Small molecule SMU-CX24 targeting toll-like receptor 3 counteracts inflammation: A novel approach to atherosclerosis therapy.

Toll-like receptor 3 (TLR3), as an important pattern recognition receptor (PRR), dominates the innate and adaptive immunity regulating many acute and chronic inflammatory diseases. Atherosclerosis is proved as an inflammatory disease, and inflammatory events involved in the entire process of initiation and deterioration. However, the contribution of TLR3 to atherosclerosis remains unclear. Herein, we identified the clinical relevance of TLR3 upregulation and disease processes in human atherosclerosis. Besides, activation of TLR3 also directly led to significant expression of atherogenic chemokines and adhesion molecules. Conversely, silencing TLR3 inhibited the uptake of oxLDL by macrophages and significantly reduced foam cell formation. Given the aberrance in TLR3 functions on atherosclerosis progression, we hypothesized that TLR3 could serve as novel target for clinical atherosclerosis therapy. Therefore, we developed the novel ellipticine derivative SMU-CX24, which specifically inhibited TLR3 (IC50 = 18.87 ± 2.21 nmol/L). In vivo, atherosclerotic burden was alleviated in Western diet fed ApoE-/- mice in response to SMU-CX24 treatment, accompanying notable reductions in TLR3 expression and inflammation infiltration within atherosclerotic lesion. Thus, for the first time, we revealed that pharmacological downregulation of TLR3 with specific inhibitor regenerated inflammatory environment to counteract atherosclerosis progression, thereby proposing a new strategy and probe for atherosclerosis therapy.

Near-Infrared Fluorescent and Photoacoustic Dual-Mode Probe for Highly Sensitive and Selective Imaging of Cysteine In Vivo.

Cysteine (Cys) plays an important role in many physiological activities of human beings. Various diseases are always accompanied by abnormal levels of Cys. A series of Cys-responsive probes were recently developed. However, most fluorescent probes have many disadvantages and exhibit poor in vivo imaging. Therefore, a near-infrared fluorescence (NIRF)/photoacoustic (PA) dual-mode probe with high selectivity and sensitivity (limit of detection = 10.6 nM) toward Cys was developed in this study. The new Probe I interacted with Cys to activate NIRF/PA signals, detecting Cys in vitro with a large emission wavelength (851 nm) and Stokes shift (191 nm), monitoring the occurrence of liver cancer in vivo. This work not only presented an effective NIRF/PA dual-mode dicyanoisophorone probe for the first time in the imaging of Cys but also provided a comprehensive and accurate tool for detecting different analytes and tumors in deeper tissues, which could be conducive to the early diagnosis of diseases.

A highly selective and sensitive chemiluminescent probe for leucine aminopeptidase detection in vitro, in vivo and in human liver cancer tissue.

Leucine aminopeptidase (LAP) is involved in tumor cell proliferation, invasion, and angiogenesis, and is a well-known tumor marker. In recent years, chemiluminescence has been widely used in the field of biological imaging, due to it resulting in a high sensitivity and excellent signal-to-noise ratio. Here, we report the design, synthesis, and evaluation of the first LAP-activated chemiluminescent probe for LAP detection and imaging. The probe initially had no chemiluminescence but produced an extremely strong chemiluminescence after the release of the dioxetane intermediate in the presence of LAP. The probe had high selectivity over other proteases and higher signal-to-noise ratios than commercial fluorophores. Real-time imaging results indicated that the chemiluminescence was remarkably enhanced at the mice tumor site after the probe was injected. Furthermore, the chemiluminescence of this probe in the cancerous tissues of patients was obviously improved compared to that of normal tissues. Taken together, this study has developed the first LAP-activable chemiluminescent probe, which could be potentially used in protein detection, disease diagnosis, and drug development.

TLR1/2 Specific Small-Molecule Agonist Suppresses Leukemia Cancer Cell Growth by Stimulating Cytotoxic T Lymphocytes.

