Cellular Binding Research Articles

Characterization of two Plasmodium falciparum lipid transfer proteins of the Sec14/CRAL-TRIO family

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is followed by dramatic modifications of erythrocytes properties, including de novo formation of new membrane systems. Lipid transfer proteins from both the parasite and the host cell are most likely an important part of those membrane remodeling processes. Using bioinformatics and in silico structural analysis, we have identified five P. falciparum potential lipid transfer proteins containing cellular retinaldehyde binding – triple functional domain (CRAL-TRIO). Two of these proteins, C6KTD4, encoded by the PF3D7_0629900 gene and Q8II87, encoded by the PF3D7_1127600 gene, were studied in more detail. In vitro lipid transfer assays using recombinant C6KTD4 and Q8II87 confirmed that these proteins are indeed bona fide lipid transfer proteins. C6KTD4 transfers sterols, phosphatidylinositol 4,5 bisphosphate, and, to some degree, also phosphatidylcholine between two membrane compartments. Q8II87 possesses phosphatidylserine transfer activity in vitro. In the yeast model, the expression of P. falciparumQ8II87 protein partially complements the absence of Sec14p and its closest homologue, Sfh1p. C6KTD4 protein can substitute for the collective essential function of oxysterol-binding related proteins. According to published whole genome studies in P. falciparum, absence of C6KTD4 and Q8II87 proteins has severe consequences for parasite viability. Therefore, CRAL-TRIO lipid transfer proteins of P. falciparum are potential targets of novel antimalarials, in search for which the yeast model expressing these proteins could be a valuable tool.

Structural requirements for the specific binding of CRABP2 to cyclin D3.

Cellular retinoic acid binding protein 2 (CRABP2) transports retinoic acid from the cytoplasm to the nucleus where it then transfers its cargo to retinoic acid receptor-containing complexes leading to activation of gene transcription. We demonstrate using purified proteins that CRABP2 is also a cyclin D3-specific binding protein and that the CRABP2 cyclin D3 binding site and the proposed CRABP2 nuclear localization sequence overlap. Both sequences are within the helix-loop-helix motif that forms a lid to the retinoic acid binding pocket. Mutations within this sequence that block both cyclin D3 and retinoic acid binding promote formation of a CRABP2 structure in which the retinoic acid binding pocket is occupied by an alternative lid conformation. Structural and functional analysis of CRABP2 and cyclin D3 mutants combined with AlphaFold models of the ternary CDK4/6-cyclin D3-CRABP2 complex supports the identification of an α-helical protein binding site on the cyclin D3 C-terminal cyclin box fold.

Pd(II)/1,10-phenanthroline complexes bearing arene ligands: On the role of N- vs O-coordination to tune their cellular activity and binding ability towards DNA and RNA

Three Pd(II)-based complexes of 1,10-phenanthroline and N- or O-coordinating ligands have been synthesised and tested with different relevant biosubstrates like double-stranded DNA, double and triple helix of RNA, DNA G-quadruplexes of different conformations and bovine serum albumin. Here a correlation between N- vs O-coordinating elements and binding mechanism emerged, where the N-coordinating ligands proved to be the most promising. These outcomes were confirmed also in the cellular experiments. The Pd(II) complex with naphthalene-1,8-diamine is the one that is able to be carried by BSA, to strongly bind nucleic acids, to produce reactive oxygen species (ROS) and to show the best cellular performances (poorly toxic towards healthy cells and highly toxic against the cisplatin-resistant cancer cell line). On the opposite, the complex with benzene-1,2-diolate may be sequestered by BSA, weakly binds nucleic acids, does not produce ROS and shows poor cellular activity. The complex with benzene-1,2-diamine stays in between. Other mechanistic details are discussed which show that the biophysical behaviour is the sum of the contribution of aromaticity, charge and N- or O-coordination.

Oligomerization-driven avidity correlates with SARS-CoV-2 cellular binding and inhibition

Cellular processes are controlled by the thermodynamics of the underlying biomolecular interactions. Frequently, structural investigations use one monomeric binding partner, while ensemble measurements of binding affinities generally yield one affinity representative of a 1:1 interaction, despite the majority of the proteome consisting of oligomeric proteins. For example, viral entry and inhibition in SARS-CoV-2 involve a trimeric spike surface protein, a dimeric angiotensin-converting enzyme 2 (ACE2) cell-surface receptor and dimeric antibodies. Here, we reveal that cooperativity correlates with infectivity and inhibition as opposed to 1:1 binding strength. We show that ACE2 oligomerizes spike more strongly for more infectious variants, while exhibiting weaker 1:1 affinity. Furthermore, we find that antibodies use induced oligomerization both as a primary inhibition mechanism and to enhance the effects of receptor-site blocking. Our results suggest that naive affinity measurements are poor predictors of potency, and introduce an antibody-based inhibition mechanism for oligomeric targets. More generally, they point toward a much broader role of induced oligomerization in controlling biomolecular interactions.

