Synthesis and application of the first radioligand targeting the allosteric binding pocket of chemokine receptor CXCR3

Strategies for the identification of allosteric modulators of chemokine receptors largely rely on various cell‐based functional assays. Radioligand binding assays are typically not available for allosteric binding sites. We synthesized, purified, and applied the first tritium‐labeled allosteric modulator of the human chemokine receptor CXCR3 (RAMX3, [³H]N‐{1‐[3‐(4‐ethoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d]pyrimidin‐2‐yl]ethyl}‐2‐[4‐fluoro‐3‐(trifluoromethyl)phenyl]‐N‐[(1‐methylpiperidin‐4‐yl)methyl]acetamide). RAMX3 is chemically derived from 8‐azaquinazolinone‐type allosteric modulators and binds to the CXCR3 receptor with a K_d value of 1.08 nM (specific activity: 80.4 Ci mmol^?1). Radioligand displacement assays showed potent negative cooperativity between RAMX3 and chemokine CXCL11, providing a basis for the use of RAMX3 to investigate other potential allosteric modulators. Additionally, the synthesis and characterization of a number of other full and truncated 8‐azaquinazoline analogues were used to validate the binding properties of RAMX3. We demonstrate that RAMX3 can be efficiently used to facilitate the discovery and characterization of small molecules as allosteric modulators of the CXCR3 receptor.     

Evaluation of a fluorescent derivative of AMD3100 and its interaction with the CXCR4 chemokine receptor

AMD3100 is a potent and selective antagonist of the CXCR4 receptor; it has been shown to block the route of entry of HIV into host T-cells. This compound and its analogues have since been found to act as haematopoietic stem cell mobilisation agents and, more recently, as anti-cancer agents. Here, we have examined a fluorescent derivative of AMD3100, L(1), which offered the potential to assess the behaviour of AMD3100 at the cell surface by using optical imaging modalities. The binuclear Zn(II) , Cu(II) and Ni(II) complexes of L(1) have also been investigated as these metals have been previously shown to enhance the binding properties of AMD3100. Furthermore, Zn(II) and Cu(II) are known to enhance and quench, respectively, the fluorescence of similar anthracenyl-based ligands. Whilst L(1) demonstrates an ability to inhibit the binding of the anti-CXCR4 monoclonal antibody 12G5 (IC(50) =0.25-0.9 μM), the incorporation of an anthracenyl moiety resulted in a significantly reduced affinity for CXCR4 compared to AMD3100 (IC(50) =10 nM). We observed no significant increase in fluorescence intensity following incubation with murine pre-B cells overexpressing CXCR4 compared to a control cell line. This limits the usefulness of L(1) as a fluorescent imaging probe. Interestingly, the Zn(II) complex, which carries an overall +4 charge, revealed marginally higher specificity and reduced toxicity in vitro compared to the free ligand, albeit with reduced affinity for CXCR4 (IC(50) =1.8-5 μM). We suggest that the incorporation of an anthracenyl group contributes to the lipophilic character of the free ligand, thereby resulting in transport across the plasma membrane. This effect is seemingly diminished when the ligand is complexed to charged metal ions.     

Peptide-functionalized luminescent iridium complexes for lifetime imaging of CXCR4 expression

The chemokine receptor 4 (CXCR4) is over-expressed in 23 types of cancer in which it plays a role in, among others, the metastatic spread. For this reason it is a potential biomarker for the field of diagnostic oncology. The antagonistic Ac-TZ14011 peptide, which binds to CXCR4, has been conjugated to luminescent iridium dyes to allow for CXCR4 visualization. The iridium dyes are cyclometalated octahedral iridium(III) 2-phenylpyridine complexes that can be functionalized with one, two or three targeting Ac-TZ14011 peptides. Confocal microscopy and fluorescence lifetime imaging microscopy (FLIM) showed that the peptide-iridium complex conjugates can be used to visualize CXCR4 expression in tumor cells. The CXCR4 receptor affinity and specific cell binding of the mono-, di- and trimeric peptide derivatives were assessed by using flow cytometry. The three derivatives possessed nanomolar receptor affinity and could distinguish between cell lines with different CXCR4 expression levels. This yields the first example of a neutral iridium(III) complex functionalized with peptides for FLIM-based visualization of a cancer associated membrane receptor.     

