Design, synthesis and biological evaluation of new RNF126-based p300/CBP degraders.

Histone acetyltransferase CREB-binding protein (CBP) and its homologous protein p300 are key transcriptional activators that can activate oncogene transcription, which present promising targets for cancer therapy. Here, we designed and synthesized a series of p300/CBP targeted low molecular weight PROTACs by assembling the covalent ligand of RNF126 E3 ubiquitin ligase and the bromodomain ligand of the p300/CBP. The optimal molecule A8 could effectively degrade p300 and CBP through the ubiquitin-proteasome system in time- and concentration-dependent manners, with half-maximal degradation (DC50) concentrations of 208.35/454.35 nM and 82.24/79.45 nM for p300/CBP in MV4-11 and Molm13 cell lines after 72 h of treatment. And the degradation of p300/CBP by A8 is dependent on the ubiquitin-proteasome pathway and its simultaneous interactions with the target proteins and RNF126. A8 exhibits good antiproliferative activity in a series of p300/CBP-dependent cancer cells. It could transcriptionally inhibit the expression of c-Myc, induce cell cycle arrest in the G0/G1 phase and apoptosis in MV4-11 cells. This study thus provided us a new chemotype for the development of drug-like PROTACs targeting p300/CBP, which is expected to be applied in cancer therapy.

Targeting type I PRMTs as promising targets for the treatment of pulmonary disorders: Asthma, COPD, lung cancer, PF, and PH.

Pulmonary disorders, including asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (PF), pulmonary hypertension (PH), and lung cancer, seriously impair the quality of lives of patients. A deeper understanding of the occurrence and development of the above diseases may inspire new strategies to remedy the scarcity of treatments. Type I protein arginine methyltransferases (PRMTs) can affect processes of inflammation, airway remodeling, fibroblast proliferation, mitochondrial mass, and epithelial dysfunction through substrate methylation and non-enzymatic activity, thus affecting the occurrence and development of asthma, COPD, lung cancer, PF, and PH. As potential therapeutic targets, inhibitors of type I PRMTs are developed, moreover, representative compounds such as GSK3368715 and MS023 have also been used for early research. Here, we collated structures of type I PRMTs inhibitors and compared their activity. Finally, we highlighted the physiological and pathological associations of type I PRMTs with asthma, COPD, lung cancer, PF, and PH. The developing of type I PRMTs modulators will be beneficial for the treatment of these diseases.

Pharmacological inhibition of EZH2 using a covalent inhibitor suppresses human ovarian cancer cell migration and invasion.

Metastasis is the cause of poor prognosis in ovarian cancer (OC). Enhancer of Zeste homolog 2 (EZH2), a histone-lysine N-methyltransferase enzyme, promotes OC cell migration and invasion by regulating the expression of tissue inhibitor of metalloproteinase-2 (TIMP2) and matrix metalloproteinases-9 (MMP9). Hence, we speculated that EZH2-targeting therapy might suppress OC migration and invasion. In this study, the expression of EZH2, TIMP2, and MMP9 in OC tissues and cell lines was analyzed using The Cancer Genome Atlas (TCGA) database and western blotting, respectively. The effects of SKLB-03220, an EZH2 covalent inhibitor, on OC cell migration and invasion were investigated using wound-healing assays, Transwell assays, and immunohistochemistry. TCGA database analysis confirmed that the EZH2 and MMP9 mRNA expression was significantly higher in OC tissues, whereas TIMP2 expression was significantly lower than that in normal ovarian tissues. Moreover, EZH2 negatively correlated with TIMP2 and positively correlated with MMP9 expression. In addition to the anti-tumor activity of SKLB-03220 in a PA-1 xenograft model, immunohistochemistry results showed that SKLB-03220 markedly increased the expression of TIMP2 and decreased the expression of MMP9. Additionally, wound-healing and Transwell assays showed that SKLB-03220 significantly inhibited the migration and invasion of both A2780 and PA-1 cells in a concentration-dependent manner. SKLB-03220 inhibited H3K27me3 and MMP9 expression and increased TIMP2 expression in PA-1 cells. Taken together, these results indicate that the EZH2 covalent inhibitor SKLB-03220 inhibits metastasis of OC cells by upregulating TIMP2 and downregulating MMP9, and could thus serve as a therapeutic agent for OC.