L-Type amino acid transporter 1-targeting nanoparticles for antisense oligonucleotide delivery to the CNS

L-type amino acid transporter 1 (LAT1)-specific ligands and polyion complexes are used as brain-specific targets to deliver RNA-based drugs across the blood-brain barrier. We characterized a LAT1-targeting antisense oligonucleotide (ASO) encapsulated nanoparticle, “Phe-NPs/ASO.” A 25% density of phenylalanine effectively binds to the surface of LAT1 targeting nanoparticles in the GL261-Luc cells and Phe-NPs/ASO showed higher binding affinity compared to that without phenylalanine by cellular binding assay. To further characterize the blood-brain barrier-targeting effect and tissue distribution following a single-dose intravenous injection in mice, we performed in vivo biodistribution studies using fluorescence imaging. The Phe-NPs/ASO were detected in the brain tissue 1 h post-intravenous at an approximately 64-fold higher ratio than that of the same ASOs administered in the absence of any nanoparticle carrier. The brain tissue delivery of antisense oligonucleotide-loaded Phe-NPs was also confirmed in a fluorescence imaging study performed in non-human primates. These results demonstrate that Phe-NPs may successfully deliver an ASO to the brain tissue across brain regions. Phe-nanoparticles loaded with RNA-based drugs have the potential to treat diseases of the central nervous system, including all forms of neurodegenerative diseases.

Searching for Protein Off-Targets of Prostate-Specific Membrane Antigen-Targeting Radioligands in the Salivary Glands.

Background: Prostate specific membrane antigen (PSMA)-targeted radioligand therapies represent a highly effective treatment for metastatic prostate cancer. However, high and sustain uptake of PSMA-ligands in the salivary glands led to dose limiting dry mouth (xerostomia), especially with α-emitters. The expression of PSMA and histologic analysis couldn't directly explain the toxicity, suggesting a potential off-target mediator for uptake. In this study, we set out to search for possible off-target non-PSMA protein(s) in the salivary glands. Methods: A machine-learning based quantitative structure activity relationship (QSAR) model was built for seeking the possible off-target(s). The resulting target candidates from the model prediction were subjected to further analysis for salivary protein expression and structural homology at key regions required for PSMA-ligand binding. Furthermore, cellular binding assays were performed utilizing multiple cell lines with high expression of the candidate proteins and low expression of PSMA. Finally, PSMA knockout (PSMA-/-) mice were scanned by small animal PET/MR using [68Ga]Ga-PSMA-11 for in-vivo validation. Results: The screening of the trained QSAR model did not yield a solid off-target protein, which was corroborated in part by cellular binding assays. Imaging using PSMA-/- mice further demonstrated markedly reduced PSMA-radioligand uptake in the salivary glands. Conclusion: Uptake of the PSMA-targeted radioligands in the salivary glands remains primarily PSMA-mediated. Further investigations are needed to illustrate a seemingly different process of uptake and retention in the salivary glands than that in prostate cancer.

Facile Rebridging Conjugation Approach to Attain Monoclonal Antibody-Targeted Nanoparticles with Enhanced Antigen Binding and Payload Delivery.

