Recharacterization of RSL3 reveals that the selenoproteome is a druggable target in colorectal cancer.

Ferroptosis is an iron-dependent, non-apoptotic form of cell death resulting from the accumulation of lipid peroxides. Colorectal cancer (CRC) accumulates high levels of intracellular iron and reactive oxygen species (ROS), thereby sensitizing cells to ferroptosis. The selenoprotein glutathione peroxidase (GPx4) is a key enzyme in the detoxification of lipid peroxides and can be inhibited by the compound (S)-RSL3 ([1S,3R]-RSL3). However, the stereoisomer (R)-RSL3 ([1R,3R]-RSL3), which does not inhibit GPx4, exhibits equipotent activity to (S)-RSL3 across a panel of CRC cell lines. Utilizing CRC cell lines with an inducible knockdown of GPx4, we demonstrate that (S)-RSL3 sensitivity does not align with GPx4 dependency. Subsequently, a biotinylated (S)-RSL3 was then synthesized to perform affinity purification-mass spectrometry (AP-MS), revealing that (S)-RSL3 acts as a pan-inhibitor of the selenoproteome, targeting both the glutathione and thioredoxin peroxidase systems as well as multiple additional selenoproteins. To investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed further chemical and genetic approaches to disrupt selenoprotein function. The findings demonstrate that the selenoprotein inhibitor Auranofin can induce ferroptosis and/or oxidative cell death both in-vitro and in-vivo. Consistent with this data we observe that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translational incorporation of selenocysteine, is essential for CRC growth. In summary, our research elucidates the complex mechanisms underlying ferroptosis in CRC and reveals that modulation of the selenoproteome provides multiple new therapeutic targets and opportunities in CRC.

Integrated transcriptomic and proteomic analysis reveals Guizhi-Fuling Wan inhibiting STAT3-EMT in ovarian cancer progression.

BACKGROUND: Ovarian cancer (OC) is the most lethal gynecological malignancy. Frequent peritoneal dissemination is the main cause of low survival rate. Guizhi-Fuling Wan (GZFL) is a classical traditional Chinese herbal formula that has been clinically used for treating ovarian cancer with good outcome. However, its therapeutic mechanism for treating OC has not been clearly elucidated. PURPOSE: We aim to elucidate the potential mechanisms of GZFL in treating OC with a focus on STAT3 signaling pathway. METHODS: In vivo efficacy of GZFL was assessed using an OC xenograft mouse model. Proteomics analysis in OC cells and RNA-seq analysis in mice tumors were performed to fully capture the translational and transcriptional signature of GZFL. Effects of GZFL on proliferation, spheroid formation and reactive oxygen species (ROS) were assessed using wildtype and STAT3 knockout OC cells in vitro. STAT3 activation and transcription activity, hypoxia and EMT-related protein expression were assessed to validate the biological activity of GZFL. RESULTS: GZFL suppresses tumor growth with a safety profile in mice, while prevents cell growth, spheroid formation and accumulates ROS in a STAT3-dependent manner in vitro. GZFL transcriptionally and translationally affects genes involved in inflammatory signaling, EMT, cell migration, and cellular hypoxic stress response. In depth molecular study confirmed that GZFL-induced cytotoxicity and EMT suppression in OC cells are directly corelated to inhibition of STAT3 activation and transcription activity. CONCLUSION: Our study provides the first evidence that GZFL inhibits OC progression through suppressing STAT3-EMT signaling. These results will further support its potential clinical use in OC.

SIRT5 variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology.

SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients.

First-in-Class NADH/Ubiquinone Oxidoreductase Core Subunit S7 (NDUFS7) Antagonist for the Treatment of Pancreatic Cancer.

Pancreatic cancer cells adapt to nutrient-scarce metabolic conditions by increasing their oxidative phosphorylation reserve to survive. Here, we present a first-in-class small-molecule NDUFS7 antagonist that inhibits oxidative phosphorylation (OXPHOS) for the treatment of pancreatic cancer. The lead compound, DX2-201, suppresses the proliferation of a panel of cell lines, and a metabolically stable analogue, DX3-213B, shows significant efficacy in a syngeneic model of pancreatic cancer. Exome sequencing of six out of six clones resistant to DX2-201 revealed a pV91M mutation in NDUFS7, providing direct evidence of its drug-binding site. In combination studies, DX2-201 showed synergy with multiple metabolic modulators, select OXPHOS inhibitors, and PARP inhibitors. Importantly, a combination with 2-deoxyglucose overcomes drug resistance in vivo. This study demonstrates that an efficacious treatment for pancreatic cancer can be achieved through inhibition of OXPHOS and direct binding to NDUFS7, providing a novel therapeutic strategy for this hard-to-treat cancer.

