Splice variants of CK1α and CK1α-like: Comparative analysis of subcellular localization, kinase activity and function in the Wnt signaling pathway.

Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/ß-catenin pathway, which promotes the degradation of ß-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/ß-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of ß-catenin regulation. Rescue experiments showed that only 2 out of 10 naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/ß-catenin signaling caused by CK1α-deficiency in cells. Importantly, the ability to phosphorylate ß-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/ß knock out models suggest that the additional non-redundant function of CK1α in the Wnt pathway beyond Ser45-ß-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/ß-catenin pathway at the level of ß-catenin and Axin.

Back-pocket optimization of 2-aminopyrimidine-based macrocycles leads to potent dual EPHA2/GAK kinase inhibitors with antiviral activity.

Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here, we developed a 2-aminopyrimidine-based macrocyclic dual EPHA2/GAK kinase inhibitor as a chemical tool to study the role of these two kinases in viral entry and assembly. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55 as a potent dual EPHA2/GAK inhibitor. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells, mainly via its EPHA2 activity, and is therefore a promising candidate for further optimization of its activity against dengue virus.

Development of Selective Pyrido[2,3-d]pyrimidin-7(8H)-one-Based Mammalian STE20-Like (MST3/4) Kinase Inhibitors.

Mammalian STE20-like (MST) kinases 1-4 play key roles in regulating the Hippo and autophagy pathways, and their dysregulation has been implicated in cancer development. In contrast to the well-studied MST1/2, the roles of MST3/4 are less clear, in part due to the lack of potent and selective inhibitors. Here, we re-evaluated literature compounds, and used structure-guided design to optimize the p21-activated kinase (PAK) inhibitor G-5555 (8) to selectively target MST3/4. These efforts resulted in the development of MR24 (24) and MR30 (27) with good kinome-wide selectivity and high cellular potency. The distinct cellular functions of closely related MST kinases can now be elucidated with subfamily-selective chemical tool compounds using a combination of the MST1/2 inhibitor PF-06447475 (2) and the two MST3/4 inhibitors developed. We found that MST3/4-selective inhibition caused a cell-cycle arrest in the G1 phase, whereas MST1/2 inhibition resulted in accumulation of cells in the G2/M phase.

Development of Potent Dual BET/HDAC Inhibitors via Pharmacophore Merging and Structure-Guided Optimization.

Bromodomain and extra-terminal domain (BET) proteins and histone deacetylases (HDACs) are prime targets in cancer therapy. Recent research has particularly focused on the development of dual BET/HDAC inhibitors for hard-to-treat tumors, such as pancreatic cancer. Here, we developed a new series of potent dual BET/HDAC inhibitors by choosing starting scaffolds that enabled us to optimally merge the two functionalities into a single compound. Systematic structure-guided modification of both warheads then led to optimized binders that were superior in potency to both parent compounds, with the best molecules of this series binding to both BRD4 bromodomains as well as HDAC1/2 with EC50 values in the 100 nM range in cellular NanoBRET target engagement assays. For one of our lead molecules, we could also show the selective inhibition of HDAC1/2 over all other zinc-dependent HDACs. Importantly, this on-target activity translated into promising efficacy in pancreatic cancer and NUT midline carcinoma cells. Our lead molecules effectively blocked histone H3 deacetylation in pancreatic cancer cells and upregulated the tumor suppressor HEXIM1 and proapoptotic p57, both markers of BET inhibition. In addition, they have the potential to downregulate the oncogenic drivers of NUT midline carcinoma, as demonstrated for MYC and TP63 mRNA levels. Overall, this study expands the portfolio of available dual BET/class I HDAC inhibitors for future translational studies in different cancer models.

Synthesis of Pyrazole-Based Macrocycles Leads to a Highly Selective Inhibitor for MST3.