Adopting conventional conjugation approaches to construct antibody-targeted nanoparticles (NPs) has demonstrated suboptimal control over the binding orientation and the structural stability of monoclonal antibodies (mAbs). Hitherto, the developed antibody-targeted NPs have shown proof of concept but lack product homogeneity, batch-to-batch reproducibility, and stability, precluding their advancement toward the clinic. To circumvent these limitations and advance toward clinical application, herein, a refined approach based on site-specific construction of mAb-immobilized NPs will be appraised. Initially, the conjugation of atezolizumab (anti-PDL1 antibody, Amab) with polymeric NPs was developed using bis-haloacetamide (BisHalide) rebridging chemistry, followed by click chemistry (NP-Fab BisHalide Ab and NP-Fc BisHalide Ab). For comparison purposes, mAb-immobilized NPs developed utilizing conventional conjugation methods, namely, N-hydroxysuccinimide (NHS) coupling and maleimide chemistry (NP-NHS Ab and NP-Mal Ab), were included. Next, flow cytometry and confocal microscopy experiments evaluated the actively targeted NPs (loaded with fluorescent dye) for cellular binding and uptake. Our results demonstrated the superior and selective binding and uptake of NP-Fab BisHalide Ab and NP-Fc BisHalide Ab into EMT6 cells by 19-fold and 13-fold, respectively. To evaluate the PDL1-dependent cell uptake and the selectivity of the treatments, a blocking step of the PDL1 receptor with Amab was performed prior to incubation with NP-Fab BisHalide Ab and NP-Fc BisHalide Ab. To our delight, the binding and uptake of fluorescent NPs were reduced significantly by 3-fold for NP-Fab BisHalide Ab, demonstrating the PDL1-mediated uptake. Moreover, NP-Fab BisHalide Ab and NP-Fc BisHalide Ab were entrapped with the paclitaxel payload, and their cytotoxicity was evaluated. They showed significant enhancements compared to free paclitaxel and NP-NHS Ab. Overall, this work will provide a facile conjugation method that could be implemented to actively target NPs with a plethora of therapeutic mAbs approved for various malignancies.

The importance of radiochemical purity: Cellular binding and internalization of different radiometal chlorides in prostate cancer cells

Radiometals play an important role in nuclear medicine, both for imaging and therapy. Binding studies represent an important step in the development of new radiolabeled ligands, as valuable insights into the binding properties can be gained. However, this technique requires high radiochemical purity, otherwise results may lead to wrong assumptions or misinterpretations of affinities or uptake rates.Therefore, this in vitro study aimed at investigating the cell binding and internalization characteristics of different radiometal chlorides ([111In]InCl3, [68Ga]GaCl3 and [177Lu]LuCl3) commonly applied in nuclear medicine, as well as the clinically applied [177Lu]Lu-PSMA-I&T in comparison, by using prostate cancer cells. PC-3 and LNCaP cells were incubated with 100 kBq of the respective radiometal chloride or [177Lu]Lu-PSMA-I&T for 1 h. For [177Lu]LuCl3, nuclei isolations and colloid determinations in saline and cell medium were also performed.Results showed that [111In]InCl3 and [68Ga]GaCl3 bind and are internalized up to 3 % to PC-3 and LNCaP cells, whereas [177Lu]LuCl3 showed cell binding of up to 25 %, internalization up to 2.5 % and a nuclear uptake below 0.3 %. In comparison, [177Lu]Lu-PSMA-I&T showed only 3 % total cell binding to LNCaP cells. Further analysis of [177Lu]LuCl3 stability in NaCl and cell medium showed only low amounts of colloids, which are not increasing over time, and negligible unspecific binding to the used cell culture plates.In conclusion, the results demonstrate the importance of high radiochemical purity, especially with regard to Lu-177 labeled compounds. Even if radiopharmaceuticals comply with common release-criteria, significant uptake can be derived from [177Lu]LuCl3 impurities and lead to wrong estimations of a compound's uptake behavior. Assuming an experimental result of 2 % membrane binding of the applied product, and 5 % residual [177Lu]LuCl3 in the final product (thereof 25 % membrane binding, as described above), would lead to 1.25 % membrane binding resulting from [177Lu]LuCl3 and only 0.75 % from the radiopharmaceutical.

A Multifunctional Nanostructured Hydrogel as a Platform for Deciphering Niche Interactions of Hematopoietic Stem and Progenitor Cells.

For over half a century, hematopoietic stem cells (HSCs) have been used for transplantation therapy to treat severe hematologic diseases. Successful outcomes depend on collecting sufficient donor HSCs as well as ensuring efficient engraftment. These processes are influenced by dynamic interactions of HSCs with the bone marrow niche, which can be revealed by artificial niche models. Here, a multifunctional nanostructured hydrogel is presented as a 2D platform to investigate how the interdependencies of cytokine binding and nanopatterned adhesive ligands influence the behavior of human hematopoietic stem and progenitor cells (HSPCs). The results indicate that the degree of HSPC polarization and motility, observed when cultured on gels presenting the chemokine SDF-1α and a nanoscale-defined density of a cellular (IDSP) or extracellular matrix (LDV) α4β1 integrin binding motif, are differentlyinfluenced on hydrogels functionalized with the different ligand types. Further, SDF-1α promotes cell polarization but not motility. Strikingly, the degree of differentiation correlates negatively with the nanoparticle spacing, which determines ligand density, but only for the cellular-derived IDSP motif. This mechanism potentially offers a means of predictably regulating early HSC fate decisions. Consequently, the innovative multifunctional hydrogel holds promise for deciphering dynamic HSPC-niche interactions and refining transplantation therapy protocols.

