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2021 ◽  
Author(s):  
Kuang-Yu Chen ◽  
Tim Krischuns ◽  
Laura Ortega Varga ◽  
Emna Harigua-Souiai ◽  
Sylvain Paisant ◽  
...  

Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC50 values in the 4-21 micromolar range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2668-2668
Author(s):  
Yuan Xiao Zhu ◽  
Laura Ann Bruins ◽  
Joseph Ahmann ◽  
Cecilia Bonolo De Campos ◽  
Esteban Braggio ◽  
...  

Abstract Venetoclax (VTX) is a selective small-molecule inhibitor of BCL-2 that exhibits antitumoral activity against MM cells presenting lymphoid features and those with translocation t(11;14). Despite its impressive clinical activity, VTX therapy for a prolonged duration can lead to drug resistance. Therefore, it is important to understand the underlying mechanisms of resistance in order to develop strategies to prevent or overcome resistance. In the present study, we established four VTX resistant human myeloma cell lines (HMCLs) from four sensitive HMCLs, including three with t(11;14), in culture with a stepwise increase in treatment dose with VTX. To identify the molecular basis of acquired VTX resistance, whole exon sequencing (WES), mRNA-sequencing (mRNAseq), and protein expression assays were performed in the four isogenic VTX-sensitive/resistant HMCLs and three MM patients with samples collected before VTX administration and after clinical resistance to the drug. Compared with sensitive cell lines and patient samples collected before VTX administration, mRNAseq analysis identified downregulation of BIM and upregulation of BCLXL in both resistant cell lines and MM cells from relapse patients. Other transcriptional changes detected included upregulation of AURKA, BIRC3, BIRC5, and IL32. Enrichment analysis of differentially expressed genes suggested involvement of PI3K and MAPK signaling, likely associated with cytokines, growth factors (EGF, FGF and IGF family members), and receptor tyrosine kinase (EGF and FGF). Western blot analysis was performed to compare BCL2 family expression in resistant cell lines versus sensitive cell lines and it showed upregulation of BCL2 survival members (such as MCL-1 and BCLXL), and downregulation of pro-apoptotic BH3 members (such as BIM and PUMA). BIM expression was completely lost in one resistant cell line, and introduction of exogenous BIM into this cell line enhanced VTX sensitivity. Interestingly, BCL2 was upregulated in some resistant cell lines generated after a long-term treatment with VTX, suggesting BCL2 expression level may not be suitable as a marker of VTX sensitivity for acquired resistance. Unlike in CLL, BCL2 mutations were not identified through WES in any resistant cell lines or primary patient sample harvested after relapse. While 8 genes were mutated in two resistant samples , no clear mutational pattern emerged . Based on the above, we further tested some specific inhibitors in in vitro or ex vivo cell models to help understanding resistant mechanism and identify strategies to overcome VTX resistance. We found that inhibition of MCL-1, with the compound S68345, substantially enhanced VTX sensitivity in three resistant HMCLs and in primary cells from one relapsed MM patient. A BCLXL inhibitor (A155463) only significantly enhanced VTX sensitivity in one resistant cell line after co-treatment with VTX. Co-treatment of the other three resistant cell lines with VTX, S68345 and A155463 resulted in the most synergistic anti-myeloma activity, suggesting those cell lines are co-dependent on MCL-1, BCLXL, and BCL2 for survival, although they are more dependent on MCL-1. We also found that inhibition of PI3K signaling, IGF1, RTK (EGF and FGF) and AURKA significantly increased VTX sensitivity, partially through downregulation of MCL-1, and BCLXL, and upregulation of BIM. Conventional anti-MM drugs such as dexamethasone, bortezomib and lenalidomide, were shown to have little activity on augmenting VTX sensitivity in most resistant cell lines. In summary, we find that acquired resistance to VTX in MM is largely associated with BCL2 family regulation, including upregulation of survival members such as MCL-1, BCLXL, BCL2, and downregulation of pro-apoptotic members, especially BIM. Our study also indicates that upstream signaling involved in BCL2 family regulation during acquired resistance is likely related to cytokine, growth factor, and/or RTK-induced cell signaling such as PI3K. Co-inhibition of MCL-1, or BCLXL, as well as the upstream PI3K, RTK (FGF and EGF), IGF-1 mediated signaling were effective in overcoming VTX resistance. Disclosures Fonseca: Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Juno: Consultancy; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; AbbVie: Consultancy; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4356-4356
Author(s):  
John S Manavalan ◽  
Ipsita Pal ◽  
Aidan Pursley ◽  
George A. Ward ◽  
Tomoko Smyth ◽  
...  

Abstract Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). Figure 1 Figure 1. Disclosures Smyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3446-3446
Author(s):  
Jennifer Lauren Kamens ◽  
Anitria Cotton ◽  
Jeannie W Lam ◽  
Jinjun Dang ◽  
Aman Seth ◽  
...  

