Synthesis and Inhibitory Activity of Machaeridiol-Based Novel Anti-MRSA and Anti-VRE Compounds and Their Profiling for Cancer-Related Signaling Pathways

Three unique 5,6-seco-hexahydrodibenzopyrans (seco-HHDBP) machaeridiols A–C, reported previously from Machaerium Pers., have displayed potent activities against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium, and E. faecalis (VRE). In order to enrich the pipeline of natural product-derived antimicrobial compounds, a series of novel machaeridiol-based analogs (1–17) were prepared by coupling stemofuran, pinosylvin, and resveratrol legends with monoterpene units R-(−)-α-phellandrene, (−)-p-mentha-2,8-diene-1-ol, and geraniol, and their inhibitory activities were profiled against MRSA ATCC 1708, VRE ATCC 700221, and cancer signaling pathways. Compounds 5 and 11 showed strong in vitro activities with MIC values of 2.5 μg/mL and 1.25 μg/mL against MRSA, respectively, and 2.50 μg/mL against VRE, while geranyl analog 14 was found to be moderately active (MIC 5 μg/mL). The reduction of the double bonds of the monoterpene unit of compound 5 resulted in 17, which had the same antibacterial potency (MIC 1.25 μg/mL and 2.50 μg/mL) as its parent, 5. Furthermore, a combination study between seco-HHDBP 17 and HHDBP machaeriol C displayed a synergistic effect with a fractional inhibitory concentrations (FIC) value of 0.5 against MRSA, showing a four-fold decrease in the MIC values of both 17 and machaeriol C, while no such effect was observed between vancomycin and 17. Compounds 11 and 17 were further tested in vivo against nosocomial MRSA at a single intranasal dose of 30 mg/kg in a murine model, and both compounds were not efficacious under these conditions. Finally, compounds 1–17 were profiled against a panel of luciferase genes that assessed the activity of complex cancer-related signaling pathways (i.e., transcription factors) using T98G glioblastoma multiforme cells. Among the compounds tested, the geranyl-substituted analog 14 exhibited strong inhibition against several signaling pathways, notably Smad, Myc, and Notch, with IC50 values of 2.17 μM, 1.86 μM, and 2.15 μM, respectively. In contrast, the anti-MRSA actives 5 and 17 were found to be inactive (IC50 \u3e 20 μM) across the panel of these cancer-signaling pathways

Analyzing the Mechanisms of Interferon-Induced Apoptosis Using CrmA and Hepatitis C Virus NS5A

AbstractThe dsRNA-dependent protein kinase, PKR, is a key component of interferon (IFN)-mediated anti-viral action and is frequently inhibited by many viruses following infection of the cell. Recently, we have demonstrated that IFN and PKR can sensitize cells to apoptosis predominantly through the FADD/caspase-8 pathway (S. Balachandran, P. C. Roberts, T. Kipperman, K. N. Bhalla, R. W. Compans, D. R. Archer, and G. N. Barber. (2000b) J. Virol. 74, 1513–1523). Given these findings, it is thus plausible that rather than specifically target IFN-inducible genes such as PKR, viruses could also subvert the mechanisms of IFN action, in part, at locations that could block the apoptotic cascade. To explore this possibility, we analyzed whether the poxvirus caspase-8 inhibitor, CrmA, was able to inhibit IFN or PKR/dsRNA-mediated apoptosis. Our findings indicated that CrmA could indeed inhibit apoptosis induced by both viral infection and dsRNA without blocking PKR activity or inhibiting IFN signaling. In contrast HCV-encoded NS5A, a putative inhibitor of PKR, did not appear to inhibit cell death mediated by a number of apoptotic stimuli, including IFN, TRAIL, and etoposide. Our data imply that viral-encoded inhibitors of apoptosis, such as CrmA, can block the innate arms of the immune response, including IFN-mediated apoptosis, and therefore potentially constitute an alternative family of inhibitors of IFN action in the cell

The interferon-inducible antiviral protein Daxx is not essential for interferon-mediated protection against avian sarcoma virus