MST1, MST2, MST3, MST4, and YSK1 are conserved members of the mammalian sterile 20-like serine/threonine (MST) family that regulate cellular functions such as proliferation and migration. The MST3 isozyme plays a role in regulating cell growth and apoptosis, and its dysregulation has been linked to high-grade tumors. To date, there are no isoform-selective inhibitors that could be used for validating the role of MST3 in tumorigenesis. We designed a series of 3-aminopyrazole-based macrocycles based on the structure of a promiscuous inhibitor. By varying the moieties targeting the solvent-exposed region and optimizing the linker, macrocycle JA310 (21c) was synthesized. JA310 exhibited high cellular potency for MST3 (EC50 = 106 nM) and excellent kinome-wide selectivity. The crystal structure of the MST3-JA310 complex provided intriguing insights into the binding mode, which is associated with large-scale structural rearrangements. In summary, JA310 demonstrates the utility of macrocyclization for the design of highly selective inhibitors and presents the first chemical probe for MST3.

Death by a thousand cuts through kinase inhibitor combinations that maximize selectivity and enable rational multitargeting.

Kinase inhibitors are successful therapeutics in the treatment of cancers and autoimmune diseases and are useful tools in biomedical research. However, the high sequence and structural conservation of the catalytic kinase domain complicate the development of selective kinase inhibitors. Inhibition of off-target kinases makes it difficult to study the mechanism of inhibitors in biological systems. Current efforts focus on the development of inhibitors with improved selectivity. Here, we present an alternative solution to this problem by combining inhibitors with divergent off-target effects. We develop a multicompound-multitarget scoring (MMS) method that combines inhibitors to maximize target inhibition and to minimize off-target inhibition. Additionally, this framework enables optimization of inhibitor combinations for multiple on-targets. Using MMS with published kinase inhibitor datasets we determine potent inhibitor combinations for target kinases with better selectivity than the most selective single inhibitor and validate the predicted effect and selectivity of inhibitor combinations using in vitro and in cellulo techniques. MMS greatly enhances selectivity in rational multitargeting applications. The MMS framework is generalizable to other non-kinase biological targets where compound selectivity is a challenge and diverse compound libraries are available.

Design and Synthesis of Pyrazole-Based Macrocyclic Kinase Inhibitors Targeting BMPR2.

Bone morphogenetic protein (BMP) signaling is mediated by transmembrane protein kinases that form heterotetramers consisting of type-I and type-II receptors. Upon BMP binding, the constitutively active type-II receptors activate specific type-I receptors by transphosphorylation, resulting in the phosphorylation of SMAD effector proteins. Drug discovery in the receptor tyrosine kinase-like (TKL) family has largely focused on type-I receptors, with few inhibitors that have been published targeting type-II receptors. BMPR2 is involved in several diseases, most notably pulmonary arterial hypertension, but also contributes to Alzheimer's disease and cancer. Here, we report that macrocyclization of the promiscuous inhibitor 1, based on a 3-amino-1H-pyrazole hinge binding moiety, led to a selective and potent BMPR2 inhibitor 8a.

Deep Annotation of Donated Chemical Probes (DCP) in Organotypic Human Liver Cultures and Patient-Derived Organoids from Tumor and Normal Colorectum.

Well-characterized small molecules are essential tools for studying the biology and therapeutic relevance of a target protein. However, many compounds reported in the literature and routinely studied in biomedical research lack the potency and selectivity required for mechanistic cellular studies on the function of a given protein. Furthermore, commercially available compounds often do not include useful tools developed by industry as part of their research and development efforts, as they frequently remain proprietary. The freely available donated chemical probe (DCP) library, fueled by generous donations of compounds from industry and academia, enables easy access to a steadily growing collection of these valuable and well-characterized tools. Here, we provide a systematic description of the current DCP library collection and their associated comprehensive characterization data, including a variety of in vitro and cellular assays. Of note, we characterized the set in relevant human primary models by employing hepatotoxicity screening in primary human liver spheroids and viability screening in patient-derived colorectal cancer organoids and matched normal-adjacent epithelium. Taken together, the DCP library represents a well-annotated, openly available collection of tool compounds for studying a wide range of targets, including kinases, G-protein-coupled receptors, and ion channels. As such, it represents a unique resource for the biomedical research community.

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.

Death by a Thousand Cuts â€" Combining Kinase Inhibitors for Selective Target Inhibition and Rational Polypharmacology.

