Development of Potent and Selective Coactivator-Associated Arginine Methyltransferase 1 (CARM1) Degraders.

CARM1 is amplified or overexpressed in many cancer types, and its overexpression correlates with poor prognosis. Potent small-molecule inhibitors for CARM1 have been developed, but the cellular efficacy of the CARM1 inhibitors is limited. We herein report the development of the proteolysis targeting chimera (PROTAC) for CARM1, which contains a CARM1 ligand TP-064, a linker, and a VHL E3 ligase ligand. Compound 3b elicited potent cellular degradation activity (DC50 = 8 nM and Dmax > 95%) in a few hours. Compound 3b degraded CARM1 in VHL- and proteasome-dependent manner and was highly selective for CARM1 over other protein arginine methyltransferases. CARM1 degradation by 3b resulted in potent downregulation of CARM1 substrate methylation and inhibition of cancer cell migration in cell-based assays. Thus, CARM1 PROTACs can be used to interrogate CARM1's cellular functions and potentially be developed as therapeutic agents for targeting CARM1-driven cancers.

Ferroptosis signaling promotes the release of misfolded proteins via exosomes to rescue ER stress in hepatocellular carcinoma.

Dysfunction of the ubiquitin‒proteasome system can induce sustained endoplasmic reticulum stress (ERS) and subsequent cell death. However, malignant cells have evolved multiple mechanisms to evade sustained ERS. Therefore, identification of the mechanisms through which tumor cells develop resistance to ERS is important for the therapeutic exploitation of these cells for drug-resistant tumors. Herein, we found that proteasome inhibitors could induce ERS, activate ferroptosis signaling, and thereby induce the adaptive tolerance of tumor cells to ERS. Mechanistically, the activation of ferroptosis signaling was found to promote the formation and secretion of exosomes containing misfolded and unfolded proteins, which resulted in rescuing ERS and promoting tumor cell survival. The inhibition of ferroptosis signaling synergized with bortezomib, a clinically used proteasome inhibitor, to suppress the viability of hepatocellular carcinoma cells in vitro and in vivo. The present findings reveal that ERS resistance can be driven by an ERS-ferroptosis signaling-exosome pathway and have important clinical implications for intracellular signaling, ER homeostasis and drug-resistant cancer therapy.

Diptoindonesin G is a middle domain HSP90 modulator for cancer treatment.

HSP90 inhibitors can target many oncoproteins simultaneously, but none have made it through clinical trials due to dose-limiting toxicity and induction of heat shock response, leading to clinical resistance. We identified diptoindonesin G (dip G) as an HSP90 modulator that can promote degradation of HSP90 clients by binding to the middle domain of HSP90 (Kd = 0.13 ± 0.02 µM) without inducing heat shock response. This is likely because dip G does not interfere with the HSP90-HSF1 interaction like N-terminal inhibitors, maintaining HSF1 in a transcriptionally silent state. We found that binding of dip G to HSP90 promotes degradation of HSP90 client protein estrogen receptor α (ER), a major oncogenic driver protein in most breast cancers. Mutations in the ER ligand-binding domain (LBD) are an established mechanism of endocrine resistance and decrease the binding affinity of mainstay endocrine therapies targeting ER, reducing their ability to promote ER degradation or transcriptionally silence ER. Because dip G binds to HSP90 and does not bind to the LBD of ER, unlike endocrine therapies, it is insensitive to ER LBD mutations that drive endocrine resistance. Additionally, we determined that dip G promoted degradation of WT and mutant ER with similar efficacy, downregulated ER- and mutant ER-regulated gene expression, and inhibited WT and mutant cell proliferation. Our data suggest that dip G is not only a molecular probe to study HSP90 biology and the HSP90 conformation cycle, but also a new therapeutic avenue for various cancers, particularly endocrine-resistant breast cancer harboring ER LBD mutations.

Sertraline inhibits stress-induced tumor growth through regulating CD8 + T cell-mediated anti-tumor immunity.

