different kinases are currently in clinical use for treatment of various types of cancer and other diseases. A first step in preclinical development of new compounds is testing candidate substances in biochemical in-vitro assays, either based on binding of the compounds to their target or based on alterations of the in-vitro activity of the target kinase monitored in biochemical activity assays. Depending on the actual mode of action of a compound, the relevance of the results of such in-vitro assays may depend on the actual setup of the assay. Mainly for reasons of cost and technical feasibility, the use of generic substrates for in-vitro kinase activity assays is well established. While many of those substrates are highly artificial and can only be considered as generic phosphate-group acceptors, they have been successfully used in the past in the development of many approved small molecule kinase inhibitors currently in clinical use. However, even while many kinases may show activity with generic substrates, for some physiological substrates are required. Members of e.g. the RAF family and other members of the MAPK pathway are highly substrate specific and will not show kinase activity with generic substrates. Such kinases which have been established with generic substrates are rarely switched to more physiological substrates as most often this will result in increased complexity and associated cost of preclinical compound development. We have compared the in-vitro activity of WEE1 using different in-vitro activity assay readouts like autophosphorylation, phosphorylation of generic substrates and phosphorylation of its physiological substrate CDK1. In addition, we examined the substrate specificity of WEE1 towards CDK1 alone and in complex with Cyclin B1. We compared the potency of a selection of WEE1 inhibitors when different substrates were used. While in early preclinical development the use of rather artificial biochemical assays has been successfully been applied in the past, it should be taken into consideration that at least for the kinase target in focus, the identification, establishment and use of a more physiologically relevant substrate may increase the value of early-stage compound screening results significantly. Citation Format: Daniel G. Mueller, Andreas Gericke, Frank Totzke, Carolin Heidemann-Dinger, Constance Ketterer, Diane Kraemer, Thomas Weber, Michael H. Kubbutat. Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 648.

Detailed structure activity relationship of two series of quinazoline EHMT1/EHMT2 inhibitors (UNC0224 and UNC0638) have been elaborated. New and active alternatives are presented for the ubiquitous substitution patterns found in literature for the linker to the lysine mimicking region and the lysine mimic itself. These findings could allow for advancing EHMT1/EHMT2 inhibitors of that type beyond tool compounds by fine-tuning physicochemical properties making these inhibitors more drug-like.

Abstract Protein kinases belong to one of the largest families of evolutionary related proteins. More than 500 distinct kinases are encoded by approximately 2% of all human genes. Over the past decades, numerous protein kinases have been described of which deregulation contributes to many human diseases. Today, more than 30 protein kinase inhibitors have been approved for the treatment of cancer emphasizing the significance of kinases as one of the most versatile drug targets. Due to the structural similarity especially within the ATP-binding site, many kinase inhibitors show limited selectivity. Still, sufficient selectivity within the human kinases is of critical importance e.g. to reduce the risk of adverse side effects during treatment. Therefore, measuring and improving selectivity of a compound within the kinome is of pivotal importance during drug discovery and optimization phase in the development of therapeutically relevant kinase inhibitors. Broad profiling of kinase inhibitors in biochemical activity assays of several hundred kinases is nowadays well established. Commonly, kinase profiling is done using one or two concentrations of a test compound and measurement of the relative inhibition of the kinase activity compared to a high and low control. However, due to the limited dynamic range of this approach, and the challenge to select the most appropriate compound concentration, this profiling approach gives often limited information with respect to the differences in the potency of compounds against On-target- and Off-target kinases. We set up an IC50 kinase profiling approach that consists of measuring the effect of a compound on the activity of 320 human protein kinases at six different concentrations. We will present data showing the effect of compound concentration on the selectivity score in traditional profiling setting. IC50 kinase profiles of different approved and clinical stage kinase inhibitors will be presented demonstrating that an IC50 based profiling allows the accurate determination of selectivity of a compound based on the comparison of the IC50 values against the On-target- in relation to the IC50 values of the Off-target kinases providing significantly improved guidance in the further optimization of the test compound. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Marcel Pathe, Christoph Schaechtele, Michael H. Kubbutat. IC50 profiling against 320 protein kinases: Improving the accuracy of kinase inhibitor selectivity testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2388.

