Phase separation of Nur77 mediates XS561-induced apoptosis by promoting the formation of Nur77/Bcl-2 condensates.

The orphan nuclear receptor Nur77 is a critical regulator of the survival and death of tumor cells. The pro-death effect of Nur77 can be regulated by its interaction with Bcl-2, resulting in conversion of Bcl-2 from a survival to killer. As Bcl-2 is overexpressed in various cancers preventing them from apoptosis and promoting their resistance to chemotherapy, targeting the apoptotic pathway of Nur77/Bcl-2 may lead to new cancer therapeutics. Here, we report our identification of XS561 as a novel Nur77 ligand that induces apoptosis of tumor cells by activating the Nur77/Bcl-2 pathway. In vitro and animal studies revealed an apoptotic effect of XS561 in a range of tumor cell lines including MDA-MB-231 triple-negative breast cancer (TNBC) and MCF-7/LCC2 tamoxifen-resistant breast cancer (TAMR) in a Nur77-dependent manner. Mechanistic studies showed XS561 potently induced the translocation of Nur77 from the nucleus to mitochondria, resulting in mitochondria-related apoptosis. Interestingly, XS561-induced accumulation of Nur77 at mitochondria was associated with XS561 induction of Nur77 phase separation and the formation of Nur77/Bcl-2 condensates. Together, our studies identify XS561 as a new activator of the Nur77/Bcl-2 apoptotic pathway and reveal a role of phase separation in mediating the apoptotic effect of Nur77 at mitochondria.

A triple fusion tissue-type plasminogen activator (TriF-ΔtPA) enhanced thrombolysis in carotid embolism-induced stroke model.

Recombinant tissue-type plasminogen activator (rtPA, or Alteplase) is the first approved thrombolytic drug for acute ischemic stroke, but suffers from a short half-life and poor resistance to plasminogen activator inhibitor (PAI-1), limiting its clinical use. The development of novel thrombolytic agents with improved benefit/risk balance has always been of great significance. In this study, we identified a mutant of serine protease domain of tPA (named ΔtPAA146V) capable of escaping the inhibition by endogenous PAI-1 with 66-fold increased resistance compared to the wild type tPA. Based on this mutant, we generated a triple fusion ΔtPA (TriF-ΔtPA) containing albumin and fibrin binding peptide(FBP). The fusion with albumin effectively prolonged the plasma half-life of ΔtPA in mice to 144 min, which is much longer than ΔtPA and did not affect its thrombolytic activity. Furthermore, FBP rendered fibrin specificity of the fusion protein, giving a dissociation constant of âˆ¼ 25 ± 0.9 µM. In a novel murine carotid embolism-induced stroke (CES) model, i.v. administration of TriF-ΔtPA promoted vascular recanalization, reduced infarct volume, and mitigated neurobehavioral deficits more significantly compared to ΔtPA-HSA or Alteplase, showing little bleeding risk. Together, this long-acting PAI-1-resistant thrombolytic agent holds great potential for clinical applications.

Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates.

Liquid-liquid phase separation promotes the formation of membraneless condensates that mediate diverse cellular functions, including autophagy of misfolded proteins. However, how phase separation participates in autophagy of dysfunctional mitochondria (mitophagy) remains obscure. We previously discovered that nuclear receptor Nur77 (also called TR3, NGFI-B, or NR4A1) translocates from the nucleus to mitochondria to mediate celastrol-induced mitophagy through interaction with p62/SQSTM1. Here, we show that the ubiquitinated mitochondrial Nur77 forms membraneless condensates capable of sequestrating damaged mitochondria by interacting with the UBA domain of p62/SQSTM1. However, tethering clustered mitochondria to the autophagy machinery requires an additional interaction mediated by the N-terminal intrinsically disordered region (IDR) of Nur77 and the N-terminal PB1 domain of p62/SQSTM1, which confers Nur77-p62/SQSTM1 condensates with the magnitude and liquidity. Our results demonstrate how composite multivalent interaction between Nur77 and p62/SQSTM1 coordinates to sequester damaged mitochondria and to connect targeted cargo mitochondria for autophagy, providing mechanistic insight into mitophagy.

Optimization of novel oxidative DIMs as Nur77 modulators of the Nur77-Bcl-2 apoptotic pathway.

Nur77, an orphan nuclear receptor, is a member of the nuclear receptor superfamily. Nur77 plays important roles in various biological processes. Previously we reported that BI1071(DIM-C-pPhCF3+MeSO3-), an oxidized form and methanesulfonate salt of (4-CF3-Ph-C-DIM), can modulate Nur77's non-genomic apoptotic pathway through that Nur77 translocated from the nucleus to mitochondria to induce cytochrome c releasing and promote apoptosis of cancer cell. Here we report our efforts to further optimize BI1071. A series of BI1071 analogs were designed, synthesized and their apoptosis potency was systematically evaluated. Our preliminary structure-activity relationship study identified compound 10b as a better modulator with strong binding to Nur77 and enhanced apoptotic activity. Binding studies demonstrated that 10b could bind to its target Nur77 with an affinity value of 33 nM. Furthermore, mechanism studies reveal that 10b acts as an anticancer agent by utilizing the Nur77-Bcl-2 apoptotic pathway.

