Development of Potent MALT1 Inhibitors Featuring a Novel "2-Thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7(6H)-one" Scaffold for the Treatment of B Cell Lymphoma.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) has emerged as a novel and promising therapeutic target for the treatment of lymphomas and autoimmune diseases. Herein, we reported a new class of MALT1 inhibitors featuring a novel "2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7(6H)-one" scaffold developed by structure-based drug design. Structure-activity relationship studies finally led to the discovery of MALT1 inhibitor 10m, which covalently and potently inhibited MALT1 protease with the IC50 value of 1.7 µM. 10m demonstrated potent and selective antiproliferative activity against ABC-DLBCL and powerful ability to induce HBL1 apoptosis. 10m also effectively downregulated the activities of MALT1 and its downstream signal pathways. Furthermore, 10m induced upregulation of mTOR and PI3K-Akt signals and exhibited a synergistic antitumor effect with Rapamycin in HBL1 cells. More importantly, 10m remarkably suppressed the tumor growth both in the implanted HBL1 and TMD8 xenograft models. Collectively, this work provides valuable MALT1 inhibitors with a distinct core structure.

Identification of Novel Fused Heteroaromatics-Based MALT1 Inhibitors by High-Throughput Screening to Treat B Cell Lymphoma.

Development of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) inhibitors is of great value and significance in the treatment of neoplastic disorders and inflammatory and autoimmune diseases. However, there is a lack of effective MALT1 inhibitors in clinic. Herein, a novel class of potent 5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-based MALT1 inhibitors and their covalent derivatives were first identified and designed through high-throughput screening. We demonstrated that compounds 15c, 15e, and 20c effectively inhibited the MALT1 protease and displayed selective cytotoxicity to activated B cell-like diffuse large B cell lymphoma with low single-digit micromolar potency. Furthermore, compound 20c specifically repressed NF-κB signaling and induced cell apoptosis in MALT1-dependent TMD8 cells in a dose-dependent manner. More importantly, 20c showed good pharmacokinetic properties and antitumor efficacy with no significant toxicity in the TMD8 xenograft tumor model. Collectively, this study provides valuable lead compounds of MALT1 inhibitors for further structural optimization and antitumor mechanism study.

MALT1 as a promising target to treat lymphoma and other diseases related to MALT1 anomalies.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a key adaptor protein that regulates the NF-κB pathway, in which MALT1 functions as a scaffold protein and protease to trigger downstream signals. The abnormal expression of MALT1 is closely associated with lymphomagenesis and other diseases, including solid tumors and autoimmune diseases. MALT1 is the only protease in the underlying pathogenesis of these diseases, and its proteolytic activity can be pharmacologically regulated. Therefore, MALT1 is a potential and promising target for anti-lymphoma and other MALT1-related disease treatments. Currently, the development of MALT1 inhibitors is still in its early stages. This review presents an overview of MALT1, particularly its X-ray structures and biological functions, and elaborates on the pathogenesis of diseases associated with its dysregulation. We then summarize previously reported MALT1 inhibitors, focusing on their molecular structure, biological activity, structure-activity relationship, and limitations. Finally, we propose future research directions to accelerate the discovery of novel MALT1 inhibitors with clinical applications. Overall, this review provides a comprehensive and systematic overview of MALT1-related research advances and serves as a theoretical basis for drug discovery and research.

TAGLN Is Downregulated by TRAF6-Mediated Proteasomal Degradation in Prostate Cancer Cells.

Transgelin (TAGLN, also named SM22) is an actin-associated protein and affects dynamics of actin filaments. Deregulation of TAGLN contributes to the development of different cancers, and it is commonly considered to be a tumor suppressor. TAGLN is usually downregulated in prostate cancer; however, the detailed functions of TAGLN in prostate cancer and how TAGLN is regulated remains unclear. In this study, we confirmed that TAGLN is downregulated in prostate cancer tissues and demonstrated that the downregulation of TAGLN occurs through proteasomal degradation. Next, we found that the expression level of TAGLN is inversely correlated with TRAF6. We screened more than 20 E2-E3 pairs by in vitro ubiquitination assay and found that the E2A-TRAF6 pair catalyzed mono ubiquitination of TAGLN. We then identified the ubiquitination sites of TAGLN to be on K89 or K108 residues and demonstrated that ubiquitination of TAGLN on K89/K108 are important for TRAF6-mediated proteasomal degradation. Furthermore, we investigated the function of TAGLN in prostate cancer cells. We found that ablation of TAGLN promoted prostate cancer cell proliferation and suppressed their migration via activation of NF-κB and Myc signaling pathways. Overall, our study provided new insights into the mechanisms underlying TAGLN expression and activity in prostate cancer. IMPLICATIONS: E3 ligase TRAF6 mediate mono-ubiquitination and degradation of TAGLN, which leads to activation of NF-κB and Myc signaling pathways in prostate cancer cells.

