Fluorescent Ligand Research Articles

Synthesis and Pharmacological Characterization of Fluorescent Ligands Targeting the Angiotensin II Receptors Derived from Agonists, β-Arrestin-Biased Agonists, and Antagonists.

Angiotensin II (AngII) regulates cerebral circulation and binds with a similar affinity to AT1 and AT2 receptors. Biased AT1 agonists, such as TRV027, which are able to selectively activate β-arrestin while blocking the Gq pathway, appear promising as new therapeutics. New pharmacological tools are needed to further explore the impact of biased AT1 agonists on cells or tissues, such as the cerebral vessels. We designed and synthesized new fluorescent derivatives based on AngII, TRV027, or the AT1 antagonist losartan. We conducted pharmacological characterization to determine their selectivity, potency, and ability to activate or not specific AT1 transduction pathways in cells and cerebral arteries. We report the first highly AT1-selective fluorescent ligand, based on losartan, that retains its antagonist activity with high affinity. Fluorescent derivatives of TRV027 become AT2-selective and lose their AT1 β-arrestin bias. These new ligands can be applied to trace AT1 or AT2 receptors in vitro and ex vivo.

Synthesis, crystal structure and sulphide ion sensing study of a Cu(II) complex of aroyl hydrazone

Herein, we report a Cu(II) complex {[CuII(HL)(H2O)2]Cl} of a benzoylhydrazone Schiff base ligand (4-((2-benzoylhydrazineylidene)methyl)-3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-1-ium chloride) (H2L). The molar conductance in solution, magnetic susceptibility, ESI-MS and EPR spectroscopy have been performed to justify the structure of the complex. The solid-state structure of the complex has also been solved by single crystal X-ray diffractometry. Cu(II) centre is penta-coordinated with imine-N, phenoxide-O and deprotonated amide-O donor of the ligand and two H2O molecules occupying the coordination sphere forming a distorted square pyramidal geometry (τ = 0.32). We have studied the anion sensing property of the complex by monitoring the changes in the UV–Vis and fluorescence spectra of the complex in aqueous Tris-HCl buffer medium with incremental addition of various anions of tetrabutylammonium salt. The complex shows appreciable sensitivity toward sulphide ion, among various other anions, with association constant (Kb) for binding of the complex with S2− and lowest detection limit (L.O.D) value of 6.87 × 104 M−1 and 6.9 × 10−7 M, respectively. Thus, the complex can be used as an efficient S2− probe. The mechanism of sensing is found to be displacement of the fluorescent ligand from the Cu(II) complex by the sulphide ion.

Rationally Designed G-Quadruplex Selective "Turn-On" NIR Fluorescent Probe with Large Stokes Shift for Nucleic Acid Research-Based Applications.

Guanine-rich DNA/RNA sequences can form Hoogsteen bonds to adopt noncanonical secondary structures called G-quadruplexes, and these have been associated with diverse cellular processes. There has been considerable research interest in the design of G4-interacting ligands for cellular probing of the G4 structure and understanding its associated biological function. Most of the fluorescent G4 ligands either do not have significant selectivity over other nucleic acid structures, have high Stokes shift, or are not in the near-infrared (NIR) region, which limits its cellular visualization. The current work involves the rational design and synthesis of NIR fluorescent probes comprising a (Z)-1-methyl-2-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium scaffold. Among the designed molecules, 4a exhibited far-red fluorescence (λmax = 680 nm) with large Stokes shift (∼182 nm) upon selective binding to human telomeric G-quadruplexes. The dye 4a does not disturb the conformation and stability of G-quadruplexes, thereby making it suitable for nucleic acid research based applications. Interestingly, 4a showed remarkable selectivity over single- and double-stranded structures in contrast to a commercially available quadruplex binding probe, Thiazole orange (TO). The molecular docking studies indicate that 4a binds at the groove region of the telomeric DNA G-quadruplex through π-π stacking interactions with the quinoline and amine-substituted phenyl ring and with the phosphate backbone through anion-π interactions with the benzothiazole ring. The designed molecule 4a has interesting photophysical properties, cell permeability, and biocompatibility with minimal cytotoxicity. Fluorescence imaging studies in live HeLa cells showed that probe 4a binds to the transient population of the DNA G-quadruplex in the nucleus and RNA quadruplexes in the cytoplasm. In brief, G-quadruplex NIR fluorescent probe 4a with a higher signal/noise ratio has significant potential for cellular imaging studies and thus opens avenues to decipher the biological pathways for better understanding of G-quadruplex biology.