Chronic stress has been reported to be associated with tumor initiation and progression. But the underlying mechanism and the specific role of tumor immunity in this process are still unknown. Herein, we applied the repeated restrain stress model in C57BL/6J mice and found that the tumor growth in stressed mice was accelerated compared with that in control mice. In addition, serotonin, also called 5-hydroxytryptamine (5-HT), in the serum of stressed mice was also elevated. Sertraline, a selective serotonin reuptake inhibitor used in the clinic, can restore the serum 5-HT level in stressed mice and restrain tumor growth. We further explored the distribution of major immune cells, including B lymphocytes cells, T lymphocytes, natural killer cells, dendritic cells, tumor-associated macrophages (TAM) and regulatory T cells (Treg). We found that the infiltration of CD8 + T cells in the tumor microenvironment (TME) decreased significantly in stressed mice. And the extra 5-HT treatment could further decrease the infiltration of CD8 + T cells in the TME. The expression of IFN-γ and Granular enzyme B (GzmB) in CD8 + T cells were also dropped in the stressed mice group, whereas the expression of programmed cell death protein 1 (PD-1) on CD8 + T cells was increased. The T cell deficiency induced by stress can be reversed by sertraline, indicating its promising role in strengthening the efficacy of anti-PDL1/PD-1 immunotherapy. The present study provides new mechanistic insights into the impact of chronic stress on antitumor immunity and implicates a novel combined immunotherapy strategy for cancer patients with chronic stress.

Accurate prediction of microvascular invasion occurrence and effective prognostic estimation for patients with hepatocellular carcinoma after radical surgical treatment.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide, with an overall 5-year survival rate of less than 18%, which may be related to tumor microvascular invasion (MVI). This study aimed to compare the clinical prognosis of HCC patients with or without MVI after radical surgical treatment, and further analyze the preoperative risk factors related to MVI to promote the development of a new treatment strategy for HCC. METHODS: According to the postoperative pathological diagnosis of MVI, 160 study patients undergoing radical hepatectomy were divided into an MVI-negative group (n = 68) and an MVI-positive group (n = 92). The clinical outcomes and prognosis were compared between the two groups, and then the parameters were analyzed by multivariate logistic regression to construct an MVI prediction model. Then, the practicability and validity of the model were evaluated, and the clinical prognosis of different MVI risk groups was subsequently compared. RESULT: There were no significant differences between the MVI-negative and MVI-positive groups in clinical baseline, hematological, or imaging data. Additionally, the clinical outcome comparison between the two groups presented no significant differences except for the pathological grading (P = 0.002) and survival and recurrence rates after surgery (P < 0.001). The MVI prediction model, based on preoperative AFP, tumor diameter, and TNM stage, presented superior predictive efficacy (AUC = 0.7997) and good practicability (high H-L goodness of fit, P = 0.231). Compared with the MVI high-risk group, the patients in the MVI low-risk group had a higher survival rate (P = 0.002) and a lower recurrence rate (P = 0.004). CONCLUSION: MVI is an independent risk factor for a poor prognosis after radical resection of HCC. The MVI prediction model, consisting of AFP, tumor diameter, and TNM stage, exhibits superior predictive efficacy and strong clinical practicability for MVI prediction and prognostication, which provides a new therapeutic strategy for the standardized treatment of HCC patients.

Development of MDM2 degraders based on ligands derived from Ugi reactions: Lessons and discoveries.

