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.

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.

Brain Region-Specific Differences in Amyloid-ß Plaque Composition in 5XFAD Mice.

Senile plaques consisting of amyloid-beta (Aß) peptides are a major pathological hallmark of Alzheimer's disease (AD). Aß peptides are heterogeneous regarding the exact length of their amino- and carboxy-termini. Aß1-40 and Aß1-42 are often considered to represent canonical "full-length" Aß species. Using immunohistochemistry, we analyzed the distribution of Aß1-x, Aßx-42 and Aß4-x species in amyloid deposits in the subiculum, hippocampus and cortex in 5XFAD mice during aging. Overall plaque load increased in all three brain regions, with the subiculum being the area with the strongest relative plaque coverage. In the subiculum, but not in the other brain regions, the Aß1-x load peaked at an age of five months and decreased thereafter. In contrast, the density of plaques positive for N-terminally truncated Aß4-x species increased continuously over time. We hypothesize that ongoing plaque remodeling takes place, leading to a conversion of deposited Aß1-x peptides into Aß4-x peptides in brain regions with a high Aß plaque burden.

Shifting the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one inhibitors towards the salt-inducible kinase (SIK) subfamily.

Salt-inducible kinases 1-3 (SIK1-3) are key regulators of the LKB1-AMPK pathway and play an important role in cellular homeostasis. Dysregulation of any of the three isoforms has been associated with tumorigenesis in liver, breast, and ovarian cancers. We have recently developed the dual pan-SIK/group I p21-activated kinase (PAK) chemical probe MRIA9. However, inhibition of p21-activated kinases has been associated with cardiotoxicity in vivo, which complicates the use of MRIA9 as a tool compound. Here, we present a structure-based approach involving the back-pocket and gatekeeper residues, for narrowing the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one-based inhibitors towards SIK kinases, eliminating PAK activity. Optimization was guided by high-resolution crystal structure analysis and computational methods, resulting in a pan-SIK inhibitor, MR22, which no longer exhibited activity on STE group kinases and displayed excellent selectivity in a representative kinase panel. MR22-dependent SIK inhibition led to centrosome dissociation and subsequent cell-cycle arrest in ovarian cancer cells, as observed with MRIA9, conclusively linking these phenotypic effects to SIK inhibition. Taken together, MR22 represents a valuable tool compound for studying SIK kinase function in cells.

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 3-Amino-1H-pyrazole-Based Kinase Inhibitors to Illuminate the Understudied PCTAIRE Family.

The PCTAIRE subfamily belongs to the CDK (cyclin-dependent kinase) family and represents an understudied class of kinases of the dark kinome. They exhibit a highly conserved binding pocket and are activated by cyclin Y binding. CDK16 is targeted to the plasma membrane after binding to N-myristoylated cyclin Y and is highly expressed in post-mitotic tissues, such as the brain and testis. Dysregulation is associated with several diseases, including breast, prostate, and cervical cancer. Here, we used the N-(1H-pyrazol-3-yl)pyrimidin-4-amine moiety from the promiscuous inhibitor 1 to target CDK16, by varying different residues. Further optimization steps led to 43d, which exhibited high cellular potency for CDK16 (EC50 = 33 nM) and the other members of the PCTAIRE and PFTAIRE family with 20-120 nM and 50-180 nM, respectively. A DSF screen against a representative panel of approximately 100 kinases exhibited a selective inhibition over the other kinases. In a viability assessment, 43d decreased the cell count in a dose-dependent manner. A FUCCI cell cycle assay revealed a G2/M phase cell cycle arrest at all tested concentrations for 43d, caused by inhibition of CDK16.

Pharmacokinetic Optimization of Small Molecule Janus Kinase 3 Inhibitors to Target Immune Cells.

Modulation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling is a promising method of treating autoimmune diseases, and the profound potency of clinical compounds makes this mode of action particularly attractive. Other questions that remain unanswered also include: What is the ideal selectivity between JAK1 and JAK3? Which cells are most relevant to JAK blockade? And what is the ideal tissue distribution pattern for addressing specific autoimmune conditions? We hypothesized that JAK3 selectivity is most relevant to low-dose clinical effects and interleukin-10 (IL-10) stimulation in particular, that immune cells are the most important compartment, and that distribution to inflamed tissue is the most important pharmacokinetic characteristic for in vivo disease modification. To test these hypotheses, we prepared modified derivatives of JAK3 specific inhibitors that target C909 near the ATP binding site based on FM-381, first reported in 2016; a compound class that was hitherto limited in uptake and exposure in vivo. These limits appear to be due to metabolic instability of side groups binding in the selectivity pocket. We identified derivatives with improved stability and tissue exposure. Conjugation to macrolide scaffolds with medium chain linkers was sufficient to stabilize the compounds and improve transport to organs while maintaining JAK3 affinity. These conjugates are inflammation targeted JAK3 inhibitors with long tissue half-lives and high exposure to activated immune cells.

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.

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.

Inhibitors of the Hippo Pathway Kinases STK3/MST2 and STK4/MST1 Have Utility for the Treatment of Acute Myeloid Leukemia.