Developing Porous Protein Cage Nanoparticles as Cargo-Loadable and Ligand-Displayable Modular Delivery Nanoplatforms.

Protein cage nanoparticles, self-assembled from protein subunits, provide distinct exterior and interior spaces and can carry diagnostic and/or therapeutic cargo agents through chemical conjugation, in vitro disassembly/reassembly process, or assembly-mediated encapsulation. Here, we developed porous SpyCatcher-mi3 (SC-mi3) as modular delivery nanoplatforms, capable of loading cargos through pores and displaying targeting ligands using SpyCatchers (SC) as anchors for SpyTagged (ST) ligands. Fluorescent dyes (F5M and A647) and a pH-sensitive prodrug (Aldox) were conjugated to the interior surface cysteines of SC-mi3, forming F5M@SC-mi3, A647@SC-mi3, and Aldox@SC-mi3. Subsequently, EGFR-binding affibody molecules (EGFRAfb) were displayed on the exterior surface of F5M@SC-mi3 and Aldox@SC-mi3 using the SC/ST protein ligation system, forming F5M@mi3/EGFRAfb and Aldox@mi3/EGFRAfb, respectively. F5M@mi3/EGFRAfb selectively bound to EGFR-overexpressing MDA-MB-468 cells, visualizing the target cancer cells, while Aldox@mi3/EGFRAfb selectively delivered doxorubicin, leading to target-specific cancer cell death. To encapsulate large proteins within SC-mi3, biotins were initially conjugated to the interior surface (BPM@SC-mi3) and mSA2-fused protein cargo molecules (mSA2-HaloTag and mSA2-yCD) were successfully introduced through the pores and securely encapsulated, forming TMR-H@SC-mi3 and yCD@SC-mi3, respectively. Subsequent display of EGFRAfb on their surface allowed the visualization of target cancer cells using fluorescent HaloTag ligand labeling and facilitated the killing of target cancer cells by converting the prodrug 5-FC to the cytotoxic drug 5-FU. Modular functionalization of the two distinct spaces in porous SC-mi3 may offer opportunities for developing target-specific functional cargo-delivery nanoplatforms in biomedical fields.

Measuring Calcium-Sensing Receptor Agonist-Driven Insertional Signaling (ADIS) and Trafficking by TIRF Microscopy.

The calcium-sensing receptor (CaSR), which regulates parathyroid hormone secretion by sensing serum calcium concentrations, has developed unique trafficking mechanisms to respond to constant exposure to its orthosteric ligand calcium. CaSR rapidly responds to fluctuations in serum calcium by driving forward trafficking of the receptor to cell surfaces in a mechanism known as agonist-driven insertional signaling (ADIS). This increase in CaSR at cell surfaces is counterbalanced by both constitutive and agonist-driven internalization of the receptor. Deciphering these mechanisms is important to understand how mutations in the CaSR and components of its signaling and trafficking pathways cause human disorders of calcium homeostasis.This chapter describes a protocol to measure CaSR ADIS and endocytosis in parallel using total internal reflection fluorescence (TIRF) microscopy. This utilizes a mammalian expression construct comprising the full-length human CaSR with an N-terminal bungarotoxin minimal-binding site that can be labeled with commercially available fluorescent ligands to measure endocytosis, and a super-ecliptic pHluorin (SEP) to measure total cell surface expression and exocytic events. This protocol could easily be adapted to simultaneously assess forward trafficking and endocytosis of other membrane proteins by TIRF microscopy.

Novel Far-Red Fluorescent 1,4-Dihydropyridines for L-Type Calcium Channel Imaging.