Abstract Protein kinases are central to cellular signal transduction and regulation of cellular processes, and are one of the most attractive target classes in modern drug discovery. Multiple kinase inhibitors have already been approved for treatment of various diseases including such severe conditions as cancer. The first and up to today largest group of drugs that effectively inhibits their respective kinase targets belong to the class of ATP competitive compounds. They bind into or near the ATP binding site of the enzymes and inhibit kinase activity by blocking access of ATP to the active site. Although there are numerous examples of highly specific ATP competitive compounds this mode of action is limited by several factors: The ATP binding pocket structures of kinases show a high degree of similarity, which makes finding highly selective compounds challenging. Furthermore, competing with ATP for binding to the same target site, compounds have to be of very high affinity due to the high intracellular ATP concentrations. Therefore, the interest to develop non-ATP competitive inhibitors has risen considerably over the last years. Such inhibitors bind to kinases at sites apart from the ATP binding site, inhibiting their activity e.g. by stabilizing an inactive conformation (like DFG-out state binders), displacing essential cofactors (like cyclins for CDKs) or by blocking activating modifications (like phosphorylation by upstream kinases). We present data of an in-vitro biochemical kinase activity assay setup which is suited to discriminate between ATP-competitive and non-ATP competitive inhibitors. The IC50 of an ATP-competitive inhibitor will increase with increasing ATP concentrations and the IC50 value at a given ATP concentration may be calculated using the equation of Cheng and Prussof: IC50=Ki+(Ki*[ATP]/KM[ATP] (Cheng Y., Prusoff W. H. (1973) Biochem. Pharmacol. 22: 3099-3108). By determining IC50 values for an inhibitor of a specific kinase at different ATP concentrations we examined whether the IC50 value changed according to the Cheng-Prusoff equation, indicating an ATP-competitive mode of action, or if the IC50 values remained unchanged in presence of elevated ATP indicating a non-ATP competitive or mixed type mode of action. In our assay setup we determined the IC50 values at ATP concentrations in a range of 0.1 to 10 fold the ATP KM of the kinase of interest. By comparing the results obtained for the non-ATP competitive MEK1 inhibitor selumetinib and the ATP competitive inhibitor staurosporine we could verify that our assay setup is well suited to discriminate between these different types of kinase inhibitors. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Carolin Heidemann-Dinger, Constance Ketterer, Diane Krämer, Marcel Pathe, Michael H. Kubbutat. A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2017-4186

Members of the family of cyclin dependent kinases (CDKs) have been recognized as pivotal regulators of cell cycle progression for more than 20 years. Concordant to their central role in the control of cell division they have been in the focus of research of proliferation associated diseases ever since, most prominently amongst these cancer. Although initial results obtained from first and second generation, low specificity CDK inhibitors (e.g. Flavopyridol, Roscovitine, Dinaciclib, AT7519, R547) have been sobering the recent approval of the first CDK-inhibitor Palbociclib for the treatment of certain forms of breast cancer clearly demonstrates the suitability of cell cycle CDKs as targets in oncology. Furthermore, in addition to cell cycle CDKs a second group of CDKs have been shown to have important roles in the regulation of gene transcription, and several of the “transcriptional” CDKs have become interesting targets in oncology. Recent results underline the notion that for being effective in the treatment of cancer, CDK inhibition requires very high specificity towards the respective target CDK(s). For example CDK1 knockdown or CDK9 inhibition have been shown to be synthetically lethal in combination with MYC overexpression. Selectivity of compounds within the family of CDKs could so far only be tested using a quite limited number of CDK-Cyclin complexes expressed in human cells. To date there are 20 CDK genes and at least 17 different Cyclin genes described, many of which give rise to different variants, e.g. there are 3 D-type cyclins, two A- and E-type cyclins etc.. Experimental data indicates that at least 50-60 different, biologically relevant CDK-Cyclin complexes may exist, but only a limited number of these are available for biochemical testing of drug candidates so far. We have recombinantly expressed and purified 28 different CDK-Cyclin complexes, covering a significant part of the CDK family, and established in-vitro kinase-activity assays for these recombinant enzymes. The resulting CDK panel represents the most comprehensive array for biochemical testing of this enzyme group currently available. We characterized the specificity of several CDK inhibitors that have been or are currently in preclinical or clinical development with this CDK collection. Results will be presented showing the specificity of these inhibitors not only for CDKs but also for CDKs complexed to different Cyclins. In several cases we could detect signifcant differences in the inhibition of the same CDK complexed to different Cyclins, e.g. a 10fold difference was seen for CDK6 complexes with Cyclin D1-3. A >100 fold difference was detected for CDK3 complexed to either Cyclin E1 or Cyclin C. This screening panel allows generating data on compound selectivity early in development, diminishing the risk of designing a compound with suboptimal target specificity. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Christian Beisenherz-Huss, Diane Kraemer, Carolin Heidemann-Dinger, Constance Ketterer, Chris Eckert, Michael H.G. Kubbutat. Characterization of CDK inhibitors in a biochemical assay using a comprehensive panel of human CDK-cyclin complexes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2821.