Targeting BCL10 by small peptides for the treatment of B cell lymphoma.

Rationale: Constitutive activation of the NF-κB signalling pathway plays a pivotal role in the pathogenesis of activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCLs), the most aggressive and chemoresistant form of DLBCL. In ABC-DLBCLs, the CARMA1-BCL10 (CB) complex forms a filamentous structure and functions as a supramolecular organizing centre (CB-SMOC) that is required for constitutive NF-κB activation, making it an attractive drug target for ABC-DLBCL treatment. However, a pharmaceutical approach targeting CB-SMOC has been lacking. Here, we developed Bcl10 peptide inhibitors (BPIs) that specifically target the BCL10 filamentation process. Methods: Electron microscopy and immunofluorescence imaging were used to visualize the effect of the BPIs on the BCL10 filamentation process. The cytotoxicity of the tested BPIs was evaluated in DLBCL cell lines according to cell proliferation assays. Different in vitro experiments (pharmacokinetics, immunoprecipitation, western blotting, annexin V and PI staining) were conducted to determine the functional mechanisms of the BPIs. The in vivo therapeutic effect of the BPIs was examined in different xenograft DLBCL mouse models. Finally, Ki67 and TUNEL staining and histopathology analysis were used to evaluate the antineoplastic mechanisms and systemic toxicity of the BPIs. Results: We showed that these BPIs can effectively disrupt the BCL10 filamentation process, destabilize BCL10 and suppress NF-κB signalling in ABC-DLBCL cells. By examining a panel of DLBCL cell lines, we found that these BPIs selectively repressed the growth of CB-SMOC-dependent DLBCL cells by inducing apoptosis and cell cycle arrest. Moreover, by converting the BPIs to acquire a D-retro inverso (DRI) configuration, we developed DRI-BPIs with significantly improved intracellular stability and unimpaired BPI activity. These DRI-BPIs selectively repressed the growth of CB-SMOC-dependent DLBCL tumors in mouse xenograft models without eliciting discernible adverse effects. Conclusion: We developed novel BPIs to target the BCL10 filamentation process and demonstrated that targeting BCL10 by BPIs is a potentially safe and effective pharmaceutical approach for the treatment of ABC-DLBCL and other CB-SMOC-dependent malignancies.

Requirement of TGFß Signaling for Effect of Fluoride on Osteoblastic Differentiation.

Research focused on transforming growth factor ß (TGFß) signaling in osteoblast is gradually increasing, whereas literature is rare in terms of fluorosis. This work aimed to investigate how TGFß signaling participated in regulation of the osteoblast by different doses of fluoride treatment. Bone marrow stem cells (BMSCs) were developed into osteoblastic cells and exposed to 1, 4, and 16 mg/L F- with and without 10 ng/mL of TGFß. Cell viability and differentiation state of osteoblast under different settings were measured by means of cell counting kit and analysis of alkaline phosphatase (ALP) activity as well as formation of mineral nodules. Real-time PCR was utilized to test expression of ALP and Runt-related transcription factor 2 (Runx2) at gene level. The gene expression of TGFß signaling effectors was also investigated, such as TGFß receptors (TßRs), smad3, and mitogen-activated protein kinases (MAPK). Results demonstrated that fluoride treatment exhibited action on osteoblast viability and osteogenic differentiation and upregulated expression of TßR2, smad3, and MAPK in this process. Administration of TGFß strengthened ALP activity but attenuated formation of mineral nodules. Co-treatment of TGFß and low-dose fluoride increased ALP activity compared to same dose of single fluoride treatment, whereas it inhibited mineral nodule formation. Administration of TGFß reversed the suppression of high-dose fluoride on osteogenic differentiation of BMSCs. Taken together, studies revealed that TßR2 acted as a target for fluoride and TGFß treatment on BMSCs, and smad3 and MAPK were involved in the mechanism of fluoride regulating osteogenic differentiation. Together, our data indicated that TGFß receptor-mediated signaling through smad3 and MAPK was required for modulation of fluoride on osteoblast viability and differentiation, and activating TßR2-smad3 signaling pathway reversed suppression of osteoblasts differentiation by high dose of fluoride treatment.

