Sphingomyelin synthase 2 is a positive regulator of the CSF1R-STAT3 pathway in pancreatic cancer-associated macrophage.

Background: Tumor-associated macrophages (TAMs) are one of the most abundant immune cells in the pancreatic cancer stroma and are related to the poor prognosis of pancreatic ductal adenocarcinoma (PDAC) patients. Therefore, targeting tumor-associated macrophages is a possible strategy for the treatment of pancreatic cancer. Purpose: We would like to investigate the role of sphingomyelin synthase 2 (SMS2) and the effect of the synthase 2 selective inhibitor YE2 in TAMs and the pancreatic tumor microenvironment. In addition, we also would like to investigate the mechanism by which YE2 attenuates macrophage M2 polarization. Methods: YE2 was utilized to treat macrophages (in vitro) and mice (in vivo). Western blotting and real-time PCR were used to detect the protein levels and mRNA levels of macrophage M2 polarization markers and their downstream signaling pathways. Sphingomyelin synthase 2 gene knockout (KO) mice and their controls were used to establish a PANC-02 orthotopic pancreatic cancer model, and immune cell infiltration in the tumor tissue was analyzed by immunohistochemistry (IHC). Results: We found that sphingomyelin synthase 2 mRNA expression is positively correlated with tumor-associated macrophages, the immunosuppressive microenvironment, and poor prognosis in pancreatic ductal adenocarcinoma patients. Sphingomyelin synthase 2 deficiency was confirmed to have an inhibitory effect on the growth of orthotopic PANC-02 tumors in vivo. The deficiency not only reduced the infiltration of tumor-associated macrophages but also regulated other immune components in the tumor microenvironment. In tissue culture, YE2 inhibited M2 polarization in both bone marrow-derived macrophages (BMDMs) and THP-1 macrophages and eliminated the protumor effect of M2 macrophages. In the mouse model, YE2 treatment reduced the infiltration of TAMs and regulated other immune components in the tumor microenvironment, slowing the progression of PANC-02 tumors. In terms of mechanism, we found that the inhibition of sphingomyelin synthase 2 could downregulate the expression of IL4Rα and CSF1R, thereby attenuating M2 polarization. Conclusion: The sphingomyelin synthase 2 inhibitor YE2 or sphingomyelin synthase 2 deficiency can prevent macrophage M2 polarization in pancreatic cancer, and sphingomyelin synthase 2 could be a new potential target for the treatment of pancreatic cancer.

Discovery of Novel 3-Hydroxyquinazoline-2,4(1H,3H)-Dione Derivatives: A Series of Metal Ion Chelators with Potent Anti-HCV Activities.

Millions of people worldwide suffer from acute or chronic liver inflammation caused by the hepatitis C virus (HCV). Metal ion chelators have achieved widespread success in the development of antiviral drugs. Some inhibitors with metal ion chelating structures have been proven to have good inhibitory activities on non-structural protein 5B (NS5B) polymerase. However, most of the reported metal ion chelators showed poor anti-HCV potency at the cellular level. Hence, we designed and synthesized a series of 3-hydroxyquinazoline-2,4(1H,3H)-dione derivatives with novel metal ion chelating structures. Typical compounds such as 21h, 21k, and 21t showed better anti-HCV activities than ribavirin with EC50 values less than 10 µM. 21t is currently known as one of the metal ion chelators with the best anti-HCV potency (EC50 = 2.0 µM) at the cellular level and has a better therapeutic index (TI > 25) as compared to ribavirin and the reported compound 6. In the thermal shift assay, the representative compounds 21e and 21k increased the melting temperature (Tm) of NS5B protein solution by 1.6 °C and 2.1 °C, respectively, at the test concentration, indicating that these compounds may exert an anti-HCV effect by targeting NS5B. This speculation was also supported by our molecular docking studies and ultraviolet-visible (UV-Vis) spectrophotometry assay, in which the possibility of binding of 3-hydroxyquinazoline-2,4(1H,3H)-diones with Mg2+ in the NS5B catalytic center was observed.

