tracerDB: a crowdsourced fluorescent tracer database for target engagement analysis.

Investigating ligand-protein complexes is essential in the areas of chemical biology and drug discovery. However, detailed information on key reagents such as fluorescent tracers and associated data for the development of widely used bioluminescence resonance energy transfer (BRET) assays including NanoBRET, time-resolved Förster resonance energy transfer (TR-FRET) and fluorescence polarization (FP) assays are not easily accessible to the research community. We created tracerDB, a curated database of validated tracers. This resource provides an open access knowledge base and a unified system for tracer and assay validation. The database is freely available at https://www.tracerdb.org/ .

A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2.

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

A C-Degron Structure-Based Approach for the Development of Ligands Targeting the E3 Ligase TRIM7.

TRIM7 is a ubiquitin E3 ligase with key regulatory functions, mediating viral infection, tumor biology, innate immunity, and cellular processes, such as autophagy and ferroptosis. It contains a PRYSPRY domain that specifically recognizes degron sequences containing C-terminal glutamine. Ligands that bind to the TRIM7 PRYSPRY domain may have applications in the treatment of viral infections, as modulators of inflammation, and in the design of a new class of PROTACs (PROteolysis TArgeting Chimeras) that mediate the selective degradation of therapeutically relevant proteins (POIs). Here, we developed an assay toolbox for the comprehensive evaluation of TRIM7 ligands. Using TRIM7 degron sequences together with a structure-based design, we developed the first series of peptidomimetic ligands with low micromolar affinity. The terminal carboxylate moiety was required for ligand activity but prevented cell penetration. A prodrug strategy using an ethyl ester resulted in enhanced permeability, which was evaluated using confocal imaging.

Chemogenomics for NR1 nuclear hormone receptors.

Nuclear receptors (NRs) regulate transcription in response to ligand binding and NR modulation allows pharmacological control of gene expression. Although some NRs are relevant as drug targets, the NR1 family, which comprises 19 NRs binding to hormones, vitamins, and lipid metabolites, has only been partially explored from a translational perspective. To enable systematic target identification and validation for this protein family in phenotypic settings, we present an NR1 chemogenomic (CG) compound set optimized for complementary activity/selectivity profiles and chemical diversity. Based on broad profiling of candidates for specificity, toxicity, and off-target liabilities, sixty-nine comprehensively annotated NR1 agonists, antagonists and inverse agonists covering all members of the NR1 family and meeting potency and selectivity standards are included in the final NR1 CG set. Proof-of-concept application of this set reveals effects of NR1 members in autophagy, neuroinflammation and cancer cell death, and confirms the suitability of the set for target identification and validation.

A chemical probe to modulate human GID4 Pro/N-degron interactions.

The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

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 two out of ten 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 KO models suggest that the additional nonredundant 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.

Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.

Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.

Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2.

OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC). DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice. RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer. CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.

Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells.

The rational development of small-molecule degraders (e.g., proteolysis targeting chimeras) remains a challenge as the rate-limiting steps that determine degrader efficiency are largely unknown. Standard methods in the field of targeted protein degradation mostly rely on classical, low-throughput endpoint assays such as western blots or quantitative proteomics. Here we applied NanoLuciferase- and HaloTag-based screening technologies to determine the kinetics and stability of small-molecule-induced ternary complex formation between a protein of interest and a selected E3 ligase. A collection of live-cell assays were designed to probe the most critical steps of the degradation process while minimizing the number of required expression constructs, making the proposed assay pipeline flexible and adaptable to the requirements of the users. This approach evaluates the underlying mechanism of selective target degraders and reveals the exact characteristics of the developed degrader molecules in living cells. The protocol allows scientists trained in basic cell culture and molecular biology to carry out small-molecule proximity-inducer screening via tracking of the ternary complex formation within 2 weeks of establishment, while degrader screening using the HiBiT system requires a CRISPR-Cas9 engineered cell line whose generation can take up to 3 months. After cell-line generation, degrader screening and validation can be carried out in high-throughput manner within days.

Development of Highly Potent and Selective Covalent FGFR4 Inhibitors Based on SNAr Electrophiles.

Fibroblast growth factor receptor 4 (FGFR4) is thought to be a driver in several cancer types, most notably in hepatocellular carcinoma. One way to achieve high potency and isoform selectivity for FGFR4 is covalently targeting a rare cysteine (C552) in the hinge region of its kinase domain that is not present in other FGFR family members (FGFR1-3). Typically, this cysteine is addressed via classical acrylamide electrophiles. We demonstrate that noncanonical covalent "warheads" based on nucleophilic aromatic substitution (SNAr) chemistry can be employed in a rational manner to generate highly potent and (isoform-)selective FGFR4 inhibitors with a low intrinsic reactivity. Key compounds showed low to subnanomolar potency, efficient covalent inactivation kinetics, and excellent selectivity against the other FGFRs, the kinases with an equivalent cysteine, and a representative subset of the kinome. Moreover, these compounds achieved nanomolar potencies in cellular assays and demonstrated good microsomal stability, highlighting the potential of SNAr-based approaches in covalent inhibitor design.

Expanding the ligand spaces for E3 ligases for the design of protein degraders.

Targeted protein degradation (TPD) has recently emerged as an exciting new drug modality. However, the strategy of developing small molecule-based protein degraders has evolved over the past two decades and has now established molecular tags that are already in clinical use, as well as chimeric molecules, PROteolysis TArgeting Chimeras (PROTACs), based mainly on ligand systems developed for the two E3 ligases CRBN and VHL. The large size of the human E3 ligase family suggests that PROTACs can be developed by targeting a large diversity of E3 ligases, some of which have restricted expression patterns with the potential to design disease- or tissue-specific degraders. Indeed, many new E3 ligands have been published recently, confirming the druggability of E3 ligases. This review summarises recent data on E3 ligases and highlights the challenges in developing these molecules into efficient PROTACs rivalling the established degrader systems.