Upregulation of L-type calcium channels (LTCCs) is implicated in a range of cardiovascular and neurological disorders. Therefore, the development of toolboxes that unlock fast imaging protocols in live cells is coveted. Herein, we report a library of first-in-class far-red small-molecule-based fluorescent ligands (FluoDiPines), able to target LTCCs. All fluorescent ligands were evaluated in whole-cell patch-clamp and live-cell Ca2+ imaging whereby FluoDiPine 6 was found to be the best candidate for live-cell fluorescence imaging. Low concentration of FluoDiPine 6 (50 nM) and a quick labeling protocol (5 min) are successfully applied to fixed and live cells to image LTCCs with good specificity.

Dually Labeled Neurotensin NTS1R Ligands for Probing Radiochemical and Fluorescence-Based Binding Assays.

The determination of ligand-receptor binding affinities plays a key role in the development process of pharmaceuticals. While the classical radiochemical binding assay uses radioligands, fluorescence-based binding assays require fluorescent probes. Usually, radio- and fluorescence-labeled ligands are dissimilar in terms of structure and bioactivity, and can be used in either radiochemical or fluorescence-based assays. Aiming for a close comparison of both assay types, we synthesized tritiated fluorescent neurotensin receptor ligands ([3H]13, [3H]18) and their nontritiated analogues (13, 18). The labeled probes were studied in radiochemical and fluorescence-based (high-content imaging, flow cytometry, fluorescence anisotropy) binding assays. Equilibrium saturation binding yielded well-comparable ligand-receptor affinities, indicating that all these setups can be used for the screening of new drugs. In contrast, discrepancies were found in the kinetic behavior of the probes, which can be attributed to technical differences of the methods and require further studies with respect to the elucidation of the underlying mechanisms.

Flexible electrospun test strips functionalized with a graphene oxide/doxorubicin complex for dopamine detection

A novel flexible test strip has been developed for the selective and sensitive detection of dopamine (DA) using an electrospun polyacrylonitrile nanofiber membrane as a substrate and doxorubicin (DOX)-functionalized graphene oxide (GO) as a fluorescent ligand. Employing the Langmuir-Blodgett technique, the fluorescent probe of GO/DOX was controllably and flatly applied onto the surface of nanofiber membranes. The adsorption behavior of DA and DOX on GO in solution and on the surface of solid test strips has been investigated, respectively. The results showed that DA exhibited a higher affinity for GO, both in solution and on the surface of test strips, compared to DOX. Due to the competitive adsorption of DOX and DA onto GO, the addition of DA to the GO/DOX complex led to a significant fluorescence enhancement, attributed to the replacement of the GO-supported DOX with DA of a higher adsorption affinity to GO, enabling the detection of DA. This nanofiber-based method proves to be simple, sensitive, selective, and effectively minimizes interference from common biomolecules and small molecules in serum. Thus, this portable, cost-effective, and selective nanofiber strip holds great promise for early diagnosis of neurodegenerative diseases by measuring DA levels.

In Vitro Screening for ToxCast Chemicals Binding to Thyroxine-Binding Globulin.

Thyroid hormone (TH) carrier proteins play an important role in distributing TH to target tissue as well as maintaining the balance of free versus bound TH in the blood. Interference with the TH carrier proteins has been identified as a potential mechanism of thyroid system disruption. To address the lack of data regarding chemicals binding to these carrier proteins and displacing TH, a fluorescence-based in vitro screening assay was utilized to screen over 1,400 chemicals from the U.S. EPA's ToxCast phase1_v2, phase 2, and e1k libraries for competitive binding to one of the carrier proteins, thyroxine-binding globulin. Initial screening at a single high concentration of 100 μM identified 714 chemicals that decreased signal of the bound fluorescent ligand by 20% or higher. Of these, 297 produced 50% or greater reduction in fluorescence and were further tested in concentration-response (0.004 to 150 μM) to determine relative potency. Ten chemicals were found to have EC50 values <1 μM, 63 < 10 μM, and 141 chemicals between 10 and 100 μM. Utilization of this assay contributes to expanding the number of in vitro assays available for identifying chemicals with the potential to disrupt TH homeostasis. These results support ranking and prioritization of chemicals to be tested in vivo to aid in the development of a framework for predicting in vivo effects from in vitro high-throughput data.