Proteolysis targeting chimeras (PROTACs) have gained tremendous interest in both the academic and pharmaceutical communities. This opens a new way to regulate the cellular protein homeostasis, especially for disease-related proteins. In this work, we designed and synthesized a series of MDM2 degraders based on ligands that were readily prepared by a four-component Ugi reaction. After extensive optimization based on anti-proliferation and MDM2 degradation, WB214 was identified as the most potent anti-proliferative agent in various leukemia cell lines. Surprisingly, our mechanistic investigations indicated that WB214 not only effectively induced the degradation of MDM2, but also led to the degradation of p53. Further studies revealed that WB214 degraded MDM2 as a molecular glue. WB214 and its related analogues did not bind to MDM2 in the p53 binding region and MDM2 was discovered as a novel neo-substrate of the E3 ligase cereblon. Finally, we found that WB214 could potently degrade GSPT1, which could rationalize the inhibition of cell growth. A selective degrader for GSPT1 over MDM2 was then developed through systematically varying different motifs.

Evaluation of the binding affinity of E3 ubiquitin ligase ligands by cellular target engagement and in-cell ELISA assay.

The discovery of potent cell-permeable E3 ubiquitin ligase ligands can significantly facilitate the development of proteolysis targeting chimeras (PROTACs). Here, we present a protocol to determine the binding affinity of ligands toward CRBN E3 ubiquitin ligase, using a cellular target engagement mechanism and in-cell ELISA assay. This protocol is easy to establish, with relatively low cost and rapid time frame. It can also be modified to measure the level of other proteins or determine the ligand affinity toward other E3s. For complete details on the use and execution of this protocol, please refer to Yang et al. (2020).

[Clinical Characteristics and Prognostic Analysis of 59 Adult Patients with Epstein-Barr Virus-Associated Hemophagocytic Syndrome].

OBJECTIVE: To analyze the clinical characteristics and the prognostic risk factors of adult patients with Epestein-Barr virus-associated hemophagocytic syndrome (EBV-HLH) so as to enhance the understanding of EBV-HLH and diagnosis and treatment level. METHODS: The clinical manifestation and survival data of 59 adult patients with EBV-HLH admitted in our hospital from January 2013 to August 2018 were analyzed retrospectively. RESULTS: The most common clinical manifestations of 59 patients were high fever (100%), liver dysfunction (91.5%), however the skin rashes (1.7%), and neurologic abnormality (3.4%) were rare. 96.6% of the patients showed the elevation of serum ferritin and LDH level, and hypoproteinemia and sCD25≥2 400 U/ml were found in 93.2% and 92.3% of the patients, respectively. The median survival time of 59 patients was 2.5±0.7 months; overall survival rate of 1, 3, 6 and 12-month was 69.5%±6.0%, 44.7%±6.6%, 23.9%±5.8%, 19.7%±5.5%, respectively. Univariate survival analysis showed that the patients with EBV-DNA copies≥5×105/ml (P＜0.05), LDH level≥600 U/L (P＜0.05) and Plt count＜20×109/L (P＜0.05) had poor prognosis, and there was statistically difference in the overall survival rate (P＜0.01) between HLH-94/2004 group and the group treated without etoposide (not HLH-94/2004). Multivariate analysis revealed that LDH level≥600 U/L (P＜0.05), Plt count＜20×109/L (P＜0.05) and treatment protocol (not HLH-94/2004) (P＜0.01) were independent prognostic risk factors in 59 patients with EBV-HLH. CONCLUSIONS: EBV-HLH assocites with severe clinical features, high mortality rate and poor prognosis of patients. EBV-DNA copies≥5×105/ml (P＜0.05), LDH level≥600 U/L (P＜0.05) and Plt count＜20×109/L (P＜0.05) are the poor prognostic factors, and the treatment with HLH-94/2004 protocol can effectively improve the survival of EBV-HLH patients, should be applied as early as possible.

Development of Selective Histone Deacetylase 6 (HDAC6) Degraders Recruiting Von Hippel-Lindau (VHL) E3 Ubiquitin Ligase.