The application of PROTAC in HDAC.

Inducing protein degradation by proteolysis targeting chimera (PROTAC) has provided great opportunities for scientific research and industrial applications. Histone deacetylase (HDAC)-PROTAC has been widely developed since the first report of its ability to induce the degradation of SIRT2 in 2017. To date, ten of the eighteen HDACs (HDACs 1-8, HDAC10, and SIRT2) have been successfully targeted and degraded by HDAC-PROTACs. HDAC-PROTACs surpass traditional HDAC inhibitors in many aspects, such as higher selectivity, more potent antiproliferative activity, and the ability to disrupt the enzyme-independent functions of a multifunctional protein and overcome drug resistance. Rationally designing HDAC-PROTACs is a main challenge in development because slight variations in chemical structure can lead to drastic effects on the efficiency and selectivity of the degradation. In the future, HDAC-PROTACs can potentially be involved in clinical research with the support of the increased amount of in vivo data, pharmacokinetic evaluation, and pharmacological studies.

Hyaluronic acid-bilirubin nanomedicine-based combination chemoimmunotherapy.

Despite significant advances in immune checkpoint blockade (ICB), immunosuppression mediated by tumor-associated myeloid cells (TAMCs) poses a major barrier to cancer immunotherapy. In addition, while immunogenic cell death (ICD) provides a viable approach to inducing anti-tumor immune response, it remains unknown how to effectively trigger ICD while addressing immunosuppressive TAMCs. Here, we show that SC144, a gp130 inhibitor that blocks the IL-6/gp130/STAT3 pathway, induces ICD of tumor cells and polarizes macrophages to M1-phenotype in vitro. However, as SC144 also induces killing of CD8+ T-cells, we sought to deliver SC144 selectively to tumor cells and TAMCs. Toward this goal, we have developed hyaluronic acid-bilirubin nanoparticles (HABN) that accumulate in CD44hi tumor cells and TAMCs. Systemic administration of SC144 loaded in HABN (SC144@HABN) induces apoptosis and ICD of tumor cells, increases the ratio of M1-like to M2-like macrophages, and decreases the frequency of myeloid-derived suppressor cells and CD4+ regulatory T-cells, while promoting anti-tumor CD8+ T-cells. Moreover, SC144@HABN combined with anti-PD-L1 ICB efficiently eliminates MC38 tumors and ICB-resistant 4T1 tumors. Overall, our work demonstrates a therapeutic strategy based on coordinated ICD induction and TAMC modulation and highlights the potential of combination chemoimmunotherapy.

GRP78 Inhibitor YUM70 Suppresses SARS-CoV-2 Viral Entry, Spike Protein Production and Ameliorates Lung Damage.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has given rise to many new variants with increased transmissibility and the ability to evade vaccine protection. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) chaperone that has been recently implicated as an essential host factor for SARS-CoV-2 entry and infection. In this study, we investigated the efficacy of YUM70, a small molecule inhibitor of GRP78, to block SARS-CoV-2 viral entry and infection in vitro and in vivo. Using human lung epithelial cells and pseudoviral particles carrying spike proteins from different SARS-CoV-2 variants, we found that YUM70 was equally effective at blocking viral entry mediated by original and variant spike proteins. Furthermore, YUM70 reduced SARS-CoV-2 infection without impacting cell viability in vitro and suppressed viral protein production following SARS-CoV-2 infection. Additionally, YUM70 rescued the cell viability of multi-cellular human lung and liver 3D organoids transfected with a SARS-CoV-2 replicon. Importantly, YUM70 treatment ameliorated lung damage in transgenic mice infected with SARS-CoV-2, which correlated with reduced weight loss and longer survival. Thus, GRP78 inhibition may be a promising approach to augment existing therapies to block SARS-CoV-2, its variants, and other viruses that utilize GRP78 for entry and infection.

Lipidation of a bioactive cyclotide-based CXCR4 antagonist greatly improves its pharmacokinetic profile in vivo.