Toll-like receptor 2 (TLR2) expressed on antigen presenting cells evokes a series of critical cytokines, which favor the development of tumor-specific cytotoxic T lymphocytes (CTLs). Therefore, TLR2 represents an attractive cancer immunotherapeutic target. Here, a synthetic library of 14 000 compounds together with a series of newly developed compounds for NF-κB activation using HEK-Blue hTLR2 cells is initially screened. Following further screening in a variety of cells including HEK-Blue hTLRs reporter cells, murine, and human macrophage cell lines, a potent small molecule agonist 23 (SMU-Z1) is identified, which specifically activates TLR2 through its association with TLR1, with a EC50 of 4.88 ± 0.79 × 10-9 m. Toxicology studies, proinflammatory cytokines (e.g., TNF-α, IL-1ß, IL-6, and nitric oxide) and target-protein based biophysical assays demonstrate the pharmacologically relevant characteristics of SMU-Z1. In addition, SMU-Z1 promotes murine splenocyte proliferation and upregulates the expression of CD8+ T cells, NK cells and DCs, which results in a significant antitumor effect in a murine leukemia model. Finally, the induced tumors in three out of seven mice disappear after administration of SMU-Z1. Our studies thus identify a novel and potent TLR1/2 small molecule agonist, which displays promising immune adjuvant properties and antitumor immunity.

Synthesis of urea analogues bearing N-alkyl-N'-(thiophen-2-yl) scaffold and evaluation of their innate immune response to toll-like receptors.

Previous high throughput virtual screening of 10 million-compound and following cell based validation led to the discovery of a novel, nonlipopeptide-like chemotype ZINC 6662436, as toll-like receptor 2 (TLR2) agonists. In this report, compounds belonging to four areas of structural modification of ZINC6662436 were evaluated for biological activity using human HEK-Blue TLR2 reporter cells, and human THP-1 monocytic cells, yield SMU-C13 as an optimized, direct and high potent (EC50 = 160 nM) agonist of human TLR2. Moreover, preliminary mechanism studies indicated that SMU-C13 through activates TLR1 and TLR2 then stimulates the NF-κB activation to trigger the downstream cytokines, such as TNF-α and secreted alkaline phosphatase (SEAP).

Structure-based discovery of a specific TLR1-TLR2 small molecule agonist from the ZINC drug library database.

We report herein the identification of urea structure-like small molecules by structure-based virtual screening of 10.5 million compounds. Based on a variety of HEK-Blue hTLRs reporter cell assay results, we validated a TLR1/2-specific small molecule agonist, ZINC666243 (SMU127), with EC50 of 0.55 ± 0.01 µM. SMU127 stimulates NF-κB activation and promotes TNFα secretion in human macrophages and mononuclear cells. Moreover, the in vivo assay indicated that SMU127 could inhibit the growth of breast cancer tumors in BABL/c mice. This work has shown for the first time that a small molecule TLR1/2 agonist can inhibit breast cancer in vivo.

The Role of Toll-Like Receptor in Inflammation and Tumor Immunity.

Toll-like receptors (TLRs) activation enables host to recognize a large number of pathogen-associated molecule patterns (PAMPs), ignite immune cells to discriminate between self and non-self, and then promote the following innate and adaptive immune responses. Accumulated clinical/preclinical evidences have proven TLRs to be critical role in the autoimmune diseases, including inflammatory and tumor-associated diseases. Activation of TLRs is becoming or has been a target for cancer treatment. It is shown that TLRs can induce preferable anti-tumor effect by eliciting inflammatory cytokines expression and cytotoxic T lymphocytes (CTLs) response. As adjuvant, TLRs agonists can launch a strong immune response to assist cancer radiotherapy and bio-chemotherapy. On the other hand, tumor-associated antigens acting as PAMPs, can also activate TLRs and induce tumor gene-related programmed cell death, including apoptosis, autophagy and programmed necrosis. While there are also arguments that the excessive TLRs expression will promote tumor deterioration in various organisms, as the TLR-induced inflammation will accelerate the cancer cells boost in the tumor microenvironment (TME). However, the effect of TLRs acting on cancers is still not quite clear today. In this review, we will summarize the recent researches of TLRs in cancer treatment and their role in TME, giving a brief overview on future expectation.

Ter-cell, A New Target for Hepatocellular Carcinoma Therapy.