Histone deacetylase 6 (HDAC6) is involved in multiple cellular processes such as aggresome formation, protein stability, and cell motility. Numerous HDAC6-selective inhibitors have been developed as cellular chemical tools to elucidate the function of HDAC6. Since HDAC6 has multiple domains that cannot be studied by HDAC6-selective inhibitors, CRISPR-CAS9 and siRNA/shRNA have been employed to elucidate the nonenzymatic functions of HDAC6. However, these genetic methods have many limitations. Proteolysis targeting chimera (PROTAC) is an emerging technology for the development of small molecules that can quickly remove the entire protein in cells. We previously developed multifunctional HDAC6 degraders that can recruit cereblon (CRBN) E3 ubiquitin ligase. These HDAC6 degraders can degrade not only HDAC6 but also neo-substrates of CRBN. They are excellent candidates for the development of anticancer therapeutics, but the multifunctional nature of the CRBN-based HDAC6 degraders has limited their utility as specific chemical probes for the study of HDAC6-related cellular pathways. Herein we report the development of the first cell-permeable HDAC6-selective degraders employing Von Hippel-Lindau (VHL) E3 ubiquitin ligase, which does not have any known neo-substrates. The DC50's of the most potent compound 3j are 7.1 nM and 4.3 nM in human MM1S and mouse 4935 cell lines, respectively. The D max's of 3j in these two cell lines are 90% and 57%, respectively.

Two-Stage Strategy for Development of Proteolysis Targeting Chimeras and its Application for Estrogen Receptor Degraders.

Proteolysis targeting chimeras (PROTACs) have emerged as useful chemical probes and potential therapeutics by taking advantage of the ubiquitin-proteasome system to degrade intracellular disease-associated proteins. PROTACs are heterobifunctional molecules composed of a target protein ligand, E3 ubiquitin ligase ligand, and a linker between them. The generation of efficient PROTACs requires screening of many parameters, especially the lengths and types of the linkers. We report our proof-of-concept study using a two-stage strategy to facilitate the development of PROTACs against the estrogen receptor (ER). In stage one, a library of close to 100 PROTACs was synthesized by simply mixing a library of ERα ligands containing a hydrazide functional group at different positions with a preassembled library of E3 ligase ligands bearing different types and lengths of linkers with a terminal aldehyde group in a 1:1 ratio. Cell-based screening occurred without further purification, because the formation of the acylhydrazone linkage is highly efficient and produces water as the only byproduct. Compound A3 was the most potent ER degrader in two ER+ cell lines (DC50= ∼ 10 nM, Dmax= ≥ 95%). Stage two involved transformation to a more stable amide linker to generate a more drug-like molecule. The new compound, AM-A3, showed comparable biological activity (DC50 = 1.1 nM, Dmax = 98%) and induced potent antiproliferation (IC50= 13.2 nM, Imax= 69%) in MCF-7. This proof-of -concept study demonstrates that the two-stage strategy can significantly facilitate the development of PROTACs against ER without the tedious process of making large numbers of PROTACs one by one. It has the potential to be expanded to many other targets.

In utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure exacerbates urinary dysfunction in hormone-treated C57BL/6J mice through a non-malignant mechanism involving proteomic changes in the prostate that differ from those elicited by testosterone and estradiol.

A recent study directed new focus on the fetal and neonatal environment as a risk factor for urinary dysfunction in aging males. Male mice were exposed in utero and via lactation (IUL) to the persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and then administered slow-release, subcutaneous implants of testosterone and estradiol (T+E2) as adults to mimic the hormonal environment of aging men. IUL TCDD exposure worsened T+E2-induced voiding dysfunction. Mice in the previous study were genetically prone to prostatic neoplasia and it was therefore unclear whether TCDD exacerbates voiding dysfunction through a malignant or non-malignant mechanism. We demonstrate here that IUL TCDD exposure acts via a non-malignant mechanism to exacerbate T+E2-mediated male mouse voiding dysfunction characterized by a progressive increase in spontaneous void spotting. We deployed a proteomic approach to narrow the possible mechanisms. We specifically tested whether IUL TCDD exacerbates urinary dysfunction by acting through the same prostatic signaling pathways as T+E2. The prostatic protein signature of TCDD/T+E2-exposed mice differed from that of mice exposed to T+E2 alone, indicating that the mechanism of action of TCDD differs from that of T+E2. We identified 3641 prostatic proteins in total and determined that IUL TCDD exposure significantly changed the abundance of 102 proteins linked to diverse molecular and physiological processes. We shed new light on the mechanism of IUL TCDD-mediated voiding dysfunction by demonstrating that the mechanism is independent of tumorigenesis and involves molecular pathways distinct from those affected by T+E2.