Transcriptomic analysis of the response mechanisms of black rockfish (Sebastes schlegelii) under noise stress from offshore wind farms

During the operational phase of offshore wind farms, the generation of low-frequency underwater noise has received widespread attention due to its potential adverse impact on fish health. This study conducted a field survey of underwater noise at offshore wind farms located in Shandong province, China. Subsequently, a small-scale experiment was conducted to study the stress on black rockfish (Sebastes schlegelii). The fish were exposed to noise with dominant frequency of 80 Hz, 125 Hz and 250 Hz. These frequencies are same with the frequencies from wind power noise (wpn) at the actual site. After a 40-day experimental period, transcriptome sequencing was conducted on brain, liver, and kidney tissues of black rockfish to elucidate the underlying molecular mechanisms involved in the response to noise stress originating from offshore wind farms. The results revealed that the 125 Hz group exhibited the highest number of differentially expressed genes (DEGs) between the noise-exposed and control check group (CK group), with a total of 797 in the brain, 1076 in the liver, and 2468 in the kidney. Gene Ontology (GO) analysis showed that DEGs were significantly enriched in entries related to cellular processes, membrane components, binding, and metabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were enriched mainly in metabolism, immunity, apoptosis, signal transduction, and diseases. The findings indicate that prolonged exposure to underwater noise from offshore wind farms may induce metabolic imbalance, immune dysfunction, and an increased risk of myocardial diseases in black rockfish.

The role of palmitoylation of the insulin receptor in insulin resistance: A hypothesis

Palmitoylation is attachment of a palmitoyl group to the cysteine residues of a protein. The intermediary in this process is Palmitoyl CoA, which is formed during the activation of fatty acids in the beta oxidationn of the palmitic acid, consequent to the shift of the energy metabolism to beta oxidation from glycolysis, in DM2.The insulin receptor has cysteine rich domains in the cytosolic portion of the beta sub unit connected by disulfide bonds. It is hypothesized that the palmitoylation forms a thio-ester bond with cystein residues of the beta sub units of the insulin receptor. Depalmitoylation results in substitution of thiol (-SH) in place of the disulfide (S-S) bonds (S-S), due to the hydrolysis by the thio-esterase enzyme. The disulfide bonds are responsible for formation of the functional tettameric form of the insulin receptor. Consequent to the substitution of the SH group in place of S-S group, during depalmitoylation, the functional specificity of the insulin receptor is lost. This leads to the failure of conformational changes and auto- phoshorylation of the beta sub units when insulin binds to the extra cellular insulin binding receptor domain. Also failure of the cascade of phosphorylation of other signalling proteins occurs. Consequently, the failure of the intra- cellular transduction of the signals results in failure of the translocation of the GLUT 4 transporter to the cell membrane. This results in the hyperglycaemia in spite of hyper insulinemia and mo glucose enters into the cell- a state called insulin resistance in DM 2.

Quantitative proteomic analysis of PK-15 cells infected with porcine circovirus type 3 using 4D-DIA approach.

Porcine circovirus type 3 (PCV3) infection is clinically related to various diseases, including porcine dermatitis and nephrotic syndrome (PDNS)-like disease, respiratory disease, reproductive disorders, and gastrointestinal and neurological diseases. Since PCV3 infection was discovered in 2016, it has developed rapidly and has attracted much attention worldwide. However, specific preventive and therapeutic interventions are currently lacking. In this study, four-dimensional (4D) data-independent acquisition (DIA)-based quantitative proteomics detection combined with bioinformatics analysis were employed to quantitatively identify the differentially expressed proteins in PK-15 cells from the PCV3-infected group compared with those from the uninfected control group. A total of 194 cellular proteins were significantly altered in response to PCV3 infection, including 58 upregulated proteins and 136 downregulated proteins. In our Gene Ontology (GO) enrichment analysis, these differentially expressed proteins were mostly associated with cellular anatomical entities, binding, cellular processes, biological regulation, catalytic activity, metabolic processes, developmental processes, protein-containing complexes and responses to stimuli. Our Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEPs were predominantly involved in metabolic pathways, the cAMP signaling pathway, protein processing in the endoplasmic reticulum, the PI3K-Akt signaling pathway, and the calcium signaling pathway. For the experiments, Western blotting (WB) was used to confirm the changes in important molecules. The differentially expressed proteins identified should contribute to a greater understanding of the mechanism of PCV3 replication and pathogenesis, as well as the host response.