Promising role of protein arginine methyltransferases in overcoming anti-cancer drug resistance.

Drug resistance remains a major challenge in cancer treatment, necessitating the development of novel strategies to overcome it. Protein arginine methyltransferases (PRMTs) are enzymes responsible for epigenetic arginine methylation, which regulates various biological and pathological processes, as a result, they are attractive therapeutic targets for overcoming anti-cancer drug resistance. The ongoing development of small molecules targeting PRMTs has resulted in the generation of chemical probes for modulating most PRMTs and facilitated clinical treatment for the most advanced oncology targets, including PRMT1 and PRMT5. In this review, we summarize various mechanisms underlying protein arginine methylation and the roles of specific PRMTs in driving cancer drug resistance. Furthermore, we highlight the potential clinical implications of PRMT inhibitors in decreasing cancer drug resistance. PRMTs promote the formation and maintenance of drug-tolerant cells via several mechanisms, including altered drug efflux transporters, autophagy, DNA damage repair, cancer stem cell-related function, epithelial-mesenchymal transition, and disordered tumor microenvironment. Multiple preclinical and ongoing clinical trials have demonstrated that PRMT inhibitors, particularly PRMT5 inhibitors, can sensitize cancer cells to various anti-cancer drugs, including chemotherapeutic, targeted therapeutic, and immunotherapeutic agents. Combining PRMT inhibitors with existing anti-cancer strategies will be a promising approach for overcoming anti-cancer drug resistance. Furthermore, enhanced knowledge of the complex functions of arginine methylation and PRMTs in drug resistance will guide the future development of PRMT inhibitors and may help identify new clinical indications.

A patent review of EZH2 inhibitors from 2017 and beyond.

INTRODUCTION: EZH2 is an important epigenetic regulator that forms the PRC2 complex with SUZ12, EED and RbAp46/48. As the key catalytic subunit of PRC2, EZH2 regulates the trimethylation of histone H3K27, which in turn promotes chromatin condensation and represses the transcription of relevant target genes. EZH2 overexpression and mutations are strictly related to tumor proliferation, invasion and metastasis. Currently, a large number of highly specific EZH2 inhibitors have been developed and some have already been in clinical trials. AREAS COVERED: The aim of the present review is to provide an overview of the molecular mechanisms of EZH2 inhibitors and to highlight the research advances in the patent literature published from 2017 to date. A search of the literature and patents for EZH2 inhibitors and degraders was performed using the Web of Science, SCIFinder, WIPO, USPTO, EPO and CNIPA databases. EXPERT OPINION: In recent years, a great number of structurally diverse EZH2 inhibitors have been identified, including EZH2 reversible inhibitors, EZH2 irreversible inhibitors, EZH2-based dual inhibitors and EZH2 degraders. Despite the multiple challenges, EZH2 inhibitors offer promising potential for the treatment of various diseases, such as cancers.

Pharmacological inhibition of EZH2 by ZLD1039 suppresses tumor growth and pulmonary metastasis in melanoma cells in vitro and in vivo.