The CXCR4 chemokine is a key molecular regulator of many biological functions controlling leukocyte functions during inflammation and immunity, and during embryonic development. Overexpression of CXCR4 is also associated with many types of cancer where its activation promotes angiogenesis, tumor growth/survival, and metastasis. In addition, CXCR4 is involved in HIV replication, working as a co-receptor for viral entry, making CXCR4 a very attractive target for developing novel therapeutic agents. Here we report the pharmacokinetic profile in rats of a potent CXCR4 antagonist cyclotide, MCo-CVX-5c, previously developed in our group that displayed a remarkable in vivo resistance to biological degradation in serum. This bioactive cyclotide, however, was rapidly eliminated through renal clearance. Several lipidated versions of cyclotide MCo-CVX-5c showed a significant increase in the half-life when compared to the unlipidated form. The palmitoylated version of cyclotide MCo-CVX-5c displayed similar CXCR4 antagonistic activity as the unlipidated cyclotide, while the cyclotide modified with octadecanedioic (18-oxo-octadecanoic) acid exhibited a remarkable decrease in its ability to antagonize CXCR4. Similar results were also obtained when tested for its ability to inhibit growth in two cancer cell lines and HIV infection in cells. These results show that the half-life of cyclotides can be improved by lipidation although it can also affect their biological activity depending on the lipid employed.

Design and Synthesis of Orally Active Quinolyl Pyrazinamides as Sigma 2 Receptor Ligands for the Treatment of Pancreatic Cancer.

Sigma 2 receptor (σ2R) is overexpressed in select cancers and is regarded as a biomarker for tumor proliferation. σ2R ligands are emerging as promising theranostics for cancer and neurodegenerative diseases. Herein, we describe the design and synthesis of a series of novel quinolyl pyrazinamides as selective and potent σ2R ligands that show sub-micromolar potency in pancreatic cancer cell lines. Compounds 14 (JR1-157) and 17 (JR2-298) bind σ2R with Ki of 47 and 10 nM, respectively. Importantly, compound 14 has an oral bioavailability of 60% and shows significant in vivo efficacy without obvious toxicity in a syngeneic model of pancreatic cancer. The cytotoxicity of the quinolyl pyrazinamides significantly enhanced in the presence of copper and diminished in the presence of the copper-chelator tetrathiomolybdate. In conclusion, compound 14 is water-soluble, metabolically stable, orally active, and increases the expression of the autophagy marker LC3B and warrants further development for the treatment of pancreatic cancer.

Inhibition of Protein Disulfide Isomerase (PDIA1) Leads to Proteasome-Mediated Degradation of Ubiquitin-like PHD and RING Finger Domain-Containing Protein 1 (UHRF1) and Increased Sensitivity of Glioblastoma Cells to Topoisomerase II Inhibitors.

Glioblastoma (GBM) is the most aggressive brain tumor, and the prognosis remains poor with current available treatments. PDIA1 is considered a promising therapeutic target in GBM. In this study, we demonstrate that targeting PDIA1 results in increased GBM cell death by topoisomerase II (Top-II) inhibitors resulting in proteasome-mediated degradation of the oncogenic protein UHRF1. Combination of the PDIA1 inhibitor, bepristat-2a, produces strong synergy with doxorubicin, etoposide, and mitoxantrone in GBM and other cancer cell lines. Our bioinformatics analysis of multiple datasets revealed downregulation of UHRF1, upon PDIA1 inhibition. In addition, PDIA1 inhibition results in proteasome-mediated degradation of UHRF1 protein. Interestingly, treatment of GBM cells with bepristat-2a results in increased apoptosis and resistance to ferroptosis. Our findings emphasize the importance of PDIA1 as a therapeutic target in GBM and present a promising new therapeutic approach using Top-II inhibitors for GBM treatment.

Pyrazolone derivatives as potent and selective small-molecule SIRT5 inhibitors.