Discovery of a highly potent CECR2 bromodomain inhibitor with 7H-pyrrolo[2,3-d] pyrimidine scaffold.

Cat eye syndrome chromosome region candidate 2 (CECR2) bromodomain is a module of CECR2-containing remodeling factor (CERF), which is a chromatin remodeling complex correlating with transcriptional control and adjustment of chromatin architecture. Potent chemical probes would be beneficial to gain insights into the biochemical and pharmacological functions of CECR2 BRD. Herein, we report the discovery of a series of CECR2 BRD inhibitors with 7H-pyrrolo[2,3-d] pyrimidine scaffold based on molecular docking model of TP-248 and CECR2 BRD. The most potent inhibitor of this series, DC-CBi-22 with IC50 of 8.0 ± 1.4 nM against CECR2 BRD and selectivity over BPTF BRD up to 24.9-fold. The SARs were detailed according to molecular docking. DC-CBi-22 would serve as a useful chemical probe for the study of CECR2.

Efficient preparation of (R)-2-(2,5-difluorophenyl)pyrrolidine via a recycle process of resolution.

An efficient preparation of (R)-2-(2,5-difluorophenyl)pyrrolidine ((R)-1) from the racemate based on a recycle process of resolution/racemization was described. In the process, the desired (R)-1 was obtained by resolution with D-malic acid in 95% EtOH. Meanwhile, the undesired (S)-1 could be racemized in the presence of potassium hydroxide in DMSO. After three times of recycle process, the desired freebase (R)-1 was obtained in a yield of 61.7% with excellent ee (98.4%).

Discovery of styrylaniline derivatives as novel alpha-synuclein aggregates ligands.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Early diagnosis is the key to treatment but is still a great challenge in the clinic now. The discovery of alpha-synuclein (α-syn) aggregates ligands has become an attractive strategy to meet the early diagnosis of PD. Herein, we designed and synthesized a series of styrylaniline derivatives as novel α-syn aggregates ligands. Several compounds displayed good potency to α-syn aggregates with Kd values less than 0.1 µM. The docking study revealed that the hydrogen bonds and cation-pi interaction between ligands and α-syn aggregates would be crucial for the activity. The representative compound 7-16 not only detected α-syn aggregates in both SH-SY5Y cells and brain tissues prepared from two kinds of α-syn preformed-fibrils-injected mice models but also showed good blood-brain barrier penetration characteristics in vivo with a brain/plasma ratio over 1.0, which demonstrates its potential as a lead compound for further development of in vivo imaging agents.

Discovery of Novel Benzothiazepinones as Irreversible Covalent Glycogen Synthase Kinase 3ß Inhibitors for the Treatment of Acute Promyelocytic Leukemia.

Recently, irreversible inhibitors have attracted great interest in antitumors due to their advantages of forming covalent bonds to target proteins. Herein, some benzothiazepinone compounds (BTZs) have been designed and synthesized as novel covalent GSK-3ß inhibitors with high selectivity for the kinase panel. The irreversible covalent binding mode was identified by kinetics and mass spectrometry, and the main labeled residue was confirmed to be the unique Cys14 that exists only in GSK-3ß. The candidate 4-3 (IC50 = 6.6 µM) showed good proliferation inhibition and apoptosis-inducing ability to leukemia cell lines, low cytotoxicity on normal cell lines, and no hERG inhibition, which hinted the potential efficacy and safety. Furthermore, 4-3 exhibited decent pharmacokinetic properties in vivo and remarkably inhibited tumor growth in the acute promyelocytic leukemia (APL) mouse model. All the results suggest that these newly irreversible BTZ compounds might be useful in the treatment of cancer such as APL.

Sphingomyelin synthase 2 facilitates M2-like macrophage polarization and tumor progression in a mouse model of triple-negative breast cancer.