Design,Synthesis, and Characterization of Novel CXCR4 Antagonists Featuring Cyclic Amines

Chemokine receptor CXCR4 and its natural ligand CXCL12 (also known as stromal cell-derived factor-1, or SDF-1) regulate a broad range of physiological functions. Dysregulation of the CXCL12/CXCR4 axis is involved in numerous pathological conditions such as HIV infection, inflammation and cancer. Herein, we report the design, synthesis, and characterization of novel CXCR4 antagonists based on cyclic amine scaffolds. Compound 24 was identified as a potent CXCR4 receptor antagonist (competitive inhibition of 12G5 binding, IC₅₀=24 nM; functional inhibition of CXCL12-induced cytosolic calcium increase, IC₅₀=0.1 nM). In addition, compound 24 potently inhibited cell migration in CXCR4/CXCL12-mediated chemotaxis in a matrigel invasion assay. The absolute configuration of compound 24 was elucidated by X-ray crystallography.     

基于醛基取代的氟硼二吡咯类荧光母体探针的合成及其应用

荧光探针具有灵敏度高、可实时检测、精准诊断与成像可视化等优点,被广泛应用于生物医药、信息存储、化学分析等领域。氟硼二吡咯(BODIPY)类荧光探针因其优异的光物理化学特性而被广泛设计与开发使用。该文综述了醛基取代BODIPY荧光团的分子设计策略和功能化应用,包括α位醛基-BODIPY、β位醛基-BODIPY、meso位醛基-BODIPY和1,7-位醛基-BODIPY的不同位点醛基调控的BODIPY荧光母体探针及其在阴离子检测、生物硫醇识别及细胞成像等方面的研究进展。设计新型的醛基取代BODIPY探针,未来在精准诊疗上具有发展空间。     

Fluorescent probes and functional materials for biomedical applications

Due to their simplicity in preparation, sensitivity and selectivity, fluorescent probes have become the analytical tool of choice in a wide range of research and industrial fields, facilitating the rapid detection of chemical substances of interest as well as the study of important physiological and pathological processes at the cellular level. In addition, many long-wavelength fluorescent probes developed have also proven applicable for in vivo biomedical applications including fluorescence-guided disease diagnosis and theranostics (e.g., fluorogenic prodrugs). Impressive progresses have been made in the development of sensing agents and materials for the detection of ions, organic small molecules, and biomacromolecules including enzymes, DNAs/RNAs, lipids, and carbohydrates that play crucial roles in biological and disease-relevant events. Here, we highlight examples of fluorescent probes and functional materials for biological applications selected from the special issues “Fluorescent Probes” and “Molecular Sensors and Logic Gates” recently published in this journal, offering insights into the future development of powerful fluorescence-based chemical tools for basic biological studies and clinical translation.     

Synthesis,Structure–Activity Relationship Studies,and ADMET Properties of 3‐Aminocyclohex‐2‐en‐1‐ones as Chemokine Receptor 2 (CXCR2) Antagonists

Herein we describe the synthesis and structure–activity relationships of 3‐aminocyclohex‐2‐en‐1‐one derivatives as novel chemokine receptor 2 (CXCR2) antagonists. Thirteen out of 44 derivatives were found to inhibit CXCR2 downstream signaling in a Tango assay specific for CXCR2, with IC₅₀ values less than 10 μm . In silico ADMET prediction suggests that all active compounds possess drug‐like properties. None of these compounds show significant cytotoxicity, suggesting their potential application in inflammatory mediated diseases. A structure–activity relationship (SAR) map has been generated to gain better understanding of their binding mechanism to guide further optimization of these new CXCR2 antagonists.     

Low‐Molecular‐Weight CXCR4 Ligands with Variable Spacers

Low‐molecular‐weight CXCR4 ligands based on known lead compounds including the 14‐mer peptide T140, the cyclic pentapeptide FC131, peptide mimetics, and dipicolylamine‐containing compounds were designed and synthesized. Three types of aromatic spacers, 1,4‐phenylenedimethanamine, naphthalene‐2,6‐diyldimethanamine, and [1,1′‐biphenyl]‐4,4′‐diyldimethanamine, were used to build four pharmacophore groups. As pharmacophore groups, 2‐pyridylmethyl and 1‐naphthylmethyl are present in all of the compounds, and several aromatic groups and a cationic group from 1‐propylguanidine and 1,1,3,3‐tetramethyl‐2‐propylguanidine were also used. Several compounds showed significant CXCR4 binding affinity, and zinc(II) complexation of bis(pyridin‐2‐ylmethyl)amine moieties resulted in a remarkable increase in CXCR4 binding affinity.     

Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3

The CXCR3 receptor, a class A G protein‐coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure‐based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small‐molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe N‐[({1‐[3‐(4‐ethoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d]pyrimidin‐2‐yl]ethyl}‐2‐[4‐fluoro‐3‐(trifluoromethyl)phenyl]‐N‐{1‐[4‐(3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl]benzyl}piperidin‐4‐yl)methyl]acetamide ( 10 ) showed significant labeling of the CXCR3 receptor (80 %) in a [³H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry.     