The incidence and mortality rate of malignant melanoma are increasing worldwide. Metastasis reduces the efficacy of current melanoma therapies and leads to poor prognosis for patients. EZH2 is a methyltransferase that promotes the proliferation, metastasis, and drug resistance of tumor cells by regulating transcriptional activity. EZH2 inhibitors could be effective in melanoma therapies. Herein, we aimed to investigate whether the pharmacological inhibition of EZH2 by ZLD1039, a potent and selective S-adenosyl-l-methionine-EZH2 inhibitor, suppresses tumor growth and pulmonary metastasis in melanoma cells. Results showed that ZLD1039 selectively reduced H3K27 methylation in melanoma cells by inhibiting EZH2 methyltransferase activity. Additionally, ZLD1039 exerted excellent antiproliferative effects on melanoma cells in 2D and 3D culture systems. Administration of ZLD1039 (100 mg/kg) by oral gavage caused antitumor effects in the A375 subcutaneous xenograft mouse model. RNA sequencing and GSEA revealed that the ZLD1039-treated tumors exhibited changes in the gene sets enriched from the "Cell Cycle" and "Oxidative Phosphorylation", whereas the "ECM receptor interaction" gene set had a negative enrichment score. Mechanistically, ZLD1039 induced G0/G1 phase arrest by upregulating p16 and p27 and inhibiting the functions of the cyclin D1/CDK6 and cyclin E/CDK2 complexes. Moreover, ZLD1039 induced apoptosis in melanoma cells via the mitochondrial reactive oxygen species apoptotic pathway, consistent with the changes in transcriptional signatures. ZLD1039 also exhibited excellent antimetastatic effects on melanoma cells in vitro and in vivo. Our data highlight that ZLD1039 may be effective against melanoma growth and pulmonary metastasis and thus could serve as a therapeutic agent for melanoma.

Discovery of a Novel Covalent EZH2 Inhibitor Based on Tazemetostat Scaffold for the Treatment of Ovarian Cancer.

Enhancer of zeste homologue 2 (EZH2) is the enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2), which plays an important role in post-translational modifications of histones. In this study, we designed and synthesized a new series EZH2 covalent inhibitors that have rarely been reported. Biochemical studies and mass spectrometry provide information that SKLB-03220 could covalently bind to the S-adenosylmethionine (SAM) pocket of EZH2. Besides, SKLB-03220 was highly potent for EZH2MUT, while exhibiting weak activities against other tested histone methyltransferases (HMTs) and kinases. Moreover, SKLB-03220 displayed noteworthy potency against ovarian cancer cell lines and continuously abolished H3K27me3 after washing out. Furthermore, oral administration of SKLB-03220 significantly inhibited tumor growth in PA-1 xenograft model without obvious adverse effects. Taken together, SKLB-03220 is a potent, selective EZH2 covalent inhibitor with noteworthy anticancer efficacy both in vitro and in vivo.

Design, synthesis and biological evaluation of a new class of 7H-pyrrolo[2,3-d]pyrimidine derivatives as Mps1 inhibitors for the treatment of breast cancer.

Monopolar spindle kinase 1 (Mps1), a core component of the spindle assembly checkpoint (SAC), plays a crucial role in the transition of cells from mid-to late mitosis. As an attractive therapeutic target, inhibition of Mps1 induces cell cycle arrest and apoptosis in a variety of tumors, including breast cancer. However, early clinical development of Mps1 inhibitors remains unsatisfactory. Here, we designed and synthesized a new class of Mps1 inhibitors with 7H-pyrrolo[2,3-d]pyrimidine structure using a scaffold hopping approach. Structure-activity relationship (SAR) revealed that 12 is a potent Mps1 inhibitor (IC50 = 29 nM), which inhibited phosphorylation of Mps1 in vitro and in vivo. Treatment with 12 not only impeded proliferation of breast cancer cell lines, but also induced cell cycle arrest and apoptosis of MCF-7 and 4T1 cells. 12 suppressed tumor growth in vivo, and no obvious toxicities were observed. These results demonstrated the potential of Mps1 inhibitor 12 for the treatment of breast cancer.

A novel small molecule RK-019 inhibits FGFR2-amplification gastric cancer cell proliferation and induces apoptosis in vitro and in vivo.

Gastric cancer (GC) is one of the most malignant cancers and is estimated to be fifth in incidence ratio and the third leading cause of cancer death worldwide. Despite advances in GC treatment, poor prognosis and low survival rate necessitate the development of novel treatment options. Fibroblast growth factor receptors (FGFRs) have been suggested to be potential targets for GC treatment. In this study, we report a novel selective FGFR inhibitor, RK-019, with a pyrido [1, 2-a] pyrimidinone skeleton. In vitro, RK-019 showed excellent FGFR1-4 inhibitory activities and strong anti-proliferative effects against FGFR2-amplification (FGFR2-amp) GC cells, including SNU-16 and KATO III cells. Treatment with RK-019 suppressed phosphorylation of FGFR and its downstream pathway proteins, such as FRS2, PLCγ, AKT, and Erk, resulting in cell cycle arrest and induction of apoptosis. Furthermore, daily oral administration of RK-019 could attenuate tumor xenograft growth with no adverse effects. Here, we reported a novel specific FGFR inhibitor, RK-019, with potent anti-FGFR2-amp GC activity both in vitro and in vivo.

