Novel BQCA- and TBPB-Derived M1 Receptor Hybrid Ligands: Orthosteric Carbachol Differentially Regulates Partial Agonism.

Recently, investigations of the complex mechanisms of allostery have led to a deeper understanding of G protein-coupled receptor (GPCR) activation and signaling processes. In this context, muscarinic acetylcholine receptors (mAChRs) are highly relevant due to their exemplary role in the study of allosteric modulation. In this work, we compare and discuss two sets of putatively dualsteric ligands, which were designed to connect carbachol to different types of allosteric ligands. We chose derivatives of TBPB [1-(1'-(2-tolyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one] as M1 -selective putative bitopic ligands, and derivatives of benzyl quinolone carboxylic acid (BQCA) as an M1 positive allosteric modulator, varying the distance between the allosteric and orthosteric building blocks. Luciferase protein complementation assays demonstrated that linker length must be carefully chosen to yield either agonist or antagonist behavior. These findings may help to design biased signaling and/or different extents of efficacy.

3-Substituted Quinolines as RORγt Inverse Agonists.

We have previously reported the syntheses of a series of 3,6-disubstituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). These molecules are potent binders but are high molecular weight and they exhibited poor solubility at pH 2 and pH 7. This manuscript details our efforts at improving physical chemical properties for this series of compounds by increasing the diversity at the 3-position (i.e. introducing heteroatoms and lowering the molecular weight). These efforts have led to molecules which are potent binders with improved solubility.

Laquinimod, a prototypic quinoline-3-carboxamide and aryl hydrocarbon receptor agonist, utilizes a CD155-mediated natural killer/dendritic cell interaction to suppress CNS autoimmunity.

BACKGROUND: Quinoline-3-carboxamides, such as laquinimod, ameliorate CNS autoimmunity in patients and reduce tumor cell metastasis experimentally. Previous studies have focused on the immunomodulatory effect of laquinimod on myeloid cells. The data contained herein suggest that quinoline-3-carboxamides improve the immunomodulatory and anti-tumor effects of NK cells by upregulating the adhesion molecule DNAX accessory molecule-1 (DNAM-1). METHODS: We explored how NK cell activation by laquinimod inhibits CNS autoimmunity in experimental autoimmune encephalomyelitis (EAE), the most utilized model of MS, and improves immunosurveillance of experimental lung melanoma metastasis. Functional manipulations included in vivo NK and DC depletion experiments and in vitro assays of NK cell function. Clinical, histological, and flow cytometric read-outs were assessed. RESULTS: We demonstrate that laquinimod activates natural killer (NK) cells via the aryl hydrocarbon receptor and increases their DNAM-1 cell surface expression. This activation improves the cytotoxicity of NK cells against B16F10 melanoma cells and augments their immunoregulatory functions in EAE by interacting with CD155+ dendritic cells (DC). Noteworthy, the immunosuppressive effect of laquinimod-activated NK cells was due to decreasing MHC class II antigen presentation by DC and not by increasing DC killing. CONCLUSIONS: This study clarifies how DNAM-1 modifies the bidirectional crosstalk of NK cells with CD155+ DC, which can be exploited to suppress CNS autoimmunity and strengthen tumor surveillance.

Synergistic antitumor effect of NVP-BEZ235 and CAPE on MDA-MB-231 breast cancer cells.

Triple negative breast cancer (TNBC) is the most lethal and aggressive kind of breast cancer. Studies with TNBC cells suggest that tumor environmental cytokines such as Transforming Growth Factor ß1 (TGF-ß1) have important roles in tumors fate. In the present study, we aimed to investigate, the effect of phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway dual inhibitor, NVP-BEZ235 and Caffeic acid phenyl ester (CAPE) on TNBC cell line (MDA-MB-231), stimulated with TGF-ß1 for 14days in vitro. We found that TGF-ß1 as a local tumor environmental cytokine plays important role in the progression and invasiveness of TNBC cells. NVP-BEZ235 inhibited the enhanced cell viability and CXCR4 expression induced by TGF-ß1. In addition, the combined treatment of TNBC cell lines with CAPE and NVP-BEZ235 synergistically inhibited cell growth and reduced CXCR4 expression. Also, treatment of MDA-MB-231 cells with CAPE and NVP-BEZ235 led to decreasing the expression levels of p-FOXO3a in a time-dependent manner. Overall, these results suggest that tumor metastasis and progression in TNBC cells can be effectively reduced through the concurrent use of NVP-BEZ235 and CAPE. This could be of particular interest in assessing the effects of this therapy in the reduction of tumor metastasis and progression in other tumor types.

Synergism between bosutinib (SKI-606) and the Chk1 inhibitor (PF-00477736) in highly imatinib-resistant BCR/ABL⁺ leukemia cells.