Kinase inhibitors are successful therapeutics in the treatment of cancers and autoimmune diseases and are useful tools in biomedical research. The high sequence and structural conservation of the catalytic kinase domain complicates the development of specific kinase inhibitors. As a consequence, most kinase inhibitors also inhibit off-target kinases which complicates the interpretation of phenotypic responses. Additionally, inhibition of off-targets may cause toxicity in patients. Therefore, highly selective kinase inhibition is a major goal in both biomedical research and clinical practice. Currently, efforts to improve selective kinase inhibition are dominated by the development of new kinase inhibitors. Here, we present an alternative solution to this problem by combining inhibitors with divergent off-target activities. We have developed a multicompound-multitarget scoring (MMS) method framework that combines inhibitors to maximize target inhibition and to minimize off-target inhibition. Additionally, this framework enables rational polypharmacology by allowing optimization of inhibitor combinations against multiple selected on-targets and off-targets. Using MMS with previously published chemogenomic kinase inhibitor datasets we determine inhibitor combinations that achieve potent activity against a target kinase and that are more selective than the most selective single inhibitor against that target. We validate the calculated effect and selectivity of a combination of inhibitors using the in cellulo NanoBRET assay. The MMS framework is generalizable to other pharmacological targets where compound specificity is a challenge and diverse compound libraries are available.

MSC-1186, a Highly Selective Pan-SRPK Inhibitor Based on an Exceptionally Decorated Benzimidazole-Pyrimidine Core.

The highly conserved catalytic sites in protein kinases make it difficult to identify ATP competitive inhibitors with kinome-wide selectivity. Serendipitously, during a dedicated fragment campaign for the focal adhesion kinase (FAK), a scaffold that had lost its initial FAK affinity showed remarkable potency and selectivity for serine-arginine-protein kinases 1-3 (SRPK1-3). Non-conserved interactions with the uniquely structured hinge region of the SRPK family were the key drivers of the exclusive selectivity of the discovered fragment hit. Structure-guided medicinal chemistry efforts led to the SRPK inhibitor MSC-1186, which fulfills all hallmarks of a reversible chemical probe, including nanomolar cellular potency and excellent kinome-wide selectivity. The combination of MSC-1186 with CDC2-like kinase (CLK) inhibitors showed additive attenuation of SR-protein phosphorylation compared to the single agents. MSC-1186 and negative control (MSC-5360) are chemical probes available via the Structural Genomics Consortium chemical probe program (https://www.sgc-ffm.uni-frankfurt.de/).

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

Development and Characterization of Type I, Type II, and Type III LIM-Kinase Chemical Probes.

LIMKs are important regulators of actin and microtubule dynamics, and they play essential roles in many cellular processes. Deregulation of LIMKs has been linked to the development of diverse diseases, including cancers and cognitive disabilities, but well-characterized inhibitors known as chemical probes are still lacking. Here, we report the characterization of three highly selective LIMK1/2 inhibitors covering all canonical binding modes (type I/II/III) and the structure-based design of the type II/III inhibitors. Characterization of these chemical probes revealed a low nanomolar affinity for LIMK1/2, and all inhibitors 1 (LIMKi3; type I), 48 (TH470; type II), and 15 (TH257; type III) showed excellent selectivity in a comprehensive scanMAX kinase selectivity panel. Phosphoproteomics revealed remarkable differences between type I and type II inhibitors compared with the allosteric inhibitor 15. In phenotypic assays such as neurite outgrowth models of fragile X-chromosome, 15 showed promising activity, suggesting the potential application of allosteric LIMK inhibitors treating this orphan disease.

A Toolbox for the Generation of Chemical Probes for Baculovirus IAP Repeat Containing Proteins.