Overcoming retinoic acid resistance in HER2-enriched breast cancers: role of MYC.

HER2-enriched (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. Although these cancers are treated with the HER2-targeted drug, trastuzumab, resistance to treatment is common. Retinoic acid (RA) is an anti-cancer agent that has been successfully used for the treatment of leukemia and holds promise for the treatment of solid cancers, including breast cancer. The HER2 gene is frequently co-amplified with RARA, a key determinant of RA sensitivity in breast cancers. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. Here, we show that MYC mediates RA resistance by suppressing the expression of cellular retinoic acid binding protein 2 (CRABP2), resulting in RARα inactivation. CRABP2 is an intracellular RA transporter that delivers RA to the nuclear receptor RARα for its activation. Our results indicate that response to RA is enhanced by MYC depletion in HER2+ breast cancer cells and that RA treatment enhances trastuzumab responsiveness. Our findings support the use of RA and trastuzumab for the treatment of subsets of patients with breast cancers that are HER2-RARα co-amplified and have low levels of MYC.

99mTc-labeled, tofacitinib citrate encapsulated chitosan microspheres loaded in situ gel formulations for intra-articular treatment of rheumatoid arthritis.

Inflammatory diseases including rheumatoid arthritis are major health problems. Although different techniques and drugs are clinically available for the diagnosis and therapy of the disease, novel approaches regarding radiolabeled drug delivery systems are researched. Hence, in the present study, it was aimed to design, prepare, and characterize 99mTc-radiolabeled and tofacitinib citrate-encapsulated microsphere loaded poloxamer in situ gel formulations for the intra-articular treatment. Among nine different microsphere formulations, MS/TOFA-9 was chosen as the most proper one due to particle size, high encapsulation efficiency, and in vitro drug release behavior. Poloxamer 338 at a concentration of 15% was used to prepare in situ gel formulations. For intra-articular administration, microspheres were dispersed in an in situ gel containing 15% Poloxamer 338 and characterized in terms of gelation temperature, viscosity, rheological, mechanical, and spreadability properties. After the determination of the safe dose for MS/TOFA-9 and PLX-MS/TOFA-9 as 40 µL/mL in the cell culture study performed on healthy cells, the high anti-inflammatory effects were due to significant cellular inhibition of fibroblasts. In the radiolabeling studies with 99mTc, the optimum radiolabeling condition was determined as 200 ppm SnCl2 and 0.5 mg ascorbic acid, and both 99mTc-MS/TOFA-9 and 99mTc-PLX-MS/TOFA-9 exhibited high cellular binding capacity. In conclusion, although further in vivo experiments are required, PLX-MS/TOFA-9 was found to be a promising agent for intra-articular injection in rheumatoid arthritis.

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.

Regulation of Absorption and Emission in a Protein/Fluorophore Complex.

Human cellular retinol binding protein II (hCRBPII) was used as a protein engineering platform to rationally regulate absorptive and emissive properties of a covalently bound fluorogenic dye. We demonstrate the binding of a thio-dapoxyl analog via formation of a protonated imine between an active site lysine residue and the chromophore's aldehyde. Rational manipulation of the electrostatics of the binding pocket results in a 204 nm shift in absorption and a 131 nm shift in emission. The protein is readily expressed in mammalian systems and binds with exogenously delivered fluorophore as demonstrated by live-cell imaging experiments.

CRABP2 promotes cell migration and invasion by activating PI3K/AKT and MAPK signalling pathways via upregulating LAMB3 in prostate cancer.

Prostate cancer (PCa) has become a worldwide health burden among men. Previous studies have suggested that cellular retinoic acid binding protein 2 (CRABP2) significantly affects the regulation of cell proliferation, motility and apoptosis in multiple cancers; however, the effect of CRABP2 on PCa is poorly reported. CRABP2 expression in different PCa cell lines and its effect on different cellular functions varied. While CRABP2 promotes cell migration and invasion, it appears to inhibit cell proliferation specifically in PC-3 cells. However, the proliferation of DU145 and 22RV1 cells did not appear to be significantly affected by CRABP2. Additionally, CRABP2 had no influence on the cell cycle distribution of PCa cells. The RNA-seq assay showed that overexpressing CRABP2 upregulated laminin subunit beta-3 (LAMB3) mRNA expression, and the enrichment analyses revealed that the differentially expressed genes were enriched in the phosphoinositide 3-kinase (PI3K)/activated protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signalling pathways. The following western blot experiments also confirmed the upregulated LAMB3 protein level and the activation of the PI3K/AKT and MAPK signalling pathways. Moreover, overexpressing CRABP2 significantly inhibited tumour growth in vivo. In conclusion, CRABP2 facilitates cell migration and invasion by activating PI3K/AKT and MAPK signalling pathways through upregulating LAMB3 in PCa.