Interactions between the dual BCR/ABL and Src inhibitor bosutinib and the Chk1 inhibitor PF-00477736 were examined in BCR/ABL(+) leukemia cells, particularly imatinib-resistant cells, including those with the T315I mutation. Bosutinib blocked PF-00477736-induced ERK1/2 activation and sharply increased apoptosis in association with Mcl-1 inhibition, p34(cdc2) dephosphorylation, BimEL up-regulation, and DNA damage in imatinib-resistant CML or Ph(+) ALL cell lines. Inhibition of Src or MEK1 by shRNA significantly enhanced PF-0047736 lethality. Bosutinib/PF-00477736 co-treatment also potentiated cell death in CD34(+) CML patient samples, including dasatinib-resistant blast crisis cells exhibiting both T315I and E355G mutations, but was minimally toxic to normal CD34(+) cells. Finally, combined in vivo treatment significantly suppressed BaF3/T315I tumor growth and prolonged survival in an allogeneic mouse model. Together, these findings suggest that this targeted combination strategy warrants attention in IM-resistant CML or Ph(+) ALL.

Tumour growth inhibition by an imidazoquinoline is associated with c-Myc down-regulation in urothelial cell carcinoma.

OBJECTIVE: To detect and characterize the potential role of c-Myc in the inhibition of proliferation and induction of cell death of urothelial cell carcinoma by imidazoquinolines, Toll-like receptor-7 (TLR7) agonists, that are thought to exert their immunogenic effects through the MyD88/NF-kappaB pathway. MATERIALS AND METHODS: Human (T24) and murine (MBT-2) bladder cancer cell lines were cultured in normal culture medium or medium supplemented with imidazoquinoline. The effects of imidazoquinoline on gene expression, transcription and tumorigenesis were then evaluated. Effects of imidazoquinoline on in vivo bladder tumour growth and gene expression were investigated using a mouse model of orthotopic bladder cancer. RESULTS: There was a dose-dependent decrease in c-Myc expression in bladder cancer cells treated with imidazoquinoline; the transcriptional activity of c-Myc was also significantly reduced. Furthermore, the in vitro proliferation and tumorigenesis of MBT-2 cells were suppressed in a dose-dependent manner. For in vivo experiments, a third of mice with bladder cancer treated with intravesical imidazoquinoline showed evidence of residual bladder tumour, vs all the placebo-treated mice. In vivo expression of c-Myc, cyclin D2 and proliferating cell nuclear antigen in the bladder tumour tissue were also down-regulated. CONCLUSIONS: Imidazoquinolines can inhibit c-Myc expression and directly affect cell growth and tumorigenesis of bladder cancer cells, independent of an immune response. These direct effects might be synergistic with previously described immunogenic actions of imidazoquinolines. Our findings could broaden the potential application of imidazoquinoline therapy beyond dermatological malignancies, and further clinical studies are warranted.

The covalent modification and regulation of TLR8 in HEK-293 cells stimulated with imidazoquinoline agonists.

The mammalian TLRs (Toll-like receptors) mediate the rapid initial immune response to pathogens through recognition of pathogen-associated molecular patterns. The pathogen pattern to which TLR8 responds is ssRNA (single-stranded RNA) commonly associated with ssRNA viruses. TLR8 also responds to small, purine-like molecules including the imidazoquinoline IRMs (immune-response modifiers). The IRMs include molecules that selectively activate TLR7, selectively activate TLR8 or non-selectively activate both TLR7 and TLR8. Using HEK-293 cells (human embryonic kidney cells) stably expressing an NF-kappaB (nuclear factor kappaB)/luciferase promoter-reporter system as a model system, we have examined the regulation of TLR8 using the non-selective TLR7/8 agonist, 3M-003. Using conservative tyrosine to phenylalanine site-directed mutation, we show that of the 13 tyrosine residues resident in the cytosolic domain of TLR8, only three appear to be critical to TLR8 signalling. Two of these, Tyr898 and Tyr904, reside in the Box 1 motif and the third, Tyr1048, lies in a YXXM putative p85-binding motif. TLR8 is tyrosine-phosphorylated following 3M-003 treatment and TLR8 signalling is inhibited by tyrosine kinase inhibitors. Treatment with 3M-003 results in the association of the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase) with TLR8 and this association is inhibited by tyrosine to phenylalanine mutation of either the YXXM or Box 1 motifs. As a further consequence of activation by 3M-003, TLR8 is modified to yield both higher and lower molecular mass species. These species include a monoubiquitinated form as deduced from ubiquitin peptide sequencing by HPLC/MS/MS (tandem MS).

Are antihistamines useful in managing asthma?

There continues to be a great deal of interest in the anti-asthmatic role of antihistamines. Antihistamines have recently been shown to have anti-inflammatory properties that are more extensive than simply the blocking of histamine receptors. For example, new evidence suggests that the suppression of cell adhesion molecule expression occurs with these drugs. The anti-inflammatory and anti-asthmatic effects of antihistamines have been evaluated in patients with both allergic asthma and rhinitis, given the established association between allergic inflammation of the upper and lower airways, with evidence to suggest that antihistamines have clinically relevant anti-asthmatic properties. As well as conferring benefits in asthma symptom control and the measurement of lung function, studies assessing the effect of histamine receptor antagonists on bronchial hyperresponsiveness suggest that there is bronchoprotection during both methacholine and mannitol challenges. Recently, there has also been considerable interest in the effect of combining an antihistamine with a leukotriene receptor antagonist. This combination has an anti-asthmatic effect that is greater than that of either drug given alone and may be comparable to inhaled corticosteroid therapy.