BACKGROUND: The antiviral protein Daxx acts as a restriction factor of avian sarcoma virus (ASV; Retroviridae) in mammalian cells by promoting epigenetic silencing of integrated proviral DNA. Although Daxx is encoded by a type I (α/β) interferon-stimulated gene, the requirement for Daxx in the interferon anti-retroviral response has not been elucidated. In this report, we describe the results of experiments designed to investigate the role of Daxx in the type I interferon-induced anti-ASV response. FINDINGS: Using an ASV reporter system, we show that type I interferons are potent inhibitors of ASV replication. We demonstrate that, while Daxx is necessary to silence ASV gene expression in the absence of interferons, type I interferons are fully-capable of inducing an antiviral state in the absence of Daxx. CONCLUSIONS: These results provide evidence that Daxx is not essential for the anti-ASV interferon response in mammalian cells, and that interferons deploy multiple, redundant antiviral mechanisms to protect cells from ASV

Inhibitory Activity of Machaeridiol-Based Novel Anti-MRSA and Anti-VRE Compounds and Their Profiling for Cancer-Related Signaling Pathways

Corresponding author (NCNPR): Premalatha Balachandran, [email protected]://egrove.olemiss.edu/pharm_annual_posters_2022/1002/thumbnail.jp

Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

SummaryRIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective “hybrid” RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies

Interferon Impedes an Early Step of Hepatitis Delta Virus Infection

Hepatitis delta virus (HDV) infects hepatocytes, the major cell type of the liver. Infection of the liver may be either transient or chronic. The prognosis for patients with chronic HDV infection is poor, with a high risk of cirrhosis and hepatocellular carcinoma. The best antiviral therapy is weekly administration for at least one year of high doses of interferon alpha. This efficacy of interferon therapy has been puzzling in that HDV replication in transfected cell lines is reported as insensitive to administration of interferon alpha or gamma. Similarly, this study shows that even when an interferon response was induced by transfection of poly(IC) into a cell line, HDV RNA accumulation was only modestly inhibited. However, when the HDV replication was initiated by infection of primary human hepatocytes, simultaneous addition of interferons alpha or gamma at 600 units/ml, a concentration comparable to that achieved in treated patients, the subsequent HDV RNA accumulation was inhibited by at least 80%. These interferon treatments were shown to produce significant time-dependent increases of host response proteins such as for Stat-1, phosphoStat-1, Mx1/2/3 and PKR, and yet interferon pretreatment of hepatocytes did not confer an increased inhibition of HDV replication over interferon treatment at the time of (or after) infection. These and other data support the interpretation that interferon action against HDV replication can occur and is largely mediated at the level of entry into primary human hepatocytes. Thus in vivo, the success of long-term interferon therapy for chronic HDV, may likewise involve blocking HDV spread by interfering with the initiation of productive infection of naïve hepatocytes

Z-DNA enhances immunotherapy by triggering death of inflammatory cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) are a heterogeneous population of cells. At one end of the spectrum are alpha-smooth muscle actin expressing myoCAFs (myofibroblast CAFs) and at the other end are the interferon (IFN) and Janus Kinase/Signal Transducer and Activator of Transcription responsive iCAFs (inflammatory CAFs). Both types of CAFs promote tumor growth. While myoCAFs foster immune exclusion and limit tumor spread, iCAFs create a highly immunosuppressive environment and foster the seeding of distant metastases. However, iCAFs also represent a tumor vulnerability. They are competent to undergo necroptosis, a highly immunogenic form of cell death that is triggered when Z-DNA or Z-RNA (collectively called ZNA) is sensed by the IFN-induced ZNA binding protein 1 (ZBP1). The sequestering of ZNA ligands by the p150 isoform of the double-stranded RNA-specific deaminase ADAR1 protects iCAFs from cell death. ZBP1-dependent necroptosis in iCAFs can be triggered by administering an orally available small molecule that generates sufficient amounts of ZNA to bypass ADAR1 inhibition. The therapeutic approach of targeting Z-prone sequences (called flipons) is agnostic to the mutations driving cancer progression. By exploiting the tumor vulnerability posed by expression of ZBP1-dependent immunogenic cell death pathways in iCAFs, flipon therapeutics offer new hope for improved clinical outcomes