Synthesis and biological evaluation of FICZ analogues as agonists of aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor involved in multiple biological processes including immune cell differentiation, intestinal function and inflammation. Based on the scaffold of naturally occurring AhR ligand 6-formylindolo (3,2-b) carbazole (FICZ, 2), a series of analogues has been designed, synthesized and evaluated by cell-based assays. The structure-activity relationships study has successfully led to the discovery of compound 11e with extremely potent activity.

Mechanism of activation for the sirtuin 6 protein deacylase.

The histone deacetylase sirtuin 6 (SIRT6) regulates numerous biological functions, including transcriptional repression, DNA repair, and telomere maintenance. Recombinant SIRT6 displays catalytic efficiencies 2 orders of magnitude greater for long-chain deacylation than deacetylation against peptide substrates; however, deacetylation can be enhanced by allosteric small-molecule activators. Here, we investigated the mechanisms of activated lysine deacetylation and enhanced long-chain acyl-group removal by SIRT6. Activity-based screening identified compounds that activated histone peptide deacetylation 18-48-fold. Chemical optimization based on structure-activity relationships yielded an activator with improved potency and selectivity for SIRT6. Using this novel activator, we conducted biochemical and kinetic analyses revealing that SIRT6 is activated via acceleration of a catalytic step occurring after substrate binding but before NAD+ cleavage. We identified a SIRT6 variant, R65A, that maintains basal deacetylase activity but cannot be activated and failed to enhance long-chain deacylation. Additional biochemical studies revealed that Arg-65 is critical for activation by facilitating a conformational step that initiates chemical catalysis. This work suggests that SIRT6 activation of deacetylation involves a similar mechanism to improved catalysis as that of long-chain deacylation. The identification of novel SIRT6 activators and the molecular insights into activation and catalysis presented here provide a foundational understanding for physiological SIRT6 activation and for rational design of activating molecules.

[Clinical Characteristics of Liver Dysfunction in Patients with Hemophagocytic Syndrome].

OBJECTIVE: To explore the clinical feature of liver injury in patients with hemophagocytic syndrome (HPS). METHODS: The clinical data of 92 patients with HPS in our hospital were analyzed retrospectively, and the characteristics of hepatic lesion and its relationship with prognosis in HPS patients were explored. RESULT: 92 cases of HPS showed different degrees of liver dysfunction from mild to moderate. The clinical parameters of liver dysfunction included the increased level of LDH (89.13%), AST (64.13%), TBIL (59.78%) and decreased level of ALB (90.22%). Moreover, 76.09% and 67.39% of the patients had the prolonging of APTT and PT respectively. The ALB level of patients in rheumatoid immune group were higher than that in infection, maglinancy and unexplained groups, all with statistically and significant difference (P＜0.05, P＜0.05 and P＜0.01), the ALB level of patients in infection group were statistically and significantly higher than that in unexplained group (P＜0.01). The Fbg level of patients in infection group were lower than that in maglinancy group, unexplained group and rheumatoid immune group, all the differences were statistically significant (P＜0.05, P＜0.01 and P＜0.05). Child-Pugh grading was further carried out in HPS patients with liver disfunction. Survival time of the patients grade A was significantly higher than that of grade B and C of patients. Univariate analysis showed that the patients with LDH≥2000 U/L, ALB＜30 g/L and PT≥15.1 s had a survival time inferior to control patients (P＜0.05, P＜0.01 and P＜0.01, respectively). Multivariate analysis showed that ALB＜30 g/L was an independent adverse prognostic factor for these patients (P＜0.01). CONCLUSION: Patients with HPS generally have impaired liver function mainly manifested with elevated LDH and AST levels, and declined ALB level, which may correlate with the disease cause and prognosis. Patients with LDH≥2000 U/L, ALB＜30 g/L and PT≥15.1 s have a poorer prognosis and should be treated as soon as possible.