Sirtiun 5 (SIRT5) is a NAD+-dependent protein lysine deacylase. It is emerging as a promising target for the development of drugs to treat cancer and metabolism-related diseases. In this study, we screened 5000 compounds and identified a hit compound 14 bearing a pyrazolone functional group as a novel SIRT5-selective inhibitor. Structure-based optimization of 14 resulted in compound 47 with an IC50 value of 0.21 ± 0.02 µM and a 100-fold improved potency. Compound 47 showed substantial selectivity for SIRT5 over SIRT1-3 and SIRT6. Biochemical studies suggest that 47 does not occupy the NAD + -binding pocket and acts as a substrate-competitive inhibitor. The identified potent and selective SIRT5 inhibitors allow further studies as research tools and therapeutic agents.

Inflammation as a chemoprevention target in asbestos-induced malignant mesothelioma.

Malignant mesothelioma (MM) is an incurable cancer of the serosal lining that is often caused by exposure to asbestos. Therefore, novel agents for the prevention and treatment of this disease are urgently needed. Asbestos induces the release of pro-inflammatory cytokines such as IL-1ß and IL-6, which play a role in MM development. IL-6 is a component of the JAK-STAT3 pathway that contributes to inflammation-associated tumorigenesis. Glycoprotein 130 (gp130), the signal transducer of this signaling axis, is an attractive drug target because of its role in promoting neoplasia via the activation of downstream STAT3 signaling. The anticancer drug, SC144, inhibits the interaction of gp130 with the IL-6 receptor (IL6R), effectively blunting signaling from this inflammatory axis. To test whether the inflammation-related release of IL-6 plays a role in the formation of MM, we evaluated the ability of SC144 to inhibit asbestos-induced carcinogenesis in a mouse model. The ability of sulindac and anakinra, an IL6R antagonist/positive control, to inhibit MM formation in this model was tested in parallel. Asbestos-exposed Nf2+/-;Cdkn2a+/- mice treated with SC144, sulindac or anakinra showed significantly prolonged survival compared to asbestos-exposed vehicle-treated mice. STAT3 activity was markedly decreased in MM specimens from SC144-treated mice. Furthermore, SC144 inhibited STAT3 activation by IL-6 in cultured normal mesothelial cells, and in vitro treatment of MM cells with SC144 markedly decreased the expression of STAT3 target genes. The emerging availability of newer, more potent SC144 analogs showing improved pharmacokinetic properties holds promise for future trials, benefitting individuals at high risk of this disease.

Targeting GRP78 suppresses oncogenic KRAS protein expression and reduces viability of cancer cells bearing various KRAS mutations.

KRAS is the most commonly mutated oncogene in human cancers with limited therapeutic options, thus there is a critical need to identify novel targets and inhibiting agents. The 78-kDa glucose-regulated protein GRP78, which is upregulated in KRAS cancers, is an essential chaperone and the master regulator of the unfolded protein response (UPR). Following up on our recent discoveries that GRP78 haploinsufficiency suppresses both KRASG12D-driven pancreatic and lung tumorigenesis, we seek to determine the underlying mechanisms. Here, we report that knockdown of GRP78 via siRNA reduced oncogenic KRAS protein level in human lung, colon, and pancreatic cancer cells bearing various KRAS mutations. This effect was at the post-transcriptional level and is independent of proteasomal degradation or autophagy. Moreover, targeting GRP78 via small molecule inhibitors such as HA15 and YUM70 with anti-cancer activities while sparing normal cells significantly suppressed oncogenic KRAS expression in vitro and in vivo, associating with onset of apoptosis and loss of viability in cancer cells bearing various KRAS mutations. Collectively, our studies reveal that GRP78 is a previously unidentified regulator of oncogenic KRAS expression, and, as such, augments the other anti-cancer activities of GRP78 small molecule inhibitors to potentially achieve general, long-term suppression of mutant KRAS-driven tumorigenesis.

Identification of 2-hydroxybenzoic acid derivatives as selective SIRT5 inhibitors.

The sirtuin deacetylase SIRT5 plays important roles in regulating multiple metabolic pathways, and potentially represents an attractive target for the treatment of several human diseases, especially cancer. In this study, we report the identification of the hit compound 11 bearing a 2-hydroxybenzoic acid functional group as a novel SIRT5-selective inhibitor via our medium-throughput thermal shift screening assay. Hit 11 stabilizes SIRT5 in a dose-dependent manner and shows moderate inhibitory activity against SIRT5 and high subtype selectivity over SIRT1, 2, and 3 in a trypsin coupled enzyme-based assay. The carboxylic acid and the adjacent hydroxyl group of 11 are essential for maintaining activity. To further improve the potency of compound 11, a lead optimization was carried out, resulting in compound 43 with a 10-fold improved potency. Overall, compound 11 represents a promising new chemical scaffold for further investigation to develop SIRT5-selective inhibitors.