High infiltration of M2-polarized macrophages in the primary tumor indicates unfavorable prognosis and poor overall survival in the patients with triple-negative breast cancer (TNBC). Thus, reversing M2-polarized tumor-associated macrophages in the tumors has been considered as a potential therapeutic strategy for TNBC. Sphingomyelin synthase 2 (SMS2) is the key enzyme for sphingomyelin production, which plays an important role in plasma membrane integrity and function. In this study we investigated whether SMS2 inhibitor or SMS2 gene knockout could reduce macrophages M2 polarization and tumor progression in a mouse model of TNBC. We showed that SMS2 mRNA expression was linked to immunosuppressive tumor microenvironment and poor prognosis in TNBC patients. The knockout of SMS2 or application of 15w (a specific SMS2 inhibitor) markedly decreased the generation of M2-type macrophages in vitro, and reduced the tumor weight and lung metastatic niche formation in a 4T1-TNBC mouse model. We further demonstrated that the in vivo antitumor efficacy of 15w was accompanied by a multifaceted remodeling of tumor immune environment reflecting not only the suppression of M2-type macrophages but also diminished levels of regulatory T cells and myeloid-derived suppressor cells leading to a dramatically improved infiltration of antitumor CD8+ T lymphocytes. Collectively, our results reveal a novel and important role of SMS2 in the protumorigenic function and may offer a new strategy for macrophage-targeted anticancer therapy.

Site-Selective Phosphoglycerate Mutase 1 Acetylation by a Small Molecule.

Post-translational modifications play vital roles in fine-tuning a myriad of physiological processes, and one of the most important modifications is acetylation. Here, we report a ligand-directed site-selective acetylation using KHAc, a derivative of a phosphoglycerate mutase 1 (PGAM1) inhibitor. KHAc binds to PGAM1 and transfers its acetyl group to the ε-NH2 of Lys100 to inactivate the enzyme. The acetyl transfer process was visualized by time-resolved crystallography, demonstrating that the transfer is driven by proximity effects. KHAc was capable of selectively and effectively acetylating Lys100 of PGAM1 in cultured human cells, accompanied by inhibited F-actin formation. Similar strategies could be used for exogenous control of other lysine post-translational modifications.

Design, synthesis and identification of N, N-dibenzylcinnamamide (DBC) derivatives as novel ligands for α-synuclein fibrils by SPR evaluation system.

PET imaging of α-synuclein (α-syn) deposition in the brain will be an effective tool for earlier diagnosis of Parkinson's disease (PD) due to α-syn aggregation is the widely accepted biomarker for PD. However, the necessary PET radiotracer for imaging is clinically unavailable until now. The lead compound discovery is the first key step for the study. Herein, we initially established an efficient biologically evaluation system well in highthroughput based on SPR technology, and identified a novel class of N, N-dibenzylcinnamamide (DBC) compounds as α-syn ligands through the assay. These compounds were proved to have high affinities against α-syn aggregates (KD < 10 nM), which well met the requirement of binding activity for the PET probe. These DBC compounds were firstly reported as α-syn ligands herein and the preliminary obtained structure has been further modified into F-labeled ones. Among them, a high-affinity tracer (5-41) with 1.03 nM (KD) has been acquired, indicating its potential as a new lead compound for developing PET radiotracer.

A Novel Allosteric Inhibitor of Phosphoglycerate Mutase 1 Suppresses Growth and Metastasis of Non-Small-Cell Lung Cancer.

Phosphoglycerate mutase 1 (PGAM1) plays a pivotal role in cancer metabolism and tumor progression via its metabolic activity and interaction with other proteins like α-smooth muscle actin (ACTA2). Allosteric regulation is considered to be an innovative strategy to discover a highly selective and potent inhibitor targeting PGAM1. Here, we identified a novel PGAM1 allosteric inhibitor, HKB99, via structure-based optimization. HKB99 acted to allosterically block conformational change of PGAM1 during catalytic process and PGAM1-ACTA2 interaction. HKB99 suppressed tumor growth and metastasis and overcame erlotinib resistance in non-small-cell lung cancer (NSCLC). Mechanistically, HKB99 enhanced the oxidative stress and altered multiple signaling pathways including the activation of JNK/c-Jun and suppression of AKT and ERK. Collectively, the study highlights the potential of PGAM1 as a therapeutic target in NSCLC and reveals a distinct mechanism by which HKB99 inhibits both metabolic activity and nonmetabolic function of PGAM1 by allosteric regulation.