Development of Multifunctional Histone Deacetylase 6 Degraders with Potent Antimyeloma Activity.

Histone deacetylase 6 (HDAC6) primarily catalyzes the removal of acetyl group from the side chain of acetylated lysine residues in cytoplasmic proteins such as α-tubulin and HSP90. HDAC6 is involved in multiple disease-relevant pathways. Based on the proteolysis targeting chimera strategy, we previously developed the first HDAC6 degrader by tethering a pan-HDAC inhibitor with cereblon (CRBN) E3 ubiquitin ligase ligand. We herein report our new generation of multifunctional HDAC6 degraders by tethering selective HDAC6 inhibitor Nexturastat A with CRBN ligand that can synergize with HDAC6 degradation for the antiproliferation of multiple myeloma (MM). This new class of degraders exhibited improved potency and selectivity for the degradation of HDAC6. After the optimization of the linker length and linking positions, we discovered potent HDAC6 degraders with nanomolar DC50 and promising antiproliferation activity in multiple myeloma (MM) cells.

Discovery of 2,3'-diindolylmethanes as a novel class of PCSK9 modulators.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of low density lipoprotein receptor (LDLR). Anti-PCSK9 agents have been approved for the treatment of hypercholesterolemia. We recently discovered a series of small-molecule PCSK9 modulators that contains a relatively small pharmacophore of 2,3'-diindolylmethane with molecular weights around only 250. These molecules can significantly lower the amount of PCSK9 protein in a cell-based phenotypic assay. Our SAR studies yielded compound 16 with a IC50-value of 200 nM. No obvious cytotoxicity was observed at concentrations below 50 µM.

Development of selective small molecule MDM2 degraders based on nutlin.

Tumor suppressor protein p53 is important to the regulation of many cellular processes and the prevention of cancer development. In some cancer cells, the function of p53 is inhibited by murine double minute 2 protein (MDM2). To restore the function of p53, the inhibition or depletion of MDM2 has become a potential therapeutic treatment. We have successfully developed a series of small molecule MDM2 degraders that can promote the proteolysis of MDM2 oncoprotein, thus reactivating tumor suppressor p53. The superior degrader features a nutlin-based MDM2 ligand and a lenalidomide-based cereblon E3 ubiquitin ligase ligand with a short linker between the two ligands. At low nanomolar concentrations in RS4; 11 leukemia cells, this degrader promotes efficient degradation of MDM2. It also inhibits the proliferation of leukemia cells with an IC50 value of 3.2 nM and induces apoptosis effectively. All of these data indicate that MDM2 degraders are promising therapeutics for the treatment of cancers, such as leukemia.

Tissue-specific quantification and localization of androgen and estrogen receptors in prostate cancer.