Serine/threonine-protein kinases 3 and 4 (STK3 and STK4, respectively) are key components of the Hippo signaling pathway, which regulates cell proliferation and death and provides a potential therapeutic target for acute myeloid leukemia (AML). Herein, we report the structure-based design of a series of pyrrolopyrimidine derivatives as STK3 and STK4 inhibitors. In an initial screen, the compounds exhibited low nanomolar potency against both STK3 and STK4. Crystallization of compound 6 with STK4 revealed two-point hinge binding in the ATP-binding pocket. Further characterization and analysis demonstrated that compound 20 (SBP-3264) specifically inhibited the Hippo signaling pathway in cultured mammalian cells and possessed favorable pharmacokinetic and pharmacodynamic properties in mice. We show that genetic knockdown and pharmacological inhibition of STK3 and STK4 suppress the proliferation of AML cells in vitro. Thus, SBP-3264 is a valuable chemical probe for understanding the roles of STK3 and STK4 in AML and is a promising candidate for further advancement as a potential therapy.

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).

Structure-Based Design of Selective Salt-Inducible Kinase Inhibitors.

Salt-inducible kinases (SIKs) are key metabolic regulators. The imbalance in SIK function is associated with the development of diverse cancers, including breast, gastric, and ovarian cancers. Chemical tools to clarify the roles of SIK in different diseases are, however, sparse and are generally characterized by poor kinome-wide selectivity. Here, we have adapted the pyrido[2,3-d]pyrimidin-7-one-based p21-activated kinase (PAK) inhibitor G-5555 for the targeting of SIK, by exploiting differences in the back-pocket region of these kinases. Optimization was supported by high-resolution crystal structures of G-5555 bound to the known off-targets, MST3 and MST4, leading to a chemical probe, MRIA9, with dual SIK/PAK activity and excellent selectivity over other kinases. Furthermore, we show that MRIA9 sensitizes ovarian cancer cells to treatment with the mitotic agent paclitaxel, confirming earlier data from genetic knockdown studies and suggesting a combination therapy with SIK inhibitors and paclitaxel for the treatment of paclitaxel-resistant ovarian cancer.

C81-evoked inhibition of the TNFR1-NFκB pathway during inflammatory processes for stabilization of the impaired vascular endothelial barrier for leukocytes.

Chronic inflammation-related diseases are characterized by persistent leukocyte infiltration into the underlying tissue. The vascular endothelium plays a major role in this pathophysiological condition. Only few therapeutic strategies focus on the vascular endothelium as a major target for an anti-inflammatory approach. In this study, we present the natural compound-derived carbazole derivative C81 as chemical modulator interfering with leukocyte-endothelial cell interactions. An in vivo assay employing intravital microscopy to monitor leukocyte trafficking after C81 treatment in postcapillary venules of a murine cremaster muscle was performed. Moreover, in vitro assays using HUVECs and monocytes were implemented. The impact of C81 on cell adhesion molecules and the NFκB signaling cascade was analyzed in vitro in endothelial cells. Effects of C81 on protein translation were determined by incorporation of a puromycin analog-based approach and polysome profiling. We found that C81 significantly reduced TNF-activated leukocyte trafficking in postcapillary venules. Similar results were obtained in vitro when C81 reduced leukocyte-endothelial cell interactions by down-regulating cell adhesion molecules. Focusing on the NFκB signaling cascade, we found that C81 reduced the activation on multiple levels of the cascade through promoted IκBα recovery by attenuation of IκBα ubiquitination and through reduced protein levels of TNFR1 caused by protein translation inhibition. We suggest that C81 might represent a promising lead compound for interfering with inflammation-related processes in endothelial cells by down-regulation of IκBα ubiquitination on the one hand and inhibition of translation on the other hand without exerting cytotoxic effects.

Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders.

Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic functions. This study presents the design, synthesis, and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5.

The transforming growth factor beta-receptor I/activin receptor-like kinase 5 (TGFBR1/ALK5) and its close homologue ALK4 are receptor protein kinases associated with the development of diverse diseases, including cancer, fibrosis, heart diseases, and dysfunctional immune response. Therefore, ALK4/5 are among the most studied kinases, and several inhibitors have been developed. However, current commercially available inhibitors either lack selectivity or have not been comprehensively characterized, limiting their value for studying ALK4/5 function in cellular systems. To this end, we report the characterization of the 2-oxo-imidazopyridine, TP-008, a potent chemical probe with dual activity for ALK4 and ALK5 as well as the development of a matching negative control compound. TP-008 has excellent cellular potency and strongly abrogates phosphorylation of the substrate SMAD2 (mothers against decapentaplegic homologue 2). Thus, this chemical probe offers an excellent tool for mechanistic studies on the ALK4/5 signaling pathway and the contribution of these targets to disease.

SuperScent--a database of flavors and scents.

Volatiles are efficient mediators of chemical communication acting universally as attractant, repellent or warning signal in all kingdoms of life. Beside this broad impact volatiles have in nature, scents are also widely used in pharmaceutical, food and cosmetic industries, so the identification of new scents is of great industrial interest. Despite this importance as well as the vast number and diversity of volatile compounds, there is currently no comprehensive public database providing information on structure and chemical classification of volatiles. Therefore, the database SuperScent was established to supply users with detailed information on the variety of odor components. The version of the database presented here comprises the 2D/3D structures of approximately 2100 volatiles and around 9200 synonyms as well as physicochemical properties, commercial availability and references. The volatiles are classified according to their origin, functionality and odorant groups. The information was extracted from the literature and web resources. SuperScent offers several search options, e.g. name, Pubchem ID number, species, functional groups, or molecular weight. SuperScent is available online at: http://bioinformatics.charite.de/superscent.