Many receptor tyrosine kinases (RTKs) represent bona fide drug targets in oncology. Effective compounds are available, but treatment invariably leads to resistance, often due to RTK mutations. The discovery of second-generation inhibitors requires cellular models of resistant RTKs.An approach using artificial transmembrane domains (TMDs) to activate RTKs was explored for the rapid generation of simple, ligand-independent cellular RTK assays, including resistance mutants.The RTKs epidermal growth factor receptor (EGFR), MET, and KIT were chosen in a proof-of-concept study. Their intracellular domains were inserted into a series of expression vectors encoding artificial TMDs, and they were tested for autophosphorylation activity in transient transfection assays. Active constructs could be identified for MET and EGFR, but not for KIT.Rat1 cell pools were generated expressing the MET or EGFR constructs, and their sensitivity to reference tool compounds was compared to that of MKN-45 or A431 cells. A good correlation between natural and recombinant cells led us to build a panel of clinically relevant MET mutant cell pools, based on the wild-type construct, which were then profiled via MET autophosphorylation and soft agar assays.In summary, a platform was established that allows for the rapid generation of cellular models for RTKs and their resistance mutants.

Abstract The epigenetic regulation of gene expression has been of increasing interest in the last couple of years. It has been shown that methylation of DNA may pass information epigenetically even from one cellular generation to the next without changing the DNA sequence. Epigenetic information may also be transferred by acetylation, methylation and phosphorylation of DNA binding proteins, e.g. transcription factors and histones. The latter constitute the core of the nucleosomal particles, which are essential for structuring the DNA into chromatin. Histones are methylated on specific lysine or arginine residues by a family of enzymes called HMTs (Histone Methyl Transferases). It leads to either compacting or loosening of the chromatin structure which influences the transcriptional activity of the DNA bound to these histones. Histones in each nucleosomal particle may be differentially methylated and the exact information coded into such methylation patterns is not yet well understood. Overexpression of several HMTs, however, has been found in a number of pathological conditions including cancer. Therefore HMTs are a promising target class for pharmacological intervention in such diseases. To start a compound development process targeting specific HMTs, but also to enable profiling against many different HMTs, robust in-vitro assays are required that allow the generation of scientifically valid results, are preferably automatable, economically feasible and safe, and are readily transferable to all HMTs. Current methods to detect HMT activity monitor the transfer of the methyl-group from the co-substrate SAM (S-adenosyl-methionine) onto the protein substrate, either directly by tracing the 3H-labelled methyl-group from SAM to a substrate, or indirectly by using methylation site specific antibodies. Alternatively the reaction co-product SAH (S-adenosyl-homocysteine) may be detected directly by antibodies or indirectly via conversion of the SAH into other compounds which may be traced by various methods. While the use of 3H is often limited due to safety reasons, antibody based detection is frequently limited by antibody specificity, is costly and, in the case of site specific antibodies, results may only to a limited degree be compared when different sites are methylated by different HMTs. We have therefore developed an universal, non radioactive, non-antibody based in-vitro assay which detects HMT activity by its co-product SAH that is converted by a two-step, enzymatically catalysed reaction to AMP which in turn is quantified using Promegas AMP-Glo technology. Currently we are in the process of recombinantly expressing and purifying at least 35 members of the HMT family. Data is presented on the production of the HMTs, assay coupling enzymes and on the development of validated in-vitro activity assays for the human HMT proteins EHMT1 and EHMT2. Citation Format: Daniel Mueller, Christian Beisenherz-Huss, Carolin Heidemann-Dinger, Constance Ketterer, Diane Krämer, Michael H.G. Kubbutat. Development of a non-radioactive method to determine Protein-Methyl-Transferase activity in-vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5160. doi:10.1158/1538-7445.AM2014-5160