Induction of Genes Implicated in Stress Response and Autophagy by a Novel Quinolin-8-yl-nicotinamide QN523 in Pancreatic Cancer.

Using a cytotoxicity-based phenotypic screen of a highly diverse library of 20,000 small-molecule compounds, we identified a quinolin-8-yl-nicotinamide, QN519, as a promising lead. QN519 represents a novel scaffold with drug-like properties, showing potent in vitro cytotoxicity in a panel of 12 cancer cell lines. Subsequently, lead optimization campaign generated compounds with IC50 values < 1 µM. An optimized compound, QN523, shows significant in vivo efficacy in a pancreatic cancer xenograft model. QN523 treatment significantly increased the expression of HSPA5, DDIT3, TRIB3, and ATF3 genes, suggesting activation of the stress response pathway. We also observed a significant increase in the expression of WIPI1, HERPUD1, GABARAPL1, and MAP1LC3B, implicating autophagy as a major mechanism of action. Due to the lack of effective treatments for pancreatic cancer, discovery of novel agents such as the QN series of compounds with unique mechanism of action has the potential to fulfill a clear unmet medical need.

Multiparameter Optimization of Oxidative Phosphorylation Inhibitors for the Treatment of Pancreatic Cancer.

Targeting oxidative phosphorylation (OXPHOS) complexes is an emerging strategy to disrupt the metabolism of select cancer subtypes and to overcome resistance to targeted therapies. Here, we describe our lead optimization campaign on a series of benzene-1,4-disulfonamides as novel OXPHOS complex I inhibitors. This effort led to the discovery of compound 23 (DX3-213B) as one of the most potent complex I inhibitors reported to date. DX3-213B disrupts adenosine triphosphate (ATP) generation, inhibits complex I function, and results in the growth inhibition of pancreatic cancer cells in the low nanomolar range. Importantly, the oral administration of DX3-213B resulted in significant in vivo efficacy in a pancreatic cancer syngeneic model without obvious toxicity. Our data clearly demonstrate that OXPHOS inhibition can be a safe and efficacious strategy to treat pancreatic cancer.

Discovery and Lead Optimization of Benzene-1,4-disulfonamides as Oxidative Phosphorylation Inhibitors.

Inhibition of oxidative phosphorylation (OXPHOS) is a promising therapeutic strategy for select cancers that are dependent on aerobic metabolism. Here, we report the discovery, optimization, and structure-activity relationship (SAR) study of a series of novel OXPHOS inhibitors. The hit compound, benzene-1,4-disulfonamide 1, was discovered in a phenotypic screen selective for cytotoxicity in a galactose-containing medium. Our multi-parameter optimization campaign led to the discovery of 65 (DX3-235), showing nanomolar inhibition of complex I function and adenosine triphosphate (ATP) production in a galactose-containing medium resulting in significant cytotoxicity. Importantly, 64 (DX3-234), a close analogue of 65, is well tolerated in mice and shows significant single agent efficacy in a Pan02 syngeneic pancreatic cancer model, suggesting that highly potent and selective OXPHOS inhibitors can be useful for the treatment of pancreatic cancer.

Why All the Fury over Furin?

Analysis of the SARS-CoV-2 sequence revealed a multibasic furin cleavage site at the S1/S2 boundary of the spike protein distinguishing this virus from SARS-CoV. Furin, the best-characterized member of the mammalian proprotein convertases, is an ubiquitously expressed single pass type 1 transmembrane protein. Cleavage of SARS-CoV-2 spike protein by furin promotes viral entry into lung cells. While furin knockout is embryonically lethal, its knockout in differentiated somatic cells is not, thus furin provides an exciting therapeutic target for viral pathogens including SARS-CoV-2 and bacterial infections. Several peptide-based and small-molecule inhibitors of furin have been recently reported, and select cocrystal structures have been solved, paving the way for further optimization and selection of clinical candidates. This perspective highlights furin structure, substrates, recent inhibitors, and crystal structures with emphasis on furin's role in SARS-CoV-2 infection, where the current data strongly suggest its inhibition as a promising therapeutic intervention for SARS-CoV-2.