Abstract Pediatric Acute Myeloid Leukemia (AML) is a rare, but deadly cancer. Outcomes over the last 20 years have remained stagnant with an overall 5-year survival rate < 70% and relapse rates around 50%. Further, few new therapies have been successfully introduced to improve these outcomes. Here we report that exploiting deficiencies in DNA damage repair (DDR) is a potential therapeutic strategy for AML. Poly-ADP Ribose Polymerase (PARP) inhibitors were initially developed to target deficient homologous recombination (HR) in BRCA1/2 mutated cancers by blocking single stranded base repair following DNA damage, leading to an accumulation of double stranded DNA breaks, thereby inducing apoptosis. To evaluate the activity of PARP inhibition in pediatric AML, talazoparib was tested as a single agent and in combination with standard chemotherapeutic agents in human AML cell lines representing low (Kasumi-1 and ME-1), intermediate (AML193), and high-risk (CTS, CMS, MOLM-13, and CHRF288-11) disease based on their genomic mutations. Talazoparib showed the highest efficacy as a single agent in all four cell lines with genomic lesions found in high-risk AML subtypes. After combination drug screens, topotecan (synergistic) and gemcitabine (additive) were chosen to move forward to in vivo testing. Our investigational combination was tested in vivo in four murine models representing pediatric AML subtypes harboring AML1-ETO9a (low risk), MLL-AF6 (high risk), CBAF2T3-GLIS2/JAK2 V617F (high risk) and NUP98-KDM5A (high risk) oncogenes. Mice received a backbone of either current standard of care chemotherapy (SOC; anthracycline plus cytarabine) or topotecan plus gemcitabine. NUP98-KDM5A and MLL-AF6 positive mice receiving single agent talazoparib were found to have prolonged survival compared to vehicle alone (p=0.019 and p<0.0001, respectively) which was further enhanced by the addition of chemotherapy irrespective of backbone (p <0.0001). Conversely, mice with AML1-ETOa positive leukemia had no response to single agent PARP inhibitor. While a few mice benefitted from the addition of talazoparib to SOC, this result was not statistically significant (p= 0.42). Early response by bioluminescent imaging confirmed that mice with MLL-AF6 and NUP98-KDM5A driven leukemias who received talazoparib in combination with chemotherapy had the lowest leukemia burdens while the AML1-ETOa cohort did not benefit from the addition of this targeted agent. Interestingly, mice harboring CBAF2T3-GLIS2/JAK2 V617F were not responsive to PARP inhibitors, which was inconsistent with the CMS cell line that has same oncogenic fusion gene but lacks the JAK2 V617F mutation. Synergy experiments with ATM inhibitor AZD0156 demonstrated tremendous synergy with talazoparib in sensitive cell lines with almost no synergy in those that were resistant, suggesting that sensitive cell lines are unable to efficiently activate the HR pathway to repair double stranded breaks induced by PARP inhibition whereas resistant cells can overcome inhibition. To determine the HR response to DNA damage in our cell lines, we exposed them to 1uM topotecan for 2 hours and then measured γH2AX response at 0, 4 and 24 hours. γH2AX is a sensor of DNA damage and therefore increases with DNA damage and decreases with repair. PARP inhibitor sensitive cell lines had persistence of gamma H2AX at 24hrs while resistant cell lines had at least partial resolution of damage, confirming that PARP inhibitor sensitive cell lines have aberrant DNA damage response through HR. RNA sequencing of our cell lines revealed a correlation between Phosphatase and tensin homolog (PTEN) transcript levels and PARP sensitivity. Western blotting confirmed that PTEN was downregulated or absent in both cell lines and murine leukemias that were sensitive to PARP inhibitors. In contrast to the CMS cell line that carries the CBFA2T3-GLIS2 fusion, murine leukemias with CBAF2T3-GLIS2/JAK2 V617F had high levels of PTEN, supporting the hypothesis that sensitivity to PARP inhibitors is due to loss of PTEN. In conclusion, we report that a subset of pediatric AML with high- risk features are sensitive to PARP inhibition due to deficient DDR through HR. Downregulation of PTEN is a candidate biomarker of response to PARP inhibitors in these patients. This data illuminates a promising therapeutic vulnerability in a patient population where new targeted treatments are vital to improve outcomes. Disclosures No relevant conflicts of interest to declare.


FEBS Journal ◽  
2021 ◽  
Author(s):  
Kaiming Luo ◽  
Roland Stocker ◽  
Warwick J Britton ◽  
Kazu Kikuchi ◽  
Stefan H Oehlers

Author(s):  
Dawid Głów ◽  
Simon Meyer ◽  
Irene García Roldán ◽  
Lara Marie Akingunsade ◽  
Kristoffer Riecken ◽  
...  

2021 ◽  
Vol 52 (1) ◽  
pp. 63-69
Author(s):  
Z. Mahmoudi Rad ◽  
H. Nourafcan ◽  
N. Mohebalipour ◽  
A. Assadi ◽  
S. Jamshidi

Silybum marianum (L.), is an important herbal medicine. Silymarin, the active component obtained from its edible seeds, is known for its antioxidant and antimicrobial activity. This  research was aimed to study the effect of foliar application of salicylic acid (SA) at four concentrations of 1250, 2500, 5000 and 10000 μmol l-1 on the improvement of phytochemical composition, antioxidant and antimicrobial activity. Significant increase was found in the content of silibinin, silybin A and silybin B in plants treated with increasing concentrations of salicylic acid. The antioxidant activity was improved with increasing the SA concentration reaching the highest amount under 5000 μmol l-1 SA treatment (p < 0.05). Irrespective to the bacterial strain, an increasing pattern in the amount of antibacterial activity was found by increasing SA concentration. The study suggest that treatments with medium doses of SA could be a promising way to improve the health beneficial flavonolignans compounds of Silybum marianum resulting in a higher antioxidant and antimicrobial activity. Nevertheless, it should be considered that the responses to SA are highly concentration dependent and application of higher concentrations can have an adverse effect by triggering a hyper sensitive cell death pathway.


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