Identification of an ergosterol derivative with anti-melanoma effect from the sponge-derived fungus Pestalotiopsis sp. XWS03F09.

It is difficult to treat malignant melanoma because of its high malignancy. New and effective therapies for treating malignant melanoma are urgently needed. Ergosterols are known for specific biological activities and have received widespread attention in cancer therapy. Here, LH-1, a kind of ergosterol from the secondary metabolites of the marine fungus Pestalotiopsis sp., was extracted, isolated, purified, and further investigated the biological activities against melanoma. In vitro experiments, the anti-proliferation effect on tumor cells was detected by MTT and colony formation assay, and the anti-metastatic effect on tumor cells was investigated by wound healing assay and transwell assay. Subcutaneous xenograft models, histopathology, and immunohistochemistry have been used to verify the anti-tumor, toxic, and side effect in vivo. Besides, the anti-tumor mechanism of LH-1 was studied by mRNA sequencing. In vitro, LH-1 could inhibit the proliferation and migration of melanoma cells A375 and B16-F10 in a dose-dependent manner and promote tumor cell apoptosis through the mitochondrial apoptosis pathway. In vivo assays confirmed that LH-1 could suppress melanoma growth by inducing cell apoptosis and reducing cell proliferation, and it did not have any notable toxic effects on normal tissues. LH-1 may play an anti-melanoma role by upregulating OBSCN gene expression. These findings suggest that LH-1 may be a potential for the treatment of melanoma.

Targeting Oncoproteins for Degradation by Small Molecule-Based Proteolysis-Targeting Chimeras (PROTACs) in Sex Hormone-Dependent Cancers.

Sex hormone-dependent cancers, including breast, ovary, and prostate cancer, contribute to the high number of cancer-related deaths worldwide. Steroid hormones promote tumor occurrence, development, and metastasis by acting on receptors, such as estrogen receptors (ERs), androgen receptors (ARs), and estrogen-related receptors (ERRs). Therefore, endocrine therapy targeting ERs, ARs, and ERRs represents the potential and pivotal therapeutic strategy in sex hormone-dependent cancers. Proteolysis-targeting chimeras (PROTACs) are a novel strategy that can harness the potential of the endogenous ubiquitin-proteasome system (UPS) to target and degrade specific proteins, rather than simply inhibiting the activity of target proteins. Small molecule PROTACs degrade a variety of proteins in cells, mice, and humans and are an emerging approach for novel drug development. PROTACs targeting ARs, ERs, ERRs, and other proteins in sex hormone-dependent cancers have been reported and may overcome the problem of resistance to existing endocrine therapy and receptor antagonist treatments. This review briefly introduces the PROTAC strategy and summarizes the progress on the development of small molecule PROTACs targeting oncoproteins in sex hormone-dependent cancers, focusing on breast and prostate cancers.

A miniaturized analytical method based on molecularly imprinted absorbents for selective extraction of (S)-1,1'-binaphthyl-2,2'-diamine and combinatorial screening of polymer precursors by computational simulation.