PTH (1-34) affects bone turnover governed by osteocytes exposed to fluoride.

Exposure to fluoride from environmental sources remains an overlooked, but serious public health risk. In this study, we looked into the role osteocytes play on the mechanism underlying fluoride induced osteopathology. We analyzed bone formation and resorption related genes generated by osteocytes that were exposed to varied doses of fluoride with and without PTH in vitro. Correspondingly, osteogenesis and osteoclastogenesis related genes were also investigated in rats exposed to fluoride for 8 weeks, and the PTH(1-34)was applied at the last 3 weeks to observe its role in regulating bone turnover upon fluoride treatment. The data in vitro indicated that fluoride treatment inhibited Sost expression of mRNA and protein and stimulated RANKL mRNA protein expression as well as the RANKL/OPG ratio in the primary osteocytes. Single PTH treatment played the similar role on expression of these genes and proteins. The PTH combined administration enhanced the action of fluoride treatment on RNAKL/OPG and SOST/Sclerostin. The up-regulation of RANKL and decreasing of Sost induced by fluoride and/or PTH treatment was validated in vivo and suggests that osteocytes are a major source of RANKL and Sost, both of which play essential roles in fluoride affecting osteogenesis and osteoclastogenesis. Expression of Wnt/ß-catenin was up-regulated in both in vitro osteocytes treated with high dose of fluoride and bone tissue of rats in the presence of fluoride and PTH. In vivo, fluoride and single PTH stimulated bone turnover respectively, furthermore, PTH combined with low dose of fluoride treatment reinforced the osteogenesis and osteoclastogenesis genes expression, however, co-treatment of PTH reversed the effect of high dose of fluoride on osteogenesis and osteoclastogenensis related factors. In conclusion, this study demonstrated that osteocytes play a key role in fluoride activated bone turnover, and PTH participates in the process of fluoride modulating SOST/Sclerostin and RANKL expression.

The role of TGFß receptor 1-smad3 signaling in regulating the osteoclastic mode affected by fluoride.

Studies that have focused on the role TGFß signaling plays in osteoclast activity are gradually increasing; however, literature is rare in terms of fluorosis. The aim of this study is to observe the role the TßR1/Smad3 pathway plays in fluoride regulating cellsosteoclast-like cells that are under the treatment of TGFß receptor 1 kinase. The RANKL-mediated osteoclast-like cells from RAW264.7 cells were used as osteoclast precursor model. The profile of miRNA expression in fluoride-treated osteoclast-like cells exhibited 303 upregulated miRNAs, 61 downregulated miRNAs, and further drew 37 signaling pathway maps by KEGG and Biocarta pathway enrichment analysis. TGFß and its downstream effectors were included among them. Osteoclast viability, formation and function were detected via MTT method, bone resorption pit and tartrate-resistant acid phosphatase (TRACP) staining, respectively. Results demonstrated that different doses of fluoride exhibited a biphasic effect on osteoclast cell viability, differentiation, formation and function. It indicated that a low dose of fluoride treatment stimulated them, but high dose inhibited them. SB431542 acted as TßR1 kinase inhibitor and blocked viability, formation and function of osteoclast-like cells regulated by fluoride. The expression of the osteoclast marker, RANK, and TßR1/Smad3 at gene and protein level was analyzed under fluoride with and without SB431542 treatment. Fluoride treatment indicated little effect on the RANK protein expression; however it significantly influenced TRACP expression in osteoclast-like cells. The stimulation of fluoride on the expression of Smad3 gene and phosphorylated Smad3 protein exhibited dose-dependent manner. SB431542 significantly impeded phosphorylation of Smad3 protein and TRACP expression in osteoclast-like cells that were exposed to fluoride. Our work demonstrated that TGFß signaling played a key role in fluoride regulating osteoclast differentiation, formation and function. It elucidated that TßR1/Smad3 pathway participated in the mechanism of biphasic modulation of osteoclast mode regulated by fluoride.