Hepatocellular carcinoma (HCC), a malignancy of the liver, has become the second most lethal cause of cancer death globally. Recently, scientists discovered that a splenic erythroblast-like cell induced by the primary tumor, termed Ter-cell, promoted HCC progression and metastasis. These findings shed light on the inhibition of Ter-cell or artemin that can serve as a new therapeutic target for HCC.

Synthesis, structure-activity relationships and preliminary mechanism study of N-benzylideneaniline derivatives as potential TLR2 inhibitors.

Toll-like receptor 2 (TLR2) can recognize pathogen-associated molecular patterns to defense against invading organisms and has been represents an attractive therapeutic target. Until today, none TLR2 small molecule antagonist have been developed in clinical trial. Herein, we designed and synthesized 50 N-benzylideneaniline compounds with the help of CADD. And subsequent in vitro studies leading to the optimized compound SMU-A0B13 with most potent inhibitory activity to TLR2 (IC50=18.21 ±â€¯0.87 µM). Preliminary mechanism studies indicated that this TLR2 inhibitor can work through the NF-κB signaling pathway with high specificity and low toxicity, and can also efficiently downregulate inflammatory cytokines, such as SEAP, TNF-α and NO in HEK-Blue hTLR2, human PBMC and Raw 264.7 cell lines. Additionally, the docking situation also indicate SMU-A0B13 can well bind to the TLR2-TIR (PDB: 1FYW) active domain, which probably explains the bioactivity.

Targeting pattern-recognition receptors to discover new small molecule immune modulators.

Pattern recognition receptors (PRRs) are key immune receptors of the innate immune system, which recognize the conserved pathogen-associated molecular patterns (PAMPs) of the invading pathogens. Compared to the adaptive immune receptors, PRRs have three distinguishing features, viz., universal expression, fast response and recognizing many kinds of microbes. Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and NOD-like receptors (NLRs) recognize viral nucleic acid/bacterial fragments and trigger anti-microbial innate immune responses. Upon recognition of their ligand species, PRRs recruit specific intracellular adaptor proteins to initiate signaling pathways culminating in the activation of nuclear factor-κB (NF-κB), mitogen-activated protein (MAP) kinases and interferon regulatory factors (IRFs) that control the transcription of genes encoding pro-inflammatory factors including type I interferon and other inflammatory cytokines, which are critical for eliminating the potential threat to the host. Here, we summarize the effects of small molecule regulators acting on signaling pathways initiated by TLR, RLR and NLR as well as their influence on innate and adaptive immune responses leading to therapy.

Unsedation colonoscopy can be not that painful: Evaluation of the effect of "Lamaze method of colonoscopy".

AIM: To evaluate the pain relieving effect of intervention with "Lamaze method of colonoscopy" in the process of colonoscopy. METHODS: Five hundred and eighty-five patients underwent colonoscopy were randomly divided into three groups, Lamaze group, anesthetic group and control group. Two hundred and twenty-four patients of Lamaze group, the "Lamaze method of colonoscopy" were practiced in the process of colonoscopy. The Lamaze method of colonoscopy is modified from the Lamaze method of childbirth, which helped patients to relieve pain through effective breathing control. One hundred and seventy-eight patients in anesthetic group accepted sedation colonoscopy. For 183 patients in control group, colonoscopy was performed without any intervention. The satisfactory of colon cleaning, intestinal lesions, intubation time, success ratio, pain grading and complications were recorded. All data were statistically analyzed. RESULTS: There were no significant differences at base line of the three groups (P > 0.05). Anesthetic group shows advantage in intubation time than the other two groups (P < 0.05). Lamaze group shows no advantage in intubation time than that in control group (P > 0.05). The anesthetic group showed an apparent advantage in relieving pain (P < 0.01). Therefore, the "Lamaze method of colonoscopy" performed in colonoscopy could relieve pain effectively comparing with control group (P < 0.05). The patients in anesthetic group had the highest incidence of complications (P < 0.05). CONCLUSION: The performance of the "Lamaze method of colonoscopy" in the process of colonoscopy could relieve patients' pain, minimize the incidence of complications, and is worthy promotion in clinical practice.