Abstract Protein kinases regulate a variety of biological signaling pathways affecting cell proliferation, differentiation, migration and apoptosis. In tumor cells the activity of many protein kinases are frequently found to be upregulated linking deregulation of protein kinases causally to the development and progression of many human cancers and other diseases. Therefore, protein kinases have become a prime molecular target for therapeutic intervention, and up to now fourteen small molecule inhibitors have been approved for treatment of various types of cancer. These inhibitors block the activity of the target protein kinases by either blocking the ATP binding site in a direct competitive manner (type1-inhibitors) or by indirectly interfering with ATP/kinase interaction by binding to an inactive state, referred to as DFG-out (type2-inhibitors). Due to the fact that these inhibitor types bind to a region which is highly conserved among the protein kinase superfamily, achieving target selectivity represent a major challenge in the development of protein kinase inhibitors. Allosteric inhibitors, also sometimes referred to as type3-inhibitors, bind to structural sites different to the ATP-binding pocket region, and have, therefore, a significant higher potential to inhibit kinases much more selective than ATP-competitive compounds. The majority of the currently clinically approved small molecule protein kinase inhibitors are commonly classified as ATP-competitive inhibitors, and are able to inhibit different protein kinases with high potency. Interestingly, analysing the effect of the ATP-concentration on the IC50 of Sorafinib against different target kinases, we observed that the inhibitory potency against various target kinases is differently affected by the ATP concentration indicating differences in the exact mode of action of the inhibitor. To answer the question whether this is a specific property of Sorafenib, or also relevant for other inhibitors, we perfomed similar studies with seven additional clinically approved kinase inhibitors (Axitinib, Crizotinib, Erlotinib, Gefitinib, Lapatinib, Pazopanib and Sunitinib) using a panel of up to 16 different target protein kinases. We extended these studies and will in addition also present data investigating the influences of (a) the tags to which the respective recombinant kinase was fused, (b) the activation status of the kinase, and (c) of activating point mutations on the mode of action of the different inhibitors. Citation Format: Daniel Müller, Christian Beisenherz-Huss, Frank Totzke, Carolin Heidemann-Dinger, Constance Ketterer, Thomas Weber, Michael H.G. Kubbutat. Effects of point mutations, recombinant tags, activation status, and identity of target kinases on the mode of action of approved kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5168. doi:10.1158/1538-7445.AM2013-5168

Abstract Anaplastic lymphoma kinase (ALK) is a human receptor tyrosine kinase which has important functions in the development and maintenance of the peripheral and central nervous system. It has also been shown to be one causative agent in the development of several human malignancies including neuroblastoma, anaplastic lymphoma and non small cell lung cancer (NSCLC). One mechanism of pathological activation of ALK occurs by chromosomal translocation of the ALK gene to EML4 giving rise to an abnormal fusion protein. In order to develop small molecule inhibitors targeting ALK, in-vitro kinase assays have been developed which use recombinant ALK fragments fused to affinity tags to facilitate purification. Here we present data which demonstrate that biochemical parameters of recombinant ALK fusion proteins, like substrate specificity, ATP Km, Vmax and effects of chemical compounds, are significantly influenced by the type of affinity tag used. That influence partially persists even after cleavage of the affinity tag during the purification process. Since affinity-tagged recombinant proteins are widely used in drug discovery, a careful evaluation of potential tag-related effects should be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B121.