Tetrahydropyridine LIMK inhibitors: Structure activity studies and biological characterization.

LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors. Homology models were first constructed to better understand the binding mode of our preliminary compounds and to explain differences in biological activity. A library of over 60 products was generated and in vitro enzymatic activities were measured in the mid to low nanomolar range. The most promising derivatives were then evaluated in cell on cofilin phosphorylation inhibition which led to the identification of 52 which showed excellent selectivity for LIMKs in a kinase selectivity panel. We also demonstrated that 52 affected the cell cytoskeleton by disturbing actin filaments. Cell migration studies with this derivative using three different cell lines displayed a significant effect on cell motility. Finally, the crystal structure of the kinase domain of LIMK2 complexed with 52 was solved, greatly improving our understanding of the interaction between 52 and LIMK2 active site. The reported data represent a basis for the development of more efficient LIMK inhibitors for future in vivo preclinical validation.

Discovery of the sEH Inhibitor Epoxykynin as a Potent Kynurenine Pathway Modulator.

Disease-related phenotypic assays enable unbiased discovery of novel bioactive small molecules and may provide novel insights into physiological systems and unprecedented molecular modes of action (MMOA). Herein, we report the identification and characterization of epoxykynin, a potent inhibitor of the soluble epoxide hydrolase (sEH). Epoxykynin was discovered by means of a cellular assay monitoring modulation of kynurenine (Kyn) levels in BxPC-3 cells upon stimulation with the cytokine interferon-γ (IFN-γ) and subsequent target identification employing affinity-based chemical proteomics. Increased Kyn levels are associated with immune suppression in the tumor microenvironment and, thus, the Kyn pathway and its key player indoleamine 2,3-dioxygenase 1 (IDO1) are appealing targets in immuno-oncology. However, targeting IDO1 directly has led to limited success in clinical investigations, demonstrating that alternative approaches to reduce Kyn levels are in high demand. We uncover a cross-talk between sEH and the Kyn pathway that may provide new opportunities to revert cancer-induced immune tolerance.

Discovery of a Small Molecule Activator of Slack (Kcnt1) Potassium Channels That Significantly Reduces Scratching in Mouse Models of Histamine-Independent and Chronic Itch.

Various disorders are accompanied by histamine-independent itching, which is often resistant to the currently available therapies. Here, it is reported that the pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch-sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack-activating antipsychotic drug, loxapine, a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles is designed that enables to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibits negligible dopamine D2 and D3 receptor binding, unlike loxapine. Notably, compound 6 displays potent on-target antipruritic activity in multiple mouse models of acute histamine-independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.

Synthesis and biological evaluation of 1H-pyrrolo[3,2-g]isoquinolines.

A structure-activity relationship study performed on 1H-pyrrolo[3,2-g]isoquinoline scaffold identified new haspin inhibitors with nanomolar potencies and selectivity indices (SI) over 6 (inhibitory potency evaluated against 8 protein kinases). Compound 22 was the most active of the series (haspin IC50 = 76 nM). Cellular evaluation of 22 confirmed its activity for endogenous haspin in U-2 OS cells and its anti-proliferative activity against various cell lines. In addition, the binding mode of analog 22 in complex with haspin was determined by X-ray crystallography.

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.

Photosensitization enables Pauson-Khand-type reactions with nitrenes.

The Pauson-Khand reaction has in the past 50 years become one of the most common cycloaddition reactions in chemistry. Coupling two unsaturated bonds with carbon monoxide, the transformation remains limited to CO as a C1 building block. Herein we report analogous cycloaddition reactions with nitrenes as an N1 unit. The reaction of a nonconjugated diene with a nitrene precursor produces bicyclic bioisosteres of common saturated heterocycles such as piperidine, morpholine, and piperazine. Experimental and computational mechanistic studies support relaying of the diradical nature of triplet nitrene into the π-system. We showcase the reaction's utility in late-stage functionalization of drug compounds and discovery of soluble epoxide hydrolase inhibitors.

The catalytic domain of free or ligand bound histone deacetylase 4 occurs in solution predominantly in closed conformation.

Human histone deacetylase 4 (HDAC4) is a key epigenetic regulator involved in a number of important cellular processes. This makes HDAC4 a promising target for the treatment of several cancers and neurodegenerative diseases, in particular Huntington's disease. HDAC4 is highly regulated by phosphorylation and oxidation, which determine its nuclear or cytosolic localization, and exerts its function through multiple interactions with other proteins, forming multiprotein complexes of varying composition. The catalytic domain of HDAC4 is known to interact with the SMRT/NCOR corepressor complex when the structural zinc-binding domain (sZBD) is intact and forms a closed conformation. Crystal structures of the HDAC4 catalytic domain have been reported showing an open conformation of HDAC4 when bound to certain ligands. Here, we investigated the relevance of this HDAC4 conformation under physiological conditions in solution. We show that proper zinc chelation in the sZBD is essential for enzyme function. Loss of the structural zinc ion not only leads to a massive decrease in enzyme activity, but it also has serious consequences for the overall structural integrity and stability of the protein. However, the Zn2+ free HDAC4 structure in solution is incompatible with the open conformation. In solution, the open conformation of HDAC4 was also not observed in the presence of a variety of structurally divergent ligands. This suggests that the open conformation of HDAC4 cannot be induced in solution, and therefore cannot be exploited for the development of HDAC4-specific inhibitors.

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.