E3 ligases constitute a large and diverse family of proteins that play a central role in regulating protein homeostasis by recruiting substrate proteins via recruitment domains to the proteasomal degradation machinery. Small molecules can either inhibit, modulate or hijack E3 function. The latter class of small molecules led to the development of selective protein degraders, such as PROTACs (PROteolysis TArgeting Chimeras), that recruit protein targets to the ubiquitin system leading to a new class of pharmacologically active drugs and to new therapeutic options. Recent efforts have focused on the E3 family of Baculovirus IAP Repeat (BIR) domains that comprise a structurally conserved but diverse 70 amino acid long protein interaction domain. In the human proteome, 16 BIR domains have been identified, among them promising drug targets such as the Inhibitors of Apoptosis (IAP) family, that typically contain three BIR domains (BIR1, BIR2, and BIR3). To date, this target area lacks assay tools that would allow comprehensive evaluation of inhibitor selectivity. As a consequence, the selectivity of current BIR domain targeting inhibitors is unknown. To this end, we developed assays that allow determination of inhibitor selectivity in vitro as well as in cellulo. Using this toolbox, we have characterized available BIR domain inhibitors. The characterized chemical starting points and selectivity data will be the basis for the generation of new chemical probes for IAP proteins with well-characterized mode of action and provide the basis for future drug discovery efforts and the development of PROTACs and molecular glues.

Illuminating the Dark: Highly Selective Inhibition of Serine/Threonine Kinase 17A with Pyrazolo[1,5-a]pyrimidine-Based Macrocycles.

Serine/threonine kinase 17A (death-associated protein kinase-related apoptosis-inducing protein kinase 1─DRAK1) is a part of the death-associated protein kinase (DAPK) family and belongs to the so-called dark kinome. Thus, the current state of knowledge of the cellular function of DRAK1 and its involvement in pathophysiological processes is very limited. Recently, DRAK1 has been implicated in tumorigenesis of glioblastoma multiforme (GBM) and other cancers, but no selective inhibitors of DRAK1 are available yet. To this end, we optimized a pyrazolo[1,5-a]pyrimidine-based macrocyclic scaffold. Structure-guided optimization of this macrocyclic scaffold led to the development of CK156 (34), which displayed high in vitro potency (KD = 21 nM) and selectivity in kinomewide screens. Crystal structures demonstrated that CK156 (34) acts as a type I inhibitor. However, contrary to studies using genetic knockdown of DRAK1, we have seen the inhibition of cell growth of glioma cells in 2D and 3D culture only at low micromolar concentrations.

Resistance to kinase inhibition through shortened target engagement.

Imatinib, a selective inhibitor of the breakpoint cluster region (BCR)-ABL kinase, is the poster child for targeted cancer therapeutics. However, its efficacy is limited by resistance mutations. Using a quantitative bioluminescence resonance energy transfer assay in living cells, we identified ABL kinase mutations that could cause imatinib resistance by altering drug residence time.

Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells.

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.

Design of a "Two-in-One" Mutant-Selective Epidermal Growth Factor Receptor Inhibitor That Spans the Orthosteric and Allosteric Sites.

Inhibitors targeting the epidermal growth factor receptor (EGFR) are an effective therapy for patients with non-small cell lung cancer harboring drug-sensitive activating mutations in the EGFR kinase domain. Drug resistance due to treatment-acquired mutations has motivated the development of successive generations of inhibitors that bind in the ATP site. The third-generation agent osimertinib is now a first-line treatment for this disease. Recently, allosteric inhibitors have been developed to overcome drug-resistant mutations that confer a resistance to osimertinib. Here, we present the structure-guided design and synthesis of a mutant-selective lead compound, which consists of a pyridinyl imidazole-fused benzylisoindolinedione scaffold that simultaneously occupies the orthosteric and allosteric sites. The compound potently inhibits enzymatic activity in L858R/T790M/C797S mutant EGFR (4.9 nM), with a significantly lower activity for wild-type EGFR (47 nM). Additionally, this compound achieves modest cetuximab-independent and mutant-selective cellular efficacies on the L858R (1.2 µM) and L858R/T790M (4.4 µM) variants.

Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance.

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type-like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

Single tracer-based protocol for broad-spectrum kinase profiling in live cells with NanoBRET.

This protocol is used to profile the engagement of kinase inhibitors across nearly 200 kinases in a live-cell context. This protocol utilizes one single kinase tracer (NanoBRET(TM) Tracer K10) that operates quantitatively at four different concentrations. Minimizing the number of tracers offers a significant workflow improvement over the previous protocol that utilized a combination of 6 tracers. Each NanoBRET(TM) kinase assay is built using commercially available plasmids and has been optimized for NanoLuc tagging orientation, diluent DNA, and tracer concentration. For complete details on the use and execution of this protocol, please refer to Vasta et al. (2018).