IS4-FAM, a fluorescent tool to study CXCR4 affinity and competitive antagonism in native cancer cells.

The ability to accurately measure drug-target interaction is critical for the discovery of new therapeutics. Classical pharmacological bioassays such as radioligand or fluorescent ligand binding assays can define the affinity or Kd of a ligand for a receptor with the lower the Kd, the stronger the binding and the higher the affinity. However, in many drug discovery laboratories today, the target of interest if often artificially upregulated by means of transfection to modify the host cell's genetic makeup. This then potentially invalidates the assumptions of classical pharmacology affinity calculations as the receptor of interest is no longer at normal physiological densities. The CXCR4 receptor is expressed on many different cancer cell types and is associated with metastasis and poor prognosis. Therefore, the CXCR4 receptor is a desirable target for novel therapeutics. In this study, we explore the applicability of the newly developed fluorescently tagged CXCR4 antagonists, IS4-FAM as an investigative tool to study CXCR4 affinity and competitive antagonism in native, non-transfected cancer cells using confocal microscopy and flow cytometry. IS4-FAM directly labels CXCR4 in several cell lines including high CXCR4 expressing SK-MEL-28 (malignant melanoma) and PC3 (metastatic prostate cancer) and lower CXCR4 expressing THP-1 (acute monocytic leukemia) and was competitive with the established CXCR4 antagonist, AMD3100. This highlights the potential of IS4-FAM as a pharmacological tool for drug discovery in native cells lines and tissues.

Regulation on the ligand fluorescence in UiO-66-Y metal-organic frameworks for effective detection of nitro-aromatic explosives

Rapid, sensitive and highly selective detection of nitro-aromatic explosives (NAEs) has become one of the most pressing environmental and safety issues. Metal-organic frameworks (MOFs) offer new possibilities for the development of new photoactive materials with excellent sensing properties. In this paper, three robust UiO-66-type fluorescent MOFs were prepared from the self-assembly of yttrium cations (Y3+) and linear dicarboxylic acid ligands, named terephthalic acid (BDC), 2-hydroxyterephthalic acid (OHBDC) and 2,5-dihydroxyterephthalic acid (DHBDC). The solid and solution fluorescence properties of three MOFs were successfully modulated by regulating the number of –OH functional groups from zero to two. At 370 nm excitation, Y-BDC MOF without –OH group was almost non-fluorescent, Y-OHBDC MOF with one –OH group emitted blue fluorescence, and Y-DHBDC MOF with two –OH groups emitted green fluorescence. Thanks to the suitable porous structures and stable frameworks, Y-OHBDC and Y-DHBDC MOFs both can quantificationally detect at least seven nitro-aromatic explosives (NAEs) by a fluorescence quenching process. Remarkably, the detection performance for 2,4,6-trinitrophenol (TNP) is the most outstanding with the KSV values of 8.4 × 104 M−1 and 2.1 × 105 M−1. Fast response, high sensitivity, strong anti-interference ability and good reusability make UiO-66-Y MOFs potential fluorescent sensors for TNP.

Fluorescence Turn-Off Ligand for Parallel G-Quadruplexes.

Parallel-stranded G-quadruplex structures are found to be common in the human promoter sequences. We tested highly fluorescent 9-methoxyluminarine ligand (9-MeLM) binding interactions with different parallel G-quadruplexes DNA by spectroscopic methods such as fluorescence and circular dichroism (CD) titration as well as UV melting profiles. The results showed that the studied 9-MeLM ligand interacted with the intramolecular parallel G-quadruplexes (G4s) with similar affinity. The binding constants of 9-methoxyluminarine with different parallel G4s were determined. The studies upon oligonucleotides with different flanking sequences on c-MYC G-quadruplex suggest that 9-methoxyluminarine may preferentially interact with 3'end of the c-MYC promoter. The high decrease in 9-MeLM ligand fluorescence upon binding to all tested G4s indicates that 9-methoxyluminarine molecule can be used as a selective fluorescence turn-off probe for parallel G-quadruplexes.