An allosteric PGAM1 inhibitor effectively suppresses pancreatic ductal adenocarcinoma.

Glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) plays a critical role in cancer metabolism by coordinating glycolysis and biosynthesis. A well-validated PGAM1 inhibitor, however, has not been reported for treating pancreatic ductal adenocarcinoma (PDAC), which is one of the deadliest malignancies worldwide. By uncovering the elevated PGAM1 expressions were statistically related to worse prognosis of PDAC in a cohort of 50 patients, we developed a series of allosteric PGAM1 inhibitors by structure-guided optimization. The compound KH3 significantly suppressed proliferation of various PDAC cells by down-regulating the levels of glycolysis and mitochondrial respiration in correlation with PGAM1 expression. Similar to PGAM1 depletion, KH3 dramatically hampered the canonic pathways highly involved in cancer metabolism and development. Additionally, we observed the shared expression profiles of several signature pathways at 12 h after treatment in multiple PDAC primary cells of which the matched patient-derived xenograft (PDX) models responded similarly to KH3 in the 2 wk treatment. The better responses to KH3 in PDXs were associated with higher expression of PGAM1 and longer/stronger suppressions of cancer metabolic pathways. Taken together, our findings demonstrate a strategy of targeting cancer metabolism by PGAM1 inhibition in PDAC. Also, this work provided "proof of concept" for the potential application of metabolic treatment in clinical practice.

A selective sphingomyelin synthase 2 inhibitor ameliorates diet induced insulin resistance via the IRS-1/Akt/GSK-3ß signaling pathway.

Insulin resistance is a typical precursor and primary feature of type 2 diabetes mellitus (T2DM). Sphingomyelin (SM) is a kind of sphingolipid located in animal brain, liver, kidney and muscle. Sphingomyelin synthase 2 (SMS2) is the key enzyme in the synthesis of sphingomyelin, inhibition of which shows protective effects on cardiovascular and glucose metabolism. We used Ly93, a selective sphingomyelin synthase 2 inhibitor, to investigate the effect of SMS2 inhibitor on insulin resistance in vitro and in vivo. Our previous studies have shown that Ly93 is able to dose-dependently inhibit the SMS activity and attenuate the atherosclerotic lesions in apoE knock out mice. In this present study, we found that high fat diet (HFD) induced insulin-resistant C57BL/6 mice treated with Ly93 were more sensitive to insulin than untreated mice, and presented lower blood insulin levels and improved insulin tolerance. Furthermore, insulin signal pathway related protein levels were detected by western blot, which indicated that SMS2 inhibitor significantly upregulated the phosphorylation of IRS-1, Akt and GSK-3ß, thus enhanced the insulin signaling. In vitro, Ly93 enhanced the phosphorylation of Akt in HepG2 cells, which was reversed by exogenous sphingomyelin. These results suggest that SMS2 inhibitor could ameliorate insulin resistance via regulating the insulin signaling. Our findings support that SMS2 is a potential target for insulin resistance.

Development of Anthraquinone Analogues as Phosphoglycerate Mutase 1 Inhibitors.

Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis and biosynthesis to promote cancer cell proliferation, and is believed to be a promising target for cancer therapy. Herein, based on the anthraquinone scaffold, we synthesized 31 anthraquinone derivatives and investigated the structure-activity relationship (SAR). The 3-substitient of sulfonamide on the anthraquinone scaffold was essential for maintaining potency and the modifications of the hydroxyl of alizarin would cause a sharp decrease in potency. In the meantime, we determined the co-crystal structure of PGAM1 and one of the anthraquinone inhibitors 9i with IC50 value of 0.27 µM. The co-crystal structure revealed that F22, K100 and R116 of PGAM1 were critical residues for the binding of inhibitors which further validated the SAR. Consistent with the crystal structure, a competitive assay illustrated that compound 9i was a noncompetitive inhibitor. In addition, compound 9i effectively restrained different lung cancer cells proliferation in vitro. Taken together, this work provides reliable guide for future development of PGAM1 inhibitors and compound 9i may act as a new leading compound for further optimization.

Synthesis and biological evaluation of anthraquinone derivatives as allosteric phosphoglycerate mutase 1 inhibitors for cancer treatment.

Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis, pentose phosphate pathway, and serine synthesis to promote tumor growth through the regulation of its substrate 3-phosphoglycerate (3 PG) and product 2-phosphoglycerate (2 PG). Herein, based on our previously reported PGAM1 inhibitor PGMI-004A, we have developed anthraquinone derivatives as novel allosteric PGAM1 inhibitors and the structure-activity relationship (SAR) was investigated. In addition, we determined the co-crystal structure of PGAM1 and the inhibitor 8g, demonstrating that the inhibitor was located at a novel allosteric site. Among the derivatives, compound 8t was selected for further study, with IC50 values of 0.25 and approximately 5 µM in enzymatic and cell-based assays, respectively. Mechanistically, compound 8t reduced the glycolysis and oxygen consumption rate in cancer cells, which led to decreased adenosine 5'-triphosphate (ATP) production and subsequent 5' adenosine monophosphate-activated protein kinase (AMPK) activation. The inhibitor 8t also exhibited good efficacy in delaying tumor growth in H1299 xenograft model without obvious toxicity. Taken together, this proof-of-principle work further validates PGAM1 as a potential target for cancer therapy and provides useful information on anti-tumor drug discovery targeting PGAM1.

Discovery, synthesis and anti-atherosclerotic activities of a novel selective sphingomyelin synthase 2 inhibitor.

The sphingomyelin synthase 2 (SMS2) is a potential target for pharmacological intervention in atherosclerosis. However, so far, few selective SMS2 inhibitors and their pharmacological activities were reported. In this study, a class of 2-benzyloxybenzamides were discovered as novel SMS2 inhibitors through scaffold hopping and structural optimization. Among them, Ly93 as one of the most potent inhibitors exhibited IC50 values of 91 nM and 133.9 µM against purified SMS2 and SMS1 respectively. The selectivity ratio of Ly93 was more than 1400-fold for purified SMS2 over SMS1. The in vitro studies indicated that Ly93 not only dose-dependently diminished apoB secretion from Huh7 cells, but also significantly reduced the SMS activity and increased cholesterol efflux from macrophages. Meanwhile, Ly93 inhibited the secretion of LPS-mediated pro-inflammatory cytokine and chemokine in macrophages. The pharmacokinetic profiles of Ly93 performed on C57BL/6J mice demonstrated that Ly93 was orally efficacious. As a potent selective SMS2 inhibitor, Ly93 significantly decreased the plasma SM levels of C57BL/6J mice. Furthermore, Ly93 was capable of dose-dependently attenuating the atherosclerotic lesions in the root and the entire aorta as well as macrophage content in lesions, in apolipoprotein E gene knockout mice treated with Ly93. In conclusion, we discovered a novel selective SMS2 inhibitor Ly93 and demonstrated its anti-atherosclerotic activities in vivo. The preliminary molecular mechanism-of-action studies revealed its function in lipid homeostasis and inflammation process, which indicated that the selective inhibition of SMS2 would be a promising treatment for atherosclerosis.

2-Hydroxy-oleic acid does not activate sphingomyelin synthase activity.