Chiral resolution of binaphthylamine is often a toilful conundrum in the field of analytical chemistry and biomedicine. The work puts forward a selective, sensitive, and miniaturized analytical method based on molecularly imprinted polymers (MIPs) as adsorbent for miniaturized tip solid-phase extraction (MTSPE) in the separation of binaphthylamine enantiomer. This method combines the advantages of MIPs (high selectivity), MTSPE (low consumption), and high-performance liquid chromatography (HPLC, high sensitivity). A simple synthesis methodology of MIP (P2) was conducted through bulk polymerization with (S)-(-)-1,1'-binaphthyl-2,2'-diamine (S-DABN) as template together with methacrylic acid monomer, and ethylene glycol dimethacrylate as cross-linker in proper porogen, realizing a selective recognition and efficient enrichment for S-DABN. The method exhibited appreciable linearity (0.06-1.00 mg ml-1 ), low quantification limit (0.056 mg ml-1 ), good absolute recoveries (45.70%-69.29%), and high precision (relative standard deviations ≤ 3.54%), along with low consumption (0.50 ml sample solution and 25.0 mg adsorbent). Based on the density functional theory, computational simulation was used to make a preliminary prediction for rational design of MIPs and gave a reasonable elaboration involving the potential mechanism of templates interacting with functional monomers. The adsorption kinetics and thermodynamics were investigated to evaluate the recombination process of substrates. In addition, the selectivity of MIPs for S-DABN was obtained by MIP-MTSPE coupled with HPLC, which supports the feasibility of this convenient design process. The proposed method was employed for selective extraction of S-DABN and exhibited promising potential in the application of chiral analysis.

Design, synthesis and biological evaluation of 7-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-2,3-dihydro-1H-inden-1-one derivatives as potent FAK inhibitors for the treatment of ovarian cancer.

Focal adhesion kinase (FAK) promotes tumor progression by intracellular signal transduction and regulation of gene expression and protein turnover, which is a compelling therapeutic target for various cancer types, including ovarian cancer. However, the clinical responses of FAK inhibitors remain unsatisfactory. Here, we describe the discovery of FAK inhibitors using a scaffold hopping strategy. Structure-activity relationship (SAR) exploration identified 36 as a potent FAK inhibitor, which exhibited inhibitory activities against FAK signaling in vitro. Treatment with 36 not only decreased migration and invasion of PA-1 cells, but also reduced expression of MMP-2 and MMP-9. Moreover, 36 inhibited tumor growth and metastasis, and no obvious adverse effects were observed during the in vivo study. These results revealed the potential of FAK inhibitor 36 for treatment of ovarian cancer.

Interfering TUG1 Attenuates Cerebrovascular Endothelial Apoptosis and Inflammatory injury After Cerebral Ischemia/Reperfusion via TUG1/miR-410/FOXO3 ceRNA Axis.

Background Emerging studies illustrate that long non-coding RNA TUG1 (TUG1) participates in neuron death after ischemia. However, the role of TUG1 in cerebral ischemia/reperfusion (CI/R) injury through cerebrovascular pathology was undetermined yet. Methods Expression of TUG1, miRNA-410-3p (miR-410), and forkhead box O3 (FOXO3) was detected by RT-qPCR and western blot. Neural function, apoptosis, and inflammatory damage were assessed by triphenyltetrazolium chloride straining, modified neurological severity score, fluorescence-activated cell sorting method, and western blot. The relationship among TUG1, miR-410, and FOXO3 was identified by dual-luciferase reporter assay, RNA pull-down, and RNA immunoprecipitation. Results TUG1 was upregulated in middle cerebral artery occlusion/reperfusion (MCAO/R) mice and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced mouse brain microvascular endothelial cells (BMECs) in a certain of time-dependent manner. Blockage of TUG1 decreased infarct volume and increased neurological score in MCAO/R mice, accompanied with elevated Bcl-2 expression and declined expression of IL-1ß, IL-6, TNF-α, Bax, and cleaved caspase 3. Abovementioned proteins were similarly expressed in OGD/R-induced BMECs with TUG1 knockdown, paralleled with diminished apoptosis rate. Either, miR-410 overexpression and FOXO3 interference could suppress OGD/R-induced inflammatory and apoptotic responses. Of note, TUG1 and FOXO3 are competing endogenous RNAs (ceRNAs) for miR-410 via target binding. Depleting miR-410 counteracted the role of TUG1 exhaustion, and reinforcing FOXO3 abated the effect of miR-410 overexpression. Conclusion Exhausting TUG1 could alleviate CI/R-induced inflammatory injury and apoptosis in brain tissues and BMECs via targeting miR-410/FOXO3 axis, suggesting an innovative perspective from cerebrovascular endothelial cells in the pathogenesis and treatment of CI/R.