Synthesis and Characterization of Folic Acid-Conjugated Terbium Complexes as Luminescent Probes for Targeting Folate Receptor-Expressing Cells.

Several conjugates between folic acid and a series of kinetically stable lanthanide complexes have been synthesized, using amide coupling and azide-alkyne cycloaddition methodologies to link the metal-binding domain to folate through a variety of spacer groups. While all these complexes exhibit affinity for the folate receptor, it is clear that the point of attachment to folate is essential, with linkage through the γ-carboxylic acid giving rise to significantly enhanced receptor affinity. All the conjugates studied show affinities consistent with displacing biological circulating folate derivatives, 5-methyltetrahydrofolate, from folate receptors. All the complexes exhibit luminescence with a short-lived component arising from ligand fluorescence overlaid on a much longer lived terbium-centered component. These can be separated using time-gating methods. From the results obtained, the most promising approach to achieve sensitized luminescence in these systems requires incorporating a sensitizing chromophore close to the lanthanide.

Robust G‐Quadruplex Dimer‐Guided Transmembrane DNA Nanovehicles for Targeted Payload Delivery

Here we report a robust G‐quadruplex (G4) dimer‐guided transmembrane DNA nanovehicle for targeted payload delivery, based on dimeric G4‐proximized aptamers that efficiently target transferrin receptors (TfR) over‐expressed on cancer cell surface. A monomolecular G4 (mono‐G4) named 93del that stably forms the interlocked dimeric structure is employed as the controlling module, which responds to environmental K+ and thereby holds two TfR aptamers at the proximity state. It endows the designed DNA system with over 3‐fold enhancement in cellular internalization, enabling the cancer‐specific delivery of fluorescent G4 ligands and therapeutic drugs for cellular imaging and treatment.

Development of a fluorescent ligand that specifically binds to the c-MYC G-quadruplex by migrating the benzene group on a carbazole-benzothiazolium scaffold

c-MYC is one of the most important oncogenes, which is overexpressed in many cancers, and is highly related to development, metastasis, and drug resistance of cancers. The G4 structure in the promoter of c-MYC oncogene contributes a lot to the gene transcriptional mechanism. Small-molecule ligands binding to the c-MYC G4 appear to be a new class of anticancer agents. However, selective ligands for the c-MYC G4 over other G4s have been rarely reported. In this study, we reported a novel fluorescent ligand by migrating the benzene group on a carbazole-benzothiazolium scaffold, which was demonstrated to exhibit considerable specificity to the c-MYC G4, which was distinguished from other small-molecule ligands. The further cellular experiments suggested that this ligand may indeed target the promoter G4 and cause apparent transcriptional inhibition of the c-MYC oncogene instead of other G4-mediated oncogenes, which thereby resulted in cancer cell growth inhibition. Collectively, this study provided a good example for developing specific c-MYC G4 ligands, which may further develop into an effective anticancer agent that inhibit the c-MYC expression.

Subtype-Specific Ligand Binding and Activation Gating in Homomeric and Heteromeric P2X Receptors.