2-Hydroxy-oleic acid (2OHOA) is a potent anticancer drug that induces cancer cell cycle arrest and apoptosis. Previous studies have suggested that 2OHOA's anticancer effect is mediated by SMS activation in cancer cells, including A549 and U118 cells. To confirm this phenomenon, in this study, we treated both A549 and U118 cells with 2OHOA and measured SMS activity. To our surprise, we found neither 2OHOA-mediated SMS activation nor sphingomyelin accumulation in the cells. However, we noted that 2OHOA significantly reduces phosphatidylcholine in these cells. We also did not observe 2OHOA-mediated SMS activation in mouse tissue homogenates. Importantly, 2OHOA inhibited rather than activated recombinant SMS1 (rSMS1) and rSMS2 in a dose-dependent fashion. Intra-gastric treatment of C57BL/6J mice with 2OHOA for 10 days had no effects on liver and small intestine SMS activities and plasma sphingomyelin levels. The treatment inhibited lysophosphatidylcholine acyltransferase (LPCAT) activity, consistent with the aforementioned reduction in plasma phosphatidylcholine. Because total cellular phosphatidylcholine is used as a predictive biomarker for monitoring tumor responses, the previously reported 2OHOA-mediated cancer suppression could be related to this phosphatidylcholine reduction, which may influence cell membrane structure and properties. We conclude that 2OHOA is not a SMS activator and that its anticancer property may be related to an effect on phosphatidylcholine metabolism.

Discovery and anti-inflammatory evaluation of benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of glycogen synthase kinase-3ß (GSK-3ß).

Glycogen synthase kinase-3ß (GSK-3ß) has been identified to promote inflammation and its inhibitors have also been proven to treat some inflammatory mediated diseases in animal models. Non-ATP competitive inhibitors inherently have better therapeutical value due to their higher specificity than ATP competitive ones. In this paper, we designed and synthesized a series of new BTZ derivatives as non-ATP competitive GSK-3ß inhibitors. Kinetic analysis revealed two typical compounds 6j and 3j showed the different non-ATP competitive mechanism of substrate competition or allosteric modulation to GSK-3ß, respectively. As expected, the two compounds showed good specificity in a panel test of 16 protein kinases, even to the closest enzymes, like CDK-1/cyclin B and CK-II. The in vivo results proved that both compounds can greatly attenuate the LPS-induced acute lung injury (ALI) and diminish inflammation response in mice by inhibiting the mRNA expression of IL-1ß and IL-6. Western blot analysis demonstrated that they negatively regulated GSK-3ß, and the mechanism of the observed beneficial effects of the inhibitors may involve both the increased phosphorylation of the Ser9 residue on GSK-3ß and protein expression of Sirtuin 1 (SIRT1). The results support that such novel BTZ compounds have a protective role in LPS-induced ALI, and might be attractive candidates for further development of inflammation pharmacotherapy, which greatly thanks to their inherently high selectivities by the non-ATP competitive mode of action. Finally, we proposed suggesting binding modes by Docking study to well explain the impacts of compounds on the target site.

Discovery of 4-Benzyloxybenzo[ d]isoxazole-3-amine Derivatives as Highly Selective and Orally Efficacious Human Sphingomyelin Synthase 2 Inhibitors that Reduce Chronic Inflammation in db/ db Mice.

Sphingomyelin synthase 2 (SMS2) is a promising therapeutic target for several chronic inflammation-associated diseases, including atherosclerosis, fatty liver, and insulin resistance. Herein, we report the identification of 4-benzyloxybenzo[ d]isoxazole-3-amine derivatives as potent and highly selective SMS2 inhibitors through a conformational restriction strategy. After systematic structural modifications, several compounds with high selectivity and good potency in vitro were selected for further evaluation. Compound 15w demonstrated good pharmacokinetics (oral bioavailability, F = 56%) in vivo and has an inhibitory potency against sphingomyelin synthase activity when Institute of Cancer Research mice are provided with an oral dose of this compound. In addition, compound 15w attenuated chronic inflammation significantly in db/ db mice after oral dosing for 6 weeks.