The Landscape of Atypical and Eukaryotic Protein Kinases Kanev, Georgi K.; de Graaf, Chris; de Esch, Iwan J.P. ...
Trends in pharmacological sciences,
November 2019, 2019-11-00, 20191101, Volume:
40, Issue:
11
Journal Article
Peer reviewed
Open access
Kinases are attractive anticancer targets due to their central role in the growth, survival, and therapy resistance of tumor cells. This review explores the two primary kinase classes, the eukaryotic ...protein kinases (ePKs) and the atypical protein kinases (aPKs), and provides a structure-centered comparison of their sequences, structures, hydrophobic spines, mutation and SNP hotspots, and inhibitor interaction patterns. Despite the limited sequence similarity between these two classes, atypical kinases commonly share the archetypical kinase fold but lack conserved eukaryotic kinase motifs and possess altered hydrophobic spines. Furthermore, atypical kinase inhibitors explore only a limited number of binding modes both inside and outside the orthosteric binding site. The distribution of genetic variations in both classes shows multiple ways they can interfere with kinase inhibitor binding. This multilayered review provides a research framework bridging the eukaryotic and atypical kinase classes.
Despite their low sequence similarity, most atypical kinases share the same characteristic eukaryotic protein kinase fold.The atypical kinases lack the highly conserved kinase motifs (e.g., the GxGxxG motif) and contain a mirrored catalytic HRD motif.Atypical kinases have altered regulatory and catalytic hydrophobic spines compared with the eukaryotic kinases.SNPs and cancer mutations occurring in the catalytic cleft of the kinases are mutually exclusive in location in both atypical and eukaryotic kinases and can interfere with inhibitor binding.Atypical kinase inhibitors currently explore a limited number of binding modes compared with eukaryotic kinase inhibitors.Selective kinase inhibitors can be designed by rationally exploiting selectivity hotspots or by targeting alternative allosteric binding sites.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Protein kinases regulate the majority of signal transduction pathways in cells and have become important targets for the development of designer drugs. We present a systematic analysis of ...kinase–ligand interactions in all regions of the catalytic cleft of all 1252 human kinase–ligand cocrystal structures present in the Protein Data Bank (PDB). The kinase–ligand interaction fingerprints and structure database (KLIFS) contains a consistent alignment of 85 kinase ligand binding site residues that enables the identification of family specific interaction features and classification of ligands according to their binding modes. We illustrate how systematic mining of kinase–ligand interaction space gives new insights into how conserved and selective kinase interaction hot spots can accommodate the large diversity of chemical scaffolds in kinase ligands. These analyses lead to an improved understanding of the structural requirements of kinase binding that will be useful in ligand discovery and design studies.
Malaria continues to pose a significant health threat, causing thousands of deaths each year. The limited availability of vaccines and medications, combined with the emergence of drug resistance, ...further complicates the fight against this disease. In this study, we aimed to enhance the antimalarial potency of the previously reported hit compound BIPPO (pIC
5.9). Through systematic modification of pyrazolopyrimidinone analogs, we discovered the promising analog
(NPD-3547), which exhibited approximately one log unit higher in vitro potency (pIC
6.8) against
. Furthermore, we identified several other BIPPO analogs (
,
,
and
) with potent antimalarial activity (pIC
> 6.0) and favorable metabolic stability in mouse liver microsomes. These compounds can serve as new tools for further optimization towards the development of potential candidates for antimalarial studies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
•A public–private partnership targets drug-binding kinetics in a multilevel approach.•New experimental approaches for measuring drug residence time are presented.•Standardized data formats will ...guarantee sustainability of the data generated.•Progress in QSKR methods as well as mechanistic simulation approaches are discussed.•In vitro to in vivo transition is key for adoption of kinetics for decision support.
A considerable number of approved drugs show non-equilibrium binding characteristics, emphasizing the potential role of drug residence times for in vivo efficacy. Therefore, a detailed understanding of the kinetics of association and dissociation of a target–ligand complex might provide crucial insight into the molecular mechanism-of-action of a compound. This deeper understanding will help to improve decision making in drug discovery, thus leading to a better selection of interesting compounds to be profiled further. In this review, we highlight the contributions of the Kinetics for Drug Discovery (K4DD) Consortium, which targets major open questions related to binding kinetics in an industry-driven public–private partnership.
Drug residence time is becoming increasingly important for decision support in the drug discovery process. Linking industry, SMEs and academia, the K4DD consortium is addressing drug-binding kinetics following a multidisciplinary approach.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including ...the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Conformational biosensors to monitor the activation state of G protein-coupled receptors are a useful addition to the molecular pharmacology assay toolbox to characterize ligand efficacy at the level ...of receptor proteins instead of downstream signaling. We recently reported the initial characterization of a NanoBRET-based conformational histamine H3 receptor (H3R) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with H3R ligands. In the current study, we have further characterized this H3R biosensor on intact cells by monitoring the effect of consecutive ligand injections in time and evaluating its compatibility with photopharmacological ligands that contain a light-sensitive azobenzene moiety for photo-switching. In addition, we have validated the H3R biosensor in membrane preparations and found that observed potency values better correlated with binding affinity values that were measured in radioligand competition binding assays on membranes. Hence, the H3R conformational biosensor in membranes might be a ready-to-use, high-throughput alternative for radioligand binding assays that in addition can also detect ligand efficacies with comparable values as the intact cell assay.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
The histamine H
4
receptor (H
4
R) plays key role in immune cell function and is a highly valued target for treating allergic and inflammatory diseases. However, structural information of H
...4
R remains elusive. Here, we report four cryo-EM structures of H
4
R/G
i
complexes, with either histamine or synthetic agonists clobenpropit, VUF6884 and clozapine bound. Combined with mutagenesis, ligand binding and functional assays, the structural data reveal a distinct ligand binding mode where D94
3.32
and a π-π network determine the orientation of the positively charged group of ligands, while E182
5.46
, located at the opposite end of the ligand binding pocket, plays a key role in regulating receptor activity. The structural insight into H
4
R ligand binding allows us to identify mutants at E182
5.46
for which the agonist clobenpropit acts as an inverse agonist and to correctly predict inverse agonism of a closely related analog with nanomolar potency. Together with the findings regarding receptor activation and G
i
engagement, we establish a framework for understanding H
4
R signaling and provide a rational basis for designing novel antihistamines targeting H
4
R.
The modulation of biological processes with light-sensitive chemical probes promises precise temporal and spatial control. Yet, the design and synthesis of suitable probes is a challenge for ...medicinal chemists. This article introduces a photocaging strategy designed to modulate the pharmacology of histamine H3 receptors (H3R) and H4 receptors (H4R). Employing the photoremovable group BODIPY as the caging entity for two agonist scaffolds—immepip and 4-methylhistamine—for H3R and H4R, respectively, we synthesized two BODIPY-caged compounds, 5 (VUF25657) and 6 (VUF25678), demonstrating 10–100-fold reduction in affinity for their respective receptors. Notably, the caged H3R agonist, VUF25657, exhibits approximately a 100-fold reduction in functional activity. The photo-uncaging of VUF25657 at 560 nm resulted in the release of immepip, thereby restoring binding affinity and potency in functional assays. This approach presents a promising method to achieve optical control of H3R receptor pharmacology.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in ...GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monodonal antibodies. Here we report the isolation and characterization of ...functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.
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