Methylation of dual-specificity phosphatase 4 controls cell differentiation.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

Dense hyperthermic intraperitoneal chemotherapy with cisplatin in patients with stage III serous epithelial ovarian cancer: a retrospective study.

BACKGROUND: To investigate the efficacy and safety of interval debulking surgery (IDS) combined with dense hyperthermic intraperitoneal chemotherapy (HIPEC) with cisplatin in Chinese patients with FIGO stage III serous epithelial ovarian cancer (EOC). METHODS: This retrospective single-center study reviewed the demographic and clinical data of 197 patients with primary FIGO stage III serous EOC who were treated with IDS with (n = 121) or without (n = 76, control group) dense HIPEC between January 2012 and April 2017. The co-primary endpoints were progression-free survival (PFS) and overall survival (OS), and the secondary endpoint was the occurrence of adverse events. RESULTS: The median PFS was 24 months in the IDS plus dense HIPEC group, whereas it was 19 months in the IDS alone group (hazard ratio [HR] 0.46, 95% confidence interval [CI]: 0.33-0.65, p = 0.000). The median OS in patients treated with IDS plus dense HIPEC (51 months) was significantly longer than that in patients treated with IDS alone (40 months, HR 0.52, 95% CI: 0.35-0.78, p = 0.001). The demographic and preoperative clinical characteristics of these two groups were comparable (p > 0.05). In the IDS alone group, no adverse events were recorded in 42 (55.3%) of the 76 patients, and 14 (18.4%) patients were reported to have grade III/IV adverse events. In the IDS plus dense HIPEC group, no adverse events were recorded in 55 (45.5%) of the 121 patients, and 23 (19.0%) patients were reported to have grade III/IV adverse events. No postoperative deaths occurred within 30 days in either group and neither did severe fatal complications in the IDS plus dense HIPEC group. CONCLUSIONS: IDS plus dense HIPEC with cisplatin in Chinese patients with FIGO stage III serous EOC is associated with improved survival and is reasonably well tolerated by patients.

The discovery of SKLB-0335 as a paralog-selective EZH2 covalent inhibitor.

By targeting the unique Cys663 of EZH2, SKLB-0335 displays high paralog-selectivity on EZH2. Biochemical studies show that SKLB-0335 can covalently bind to EZH2 at its S-adenosylmethionine (SAM) pocket and inhibit H3K27Me3. SKLB-0335 could be an effective chemical probe with which to further investigate the specific biological functions of EZH2.

BRD4 inhibition sensitizes aggressive non-Hodgkin lymphomas to PI3Kδ inhibitors by suppressing PI3K reactivation and c-MYC expression.

Targeting phosphatidylinositol 3-kinase δ (PI3Kδ) is an important therapeutic strategy for indolent non-Hodgkin lymphomas (NHLs). However, we previously observed reactivation of phosphatidylinositol 3-kinase (PI3K) pathways in aggressive NHL cell lines following continuous exposure to PI3Kδ inhibitors (PI3Kδi), which limited their efficacy and suggests that more studies should be focused on this reactivation to improve current PI3Kδi-based treatments. Herein we conducted a drug synergy screening that combined a marketed PI3Kδi, idelalisib, with 14 well-characterized epigenetic drugs across several types of aggressive NHL cell lines. We identified BRD4 inhibitors (BRD4i) as potent partners that, in combination with idelalisib, were capable of synergistically exerting anti-proliferative activity and inducing cell apoptosis in a panel of aggressive NHL cell lines through continuous suppression of PI3K pathways. More importantly, the combination of BRD4i and PI3Kδi simultaneously inhibited transcription and translation of the oncogenic transcription factor c-MYC, downregulating the expression of c-MYC and continuously suppressing the proliferation of cancer cells in vitro, as well as the growth of tumors in vivo even after drug withdrawal. This study, thus, reveals the potential of simultaneously targeting PI3Kδ and BRD4 as a new therapeutic strategy for aggressive forms of NHL.