Androgens and estrogens, working together, promote prostate cancer (PRCA) initiation and progression, with androgens acting via androgen receptor (AR) and estrogens acting primarily through estrogen receptor α (ERα). While the interplay between these steroid hormones has been established, the interaction between steroid hormone receptors in prostatic disease remains unstudied. The goal of this study was to objectively determine the incidence, stage specificity, and tissue/cell type specificity of AR and ERα expression, both independently and simultaneously, during the progression of PRCA. Using multiplexed immunohistochemistry and multispectral imaging analysis, AR, ERα, and smooth muscle α-actin expression was detected and quantitated in benign prostate tissue (BPT), high-grade prostatic intraepithelial neoplasia (HGPIN), PRCA, and metastasis (MET) from patient specimens (n=340). Epithelial AR expression was significantly increased in HGPIN, PRCA, and MET compared with BPT, whereas ERα expression in epithelial and stromal cells was highest in HGPIN. With analysis of AR and ERα coexpression, we identified a unique population of double-positive (AR+/ERα+) cells that increased in HGPIN specimens in both the stroma and the epithelium. Double-negative (AR-/ERα-) cells significantly decreased across PRCA progression, from 65% in BPT to 30% in MET. Preliminary analysis of this AR+/ERα+ population indicates potential cell type specificity in smooth muscle α-actin-negative stromal cells. This study demonstrates stage-, tissue-, and cell type-specific AR and ERα expression changes during PRCA progression, both independently and coexpressed. A more complete understanding of steroid hormones and their receptors in the initiation and progression of prostatic disease may elucidate improved strategies for PRCA prevention or therapy.

Identification of a novel class of RIP1/RIP3 dual inhibitors that impede cell death and inflammation in mouse abdominal aortic aneurysm models.

Receptor interacting protein kinase-1 and -3 (RIP1 and RIP3) are essential mediators of cell death processes and participate in inflammatory responses. Our group recently demonstrated that gene deletion of Rip3 or pharmacological inhibition of RIP1 attenuated pathogenesis of abdominal aortic aneurysm (AAA), a life-threatening degenerative vascular disease characterized by depletion of smooth muscle cells (SMCs), inflammation, negative extracellular matrix remodeling, and progressive expansion of aorta. The goal of this study was to develop drug candidates for AAA and other disease conditions involving cell death and inflammation. We screened 1141 kinase inhibitors for their ability to block necroptosis using the RIP1 inhibitor Necrostatin-1s (Nec-1s) as a selection baseline. Positive compounds were further screened for cytotoxicity and virtual binding to RIP3. A cluster of top hits, represented by GSK2593074A (GSK'074), displayed structural similarity to the established RIP3 inhibitor GSK'843. In multiple cell types including mouse SMCs, fibroblasts (L929), bone marrow derived macrophages (BMDM), and human colon epithelial cells (HT29), GSK'074 inhibited necroptosis with an IC50 of ~3 nM. Furthermore, GSK'074, but not Nec-1s, blocked cytokine production by SMCs. Biochemical analyses identified both RIP1 and RIP3 as the biological targets of GSK'074. Unlike GSK'843 which causes profound apoptosis at high doses (>3 µM), GSK'074 showed no detectable cytotoxicity even at 20 µM. Daily intraperitoneal injection of GSK'074 at 0.93 mg/kg significantly attenuated aortic expansion in two mouse models of AAA (calcium phosphate: DMSO 66.06 ± 9.17% vs GSK'074 27.36 ± 8.25%, P < 0.05; Angiotensin II: DMSO 85.39 ± 15.76% vs GSK'074 36.28 ± 5.76%, P < 0.05). Histologically, GSK'074 treatment diminished cell death and macrophage infiltration in aneurysm-prone aortae. Together, our data suggest that GSK'074 represents a new class of necroptosis inhibitors with dual targeting ability to both RIP1 and RIP3. The high potency and minimum cytotoxicity make GSK'074 a desirable drug candidate of pharmacological therapies to attenuate AAA progression and other necroptosis related diseases.

Synthesis and antitumor activity of a series of lactone-opened camptothecin derivatives.

A series of E-ring lactone-opened camptothecin (CPT) derivatives bearing with terminal aza-heterocyclic groups were synthesized, and their antitumor activity was evaluated both in vitro and in vivo. Hydroxyl-amide analogues with morpholin-4-yl displayed excellent antitumor activity in vitro and efficient inhibition on tumor xenograph model in nude mice. Ester-amide compounds acted less active in vitro cytotoxicity and lower inhibition activity in vivo. Substitutions at 7- and 10- positions favored the antitumor activity.