Anti-TNFR2 Antibody-Conjugated PLGA Nanoparticles for Targeted Delivery of Adriamycin in Mouse Colon Cancer.

High levels of tumor necrosis factor receptor type II (TNFR2) are preferentially expressed by immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs), especially those present in the tumor microenvironment, as initially reported by us. There is compelling evidence that targeting TNFR2 markedly enhances antitumor immune responses. Furthermore, a broad spectrum of human cancers also expresses TNFR2, while its expression by normal tissue is very limited. We thus hypothesized that TNFR2 may be harnessed for tumor-targeted delivery of chemotherapeutic agents. In this study, we performed a proof-of-concept study by constructing a TNFR2-targeted PEGylated poly(dl-lactic-co-glycolic acid) (PLGA-PEG) nanodrug delivery system [designated as TNFR2-PLGA-ADR (Adriamycin)]. The results of invitro study showed that this TNFR2-targeted delivery system had the properties in cellular binding and cytotoxicity toward mouse colon cancer cells. Further, upon intravenous injection, TNFR2-PLGA-ADR could efficiently accumulate in MC38 and CT26 mouse colon tumor tissues and preferentially bind with tumor-infiltrating Tregs. Compared with ADR and ISO-PLGA-ADR, the invivo antitumor effect of TNFR2-PLGA-ADR was markedly enhanced, which was associated with a decrease of TNFR2+ Tregs and an increase of IFNγ+CD8+ cytotoxic T lymphocytes in the tumor tissue. Therefore, our results clearly show that targeting TNFR2 is a promising strategy for designing tumor-specific chemoimmunotherapeutic agent delivery system.

Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1.

In this study, we describe the structure-based development of the first fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor type 1 (CCR1), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in inflammation and immune diseases. Starting from previously reported intracellular allosteric modulators of CCR1, tetramethylrhodamine (TAMRA)-labeled ligands were designed, synthesized, and tested for their suitability as fluorescent tracers to probe binding to the IABS of CCR1. In the course of these studies, we developed LT166 (12) as a highly versatile fluorescent CCR1 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonradioactive and high-throughput manner. Besides the detection of intracellular allosteric ligands by direct competition with 12, we were also able to monitor the binding of extracellular antagonists due to their positive cooperative binding with 12. Thereby, we provide a straightforward and nonradioactive method to easily distinguish between ligands binding to the IABS of CCR1 and extracellular negative modulators. Further, we applied 12 for the identification of novel chemotypes for intracellular CCR1 inhibition that feature high binding selectivity for CCR1 over CCR2. For one of the newly identified intracellular CCR1 ligands (i.e., 23), we were able to show CCR1 over CCR2 selectivity also on a functional level and demonstrated that this compound inhibits basal β-arrestin recruitment to CCR1, thereby acting as an inverse agonist. Thus, our fluorescent CCR1 ligand 12 represents a highly promising tool for future studies of CCR1-targeted pharmacology and drug discovery.

Synthesis and evaluation of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate analogs as competitive partial agonists of butyrophilin 3A1

Phosphoantigens (pAgs) induce conformational changes after binding to the intracellular region of BTN3A1 which result in its clustering with BTN2A1, forming an activating ligand for the Vγ9Vδ2 T cell receptor. Here, we designed a small panel of bulky analogs of the prototypical pAg (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) that contain an aromatic ring attached to the C-3 position in place of methyl group. These compounds bind with high affinity to BTN3A1 but fail to fully support its interaction with BTN2A1 and only partially trigger T cell activation relative to HMBPP. Furthermore, they can compete with HMBPP for cellular binding to BTN3A1 and reduce the cellular response to HMBPP, a classic partial agonist phenotype. Trifluoromethyl analog 6e was the weakest agonist but the strongest inhibitor of HMBPP ELISA response. Our study provides a rationale for the mode of action of pAg-induced γδ T cell activation and provides insights into other naturally occurring BTN proteins and their respective ligands.