P2X receptors are ATP-activated, non-specific cation channels involved in sensory signalling, inflammation, and certain forms of pain. Investigations of agonist binding and activation are essential for comprehending the fundamental mechanisms of receptor function. This encompasses the ligand recognition by the receptor, conformational changes following binding, and subsequent cellular signalling. The ATP-induced activation of P2X receptors is further influenced by the concentration of Mg2+ that forms a complex with ATP. To explore these intricate mechanisms, two new fluorescently labelled ATP derivatives have become commercially available: 2-[DY-547P1]-AHT-ATP (fATP) and 2-[DY-547P1]-AHT-α,βMe-ATP (α,βMe-fATP). We demonstrate a subtype-specific pattern of ligand potency and efficacy on human P2X2, P2X3, and P2X2/3 receptors with distinct relations between binding and gaiting. Given the high in vivo concentrations of Mg2+, the complex formed by Mg2+ and ATP emerges as an adequate ligand for P2X receptors. Utilising fluorescent ligands, we observed a Mg2+-dependent reduction in P2X2 receptor activation, while binding remained surprisingly robust. In contrast, P2X3 receptors initially exhibited decreased activation at high Mg2+ concentrations, concomitant with increased binding, while the P2X2/3 heteromer showed a hybrid effect. Hence, our new fluorescent ATP derivatives are powerful tools for further unravelling the mechanism underlying ligand binding and activation gating in P2X receptors.

Efficient application of Pyrosin B for nano-gram level assay of antiparkinsonian medication, rasagiline: Evaluation of tablets and content uniformity.

Rasagiline (RAS) is a medication for Parkinson's disease that increases dopamine levels in the brain by inhibiting monoamine oxidase, helping to alleviate symptoms. The proposed study aims to develop an efficient, feasible, and sensitive method for RAS assay, utilizing Pyrosin B dye, a convenient fluorescent ligand. Combining the RAS analyte with Pyrosin B ligand in a mildly acidic buffered solution rapidly quenches the native fluorescence of the ligand. This quenching results from the formation of a specific ion-dipole association complex between the lone pair-bearing atoms of the ligand and the protonated amine moiety of RAS, highlighting their interactive chemistry under these conditions. The degree of this interaction demonstrated superior sensitivity compared with reported alternatives, exhibiting a linear range of 50.0 to 1000.0ng/mL. The method is characterized by a limit of detection (LOD) of 16.0ng/mL and a limit of quantification (LOQ) of 48.0ng/mL. By optimizing the RAS-Pyrosin B system, the variable parameters were finely tuned, ensuring the assay method's reliability. The method's accuracy, precision, selectivity, and robustness were validated according to International Council for Harmonization (ICH) guidelines, enabling precise and efficient analysis of RAS in the nanogram range. This method streamlines the analysis procedure and reduces environmental impact, making it a promising approach for the quality control of ParkintreatR tablets (1mg) and other analytical applications.

A 1,8-Naphthalimide-based Tripodal Fluorescent Chemosensor to Selectively Detect Copper Ions.

A 1,8-naphthalimide-based tripodal fluorescent ligand (L3) was synthesized through the copper (I) catalyzed Huisgen azide-alkyne cycloaddition reaction of 2-(2-azidoethyl)-6-morpholino-1H-benzo[de]isoquinoline-1,3(2H)-dione with triproparagylamine. Naphthalimide acts as the fluorophore while the triazole and amine nitrogens chelate the metal ion. L3 showed a selective fluorescence turn-off for Cu(II) over other metal ions in aqueous acetonitrile solution. A Job's plot, Benesi-Hildbrand plot and high-resolution mass spectrometry data confirm a 1:1 binding stoichiometry with a binding constant of 7.8 х105 M- 1 while addition of disodium EDTA demonstrates its reversibility. The structure and stability of the complex was supported by theoretical calculations. The limit of detection for Cu(II) was calculated to be 0.3 µM which is considerably lower than WHO recommended Cu(II) limit in drinking water.

Ligand-Directed Labeling of the Adenosine A1 Receptor in Living Cells.

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A1AR in living cells. We pharmacologically demonstrate covalent labeling of A1AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A1AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A1ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A1AR in more physiologically relevant environments.

Development of a NanoBRET Assay Platform to Detect Intracellular Ligands for the Chemokine Receptors CCR6 and CXCR1.

A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein-coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET-based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno-oncology. Our assay platform enables cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1/CXCR2/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1/CXCR2 antagonist and phase II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound 10, a previously reported tert-butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. Further, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF-07054894, currently evaluated in phase I clinical trials for the treatment of ulcerative colitis, thereby providing profound evidence for the existence and the pharmacological relevance of a druggable IABS at CCR6.