Novel Monocyclam Derivatives as HIV Entry Inhibitors: Design,Synthesis, Anti‐HIV Evaluation,and Their Interaction with the CXCR4 Co‐receptor

The CXCR4 receptor has been shown to interact with the human immunodeficiency virus (HIV) envelope glycoprotein gp120, leading to fusion of viral and cell membranes. Therefore, ligands that can attach to this receptor represent an important class of therapeutic agents against HIV, thus inhibiting the first step in the cycle of viral infection: the virus–cell entry/fusion. Herein we describe the in silico design, synthesis, and biological evaluation of novel monocyclam derivatives as HIV entry inhibitors. In vitro activity testing of these compounds in cell cultures against HIV strains revealed EC₅₀ values in the low micromolar range without cytotoxicity at the concentrations tested. Docking and molecular dynamics simulations were performed to predict the binding interactions between CXCR4 and the novel monocyclam derivatives. A binding mode of these compounds is proposed which is consistent with the main existing site‐directed mutagenesis data on the CXCR4 co‐receptor. Moreover, molecular modeling comparisons were performed between these novel monocyclams, previously reported non‐cyclam compounds from which the monocyclams are derived, and the well‐known AMD3100 bicyclam CXCR4 inhibitors. Our results suggest that these three structurally diverse CXCR4 inhibitors bind to overlapping but not identical amino acid residues in the transmembrane regions of the receptor.     

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.     

Comparison of the Response to the CXCR4 Antagonist AMD3100 during the Development of Retinal Organoids Derived from ES Cells and Zebrafish Retina

Retinal organoids generated from human embryonic stem cells or iPSCs recreate the key structural and functional features of mammalian retinal tissue in vitro. However, the differences in the development of retinal organoids and normal retina in vivo are not well defined. Thus, in the present study, we analyzed the development of retinal organoids and zebrafish retina after inhibition of CXCR4, a key role in neurogenesis and optic nerve development, with the antagonist AMD3100. Our data indicated that CXCR4 was mainly expressed in ganglion cells in retinal organoids and was rarely expressed in amacrine or photoreceptor cells. AMD3100 treatment reduced the retinal organoid generation ratio, impaired differentiation, and induced morphological changes. Ganglion cells, amacrine cells, and photoreceptors were decreased and abnormal locations were observed in organoids treated with AMD3100. Neuronal axon outgrowth was also damaged in retinal organoids. Similarly, a decrease of ganglion cells, amacrine cells, and photoreceptors and the distribution of neural outgrowth was induced by AMD3100 treatment in zebrafish retina. However, abnormal photoreceptor ensembles induced by AMD3100 treatment in the organoids were not detected in zebrafish retina. Therefore, our study suggests that although retinal organoids might provide a reliable model for reproducing a retinal developmental model, there is a difference between the organoids and the retina in vivo.     

Fluorescent agonists for the Torpedo nicotinic acetylcholine receptor

We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC-Gly-C6-choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl- and NBD-acylcholine analogues. Binding of DEAC-Gly-C6-choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the alphadelta-binding site and 15.0 nM for the alphagamma-binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug-screening studies.     

Design and Expeditious Synthesis of Quinoline-Pyrene-Based Ratiometric Fluorescent Probes for Targeting Lysosomal pH

Intracellular pH plays a significant role in many pathological and physiological processes. A series of quinoline-pyrene probes were synthesized in one-step fashion through an oxonium-ion-triggered alkyne carboamination sequence involving C−C, C−O and C−N bond formation for intracellular pH sensing. The quinoline-pyrenes showed significant red shifts at low pH. Fluorescence lifetime decay measurements of the probes showed decreases in lifetime at pH 4. The probes showed excellent selectivity in the presence of various potential interfering agents such as amino acids and cations/anions. Furthermore, the probes were found to show completely reversible emission behaviour in the window between pH 4 and 7. A morpholine-substituted quinoline-pyrene probe efficiently stained lysosomes with high Pearson correlation coefficients (0.86) with Lysotracker Deep Red DND-99 as a reference. A co-localization study of the probe with Lysotracker DND-99 showed selective intracellular targeting and a shift in fluorescence emission due to acidic lysosomal pH.     

线粒体靶向型离子荧光探针的研究进展

线粒体在细胞的能量代谢过程中发挥着关键作用。线粒体内含有各种生物活性物质,它们在人体各种生理和病理过程中发挥着重要作用。综述了近年来有机单、双光子线粒体靶向型离子荧光探针的研究现状,重点介绍了荧光探针的设计机理、识别机理、特点以及生物应用,并对该类探针的未来发展趋势进行了展望。