[Recent progress in developing of thrombolytic agents for ischemic stroke].

Ischemic stroke is a major health crisis causing high mortality and morbidity. The key treatment relies on the rapid intervention to dissolve thrombus, to reduce bleeding side effect and re-canalize clotted blood vessels using clot lysis drugs. Tissue plasminogen activator (tPA) is the only FDA-approved drug for ischemic stroke, but it has many limitations in clinical use. In recent years, the development of thrombolytic drugs and treatment strategies based on tPA has been progressed rapidly. Here we review the recent progress in this field, including the contributions from us and others, to promote the future development of novel thrombolytic drugs.

SAR study of celastrol analogs targeting Nur77-mediated inflammatory pathway.

Nur77, an orphan member of the nuclear receptor superfamily, plays an important role in the regulation of inflammatory processes. Our previous work found that celastrol, a pentacyclic triterpene, bound to Nur77 to inhibit inflammation in a Nur77-dependent manner. Celastrol binding to Nur77 promotes Nur77 translocation from nucleus to cytoplasm, resulting in clearance of inflamed mitochondria and then alleviation of inflammation. Here, we report the design, synthesis, SAR study and biological evaluation of a series of celastrol analogs. A total of 24 celastrol derivatives were made. Compound 3a with a Kd of 0.87 µM was found to be less toxic than celastrol and could be a hit molecule for further optimization.

A novel ELISA for the detection of active form of plasminogen activator inhibitor-1 based on a highly specific trapping agent.

Plasminogen activator inhibitor-1 (PAI-1) is a labile molecule that exists in four different forms: active, latent, cleaved and target bound form. Although there have been many methods to measure the total PAI-1, the measurement of active form of PAI-1 antigen is still challenging. Here we developed a novel ELISA to detect the active form of PAI-1 based on a highly specific PAI-1 capturing agent which binds to active PAI-1 with high affinity. We also used a highly stable PAI-1 mutant as an assay calibrator to enhance the method's reproducibility. This ELISA has the advantage of measuring both the antigen level and activity of PAI-1 at the same time. The assay had a sensitivity of 0.167 ng/ml and a working range of 0.195-25 ng/ml. The intra- and inter-assay variations were 6.7% and 11.3% respectively. The mean recovery of spiked standard was 102%. We used this strategy to measure the active PAI-1 level in plasma of healthy donors, and had an interesting observation: the PAI-1 level reduced by half after plasma storage for 6 h at room temperature. This finding represents the first observation of activity loss in plasma PAI-1 samples, and may explain large variations in PAI-1 levels (0-100 ng/ml) observed in human samples using commercial assays.

tPA Point Mutation at Autolysis Loop Enhances Resistance to PAI-1 Inhibition and Catalytic Activity.

Recombinant tissue-type plasminogen activator (r-tPA) was approved by U.S. Food and Drug Administration as a thrombolytic drug. However, a high dose of r-tPA (up to 100 mg/person) is typically used in clinical applications. Such high dosage leads to severe side effects including haemorrhage and neurotoxicity, which can be fatal. To improve the proteolytic properties of tPA to enhance thrombolytic therapy, we designed a series of mutants in tPA serine protease domain (tPA-SPD) based on the crystal structure of tPA-SPD:plasminogen activators inhibitor-1 (PAI-1) complex that we determined recently. We found that the A146Y substitution in tPA-SPD(A146Y) enhanced resistance to PAI-1 inactivation by 30-fold compared with original tPA-SPD. Interestingly, the tPA-SPD(A146Y) variant showed fivefold higher activation for plasminogen compared with tPA-SPD. The variant also demonstrated thrombolytic activity stronger than tPA-SPD in a clot lysis assay. In vivo, we showed tPA-SPD(A146Y) possessed higher thrombolytic efficacy in a pulmonary embolism model compared with original tPA-SPD. Furthermore, a mouse tail bleeding assay showed that tPA-SPD(A146Y) did not increase bleeding risk compared with clinical drug r-tPA. Together, our findings reveal novel functions of A146Y variant, which not only increases the catalytic efficiency of the enzyme, but also enhances resistance to PAI-1 inhibition, and demonstrating that tPA-SPD (A146Y) variant is a much improved agent for thrombolytic therapy.

A long-acting PAI-1 inhibitor reduces thrombus formation.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we fused PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which was the H37R variant of PAItrap fused to HSA, gave a six-fold improvement of IC50 (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC50 value of 12 nM, allowing the inhibitor to be evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis.