RNA-Dependent RNA Polymerase as a Target for COVID-19 Drug Discovery.

COVID-19 respiratory disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global health issue since it emerged in December 2019. While great global efforts are underway to develop vaccines and to discover or repurpose therapeutic agents for this disease, as of this writing only the nucleoside drug remdesivir has been approved under Emergency Use Authorization to treat COVID-19. The RNA-dependent RNA polymerase (RdRP), a viral enzyme for viral RNA replication in host cells, is one of the most intriguing and promising drug targets for SARS-CoV-2 drug development. Because RdRP is a viral enzyme with no host cell homologs, selective SARS-CoV-2 RdRP inhibitors can be developed that have improved potency and fewer off-target effects against human host proteins and thus are safer and more effective therapeutics for treating COVID-19. This review focuses on biochemical enzyme and cell-based assays for RdRPs that could be used in high-throughput screening to discover new and repurposed drugs against SARS-CoV-2.

Patient-derived glioblastoma cultures as a tool for small-molecule drug discovery.

There is a compelling need for new therapeutic strategies for glioblastoma multiforme (GBM). Preclinical target and therapeutic discovery for GBMs is primarily conducted using cell lines grown in serum-containing media, such as U-87 MG, which do not reflect the gene expression profiles of tumors found in GBM patients. To address this lack of representative models, we sought to develop a panel of patient-derived GBM models and characterize their genomic features, using RNA sequencing (RNA-seq) and growth characteristics, both when grown as neurospheres in culture, and grown orthotopically as xenografts in mice. When we compared these with commonly used GBM cell lines in the Cancer Cell Line Encyclopedia (CCLE), we found these patient-derived models to have greater diversity in gene expression and to better correspond to GBMs directly sequenced from patient tumor samples. We also evaluated the potential of these models for targeted therapy, by using the genomic characterization to identify small molecules that inhibit the growth of distinct subsets of GBMs, paving the way for precision medicines for GBM.

MDM2 and MDMX promote ferroptosis by PPARα-mediated lipid remodeling.

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.

Tumoricidal stem cell therapy enables killing in novel hybrid models of heterogeneous glioblastoma.

BACKGROUND: Tumor-homing tumoricidal neural stem cell (tNSC) therapy is a promising new strategy that recently entered human patient testing for glioblastoma (GBM). Developing strategies for tNSC therapy to overcome intratumoral heterogeneity, variable cancer cell invasiveness, and differential drug response of GBM will be essential for efficacious treatment response in the clinical setting. The aim of this study was to create novel hybrid tumor models and investigate the impact of GBM heterogeneity on tNSC therapies. METHODS: We used organotypic brain slice explants and distinct human GBM cell types to generate heterogeneous models ex vivo and in vivo. We then tested the efficacy of mono- and combination therapy with primary NSCs and fibroblast-derived human induced neural stem cells (iNSCs) engineered with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or enzyme-prodrug therapy. RESULTS: Optical imaging, molecular assays, and immunohistochemistry revealed that the hybrid models recapitulated key aspects of patient GBM, including heterogeneity in TRAIL sensitivity, proliferation, migration patterns, hypoxia, blood vessel structure, cancer stem cell populations, and immune infiltration. To explore the impact of heterogeneity on tNSC therapy, testing in multiple in vivo models showed that tNSC-TRAIL therapy potently inhibited tumor growth and significantly increased survival across all paradigms. Patterns of tumor recurrence varied with therapeutic (tNSC-TRAIL and/or tNSC-thymidine kinase), dose, and route of administration. CONCLUSIONS: These studies report new hybrid models that accurately capture key aspects of GBM heterogeneity which markedly impact treatment response while demonstrating the ability of tNSC mono- and combination therapy to overcome certain aspects of heterogeneity for robust tumor kill.

Copper-Binding Small Molecule Induces Oxidative Stress and Cell-Cycle Arrest in Glioblastoma-Patient-Derived Cells.

Transition metals are essential, but deregulation of their metabolism causes toxicity. Here, we report that the compound NSC319726 binds copper to induce oxidative stress and arrest glioblastoma-patient-derived cells at picomolar concentrations. Pharmacogenomic analysis suggested that NSC319726 and 65 other structural analogs exhibit lethality through metal binding. Although NSC319726 has been reported to function as a zinc ionophore, we report here that this compound binds to copper to arrest cell growth. We generated and validated pharmacogenomic predictions: copper toxicity was substantially inhibited by hypoxia, through an hypoxia-inducible-factor-1α-dependent pathway; copper-bound NSC319726 induced the generation of reactive oxygen species and depletion of deoxyribosyl purines, resulting in cell-cycle arrest. These results suggest that metal-induced DNA damage may be a consequence of exposure to some xenobiotics, therapeutic agents, as well as other causes of copper dysregulation, and reveal a potent mechanism for targeting glioblastomas.

Neuroprotective Effects of Annexin A1 Tripeptide after Deep Hypothermic Circulatory Arrest in Rats.

Resolution agonists, including lipid mediators and peptides such as annexin A1 (ANXA1), are providing novel approaches to treat inflammatory conditions. Surgical trauma exerts a significant burden on the immune system that can affect and impair multiple organs. Perioperative cerebral injury after cardiac surgery is associated with significant adverse neurological outcomes such as delirium and postoperative cognitive dysfunction. Using a clinically relevant rat model of cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA), we tested the pro-resolving effects of a novel bioactive ANXA1 tripeptide (ANXA1sp) on neuroinflammation and cognition. Male rats underwent 2 h CPB with 1 h DHCA at 18°C, and received vehicle or ANXA1sp followed by timed reperfusion up to postoperative day 7. Immortalized murine microglial cell line BV2 were treated with vehicle or ANXA1sp and subjected to 2 h oxygen-glucose deprivation followed by timed reoxygenation. Microglial activation, cell death, neuroinflammation, and NF-κB activation were assessed in tissue samples and cell cultures. Rats exposed to CPB and DHCA had evident neuroinflammation in various brain areas. However, in ANXA1sp-treated rats, microglial activation and cell death (apoptosis and necrosis) were reduced at 24 h and 7 days after surgery. This was associated with a reduction in key pro-inflammatory cytokines due to inhibition of NF-κB activation in the brain and systemically. Treated rats also had improved neurologic scores and shorter latency in the Morris water maze. In BV2 cells treated with ANXA1sp, similar protective effects were observed including decreased pro-inflammatory cytokines and cell death. Notably, we also found increased expression of ANXA1, which binds to NF-κB p65 and thereby inhibits its transcriptional activity. Our findings provide evidence that treatment with a novel pro-resolving ANXA1 tripeptide is neuroprotective after cardiac surgery in rats by attenuating neuroinflammation and may prevent postoperative neurologic complications.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease.

Huntington's Disease (HD) is a neurodegenerative disease caused by poly-glutamine expansion in the Htt protein, resulting in Htt misfolding and cell death. Expression of the cellular protein folding and pro-survival machinery by heat shock transcription factor 1 (HSF1) ameliorates biochemical and neurobiological defects caused by protein misfolding. We report that HSF1 is degraded in cells and mice expressing mutant Htt, in medium spiny neurons derived from human HD iPSCs and in brain samples from patients with HD. Mutant Htt increases CK2α' kinase and Fbxw7 E3 ligase levels, phosphorylating HSF1 and promoting its proteasomal degradation. An HD mouse model heterozygous for CK2α' shows increased HSF1 and chaperone levels, maintenance of striatal excitatory synapses, clearance of Htt aggregates and preserves body mass compared with HD mice homozygous for CK2α'. These results reveal a pathway that could be modulated to prevent neuronal dysfunction and muscle wasting caused by protein misfolding in HD.

Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract.

Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.

IKK/NF-κB signaling contributes to glioblastoma stem cell maintenance.

Glioblastoma multiforme (GBM) carries a poor prognosis and continues to lack effective treatments. Glioblastoma stem cells (GSCs) drive tumor formation, invasion, and drug resistance and, as such, are the focus of studies to identify new therapies for disease control. Here, we identify the involvement of IKK and NF-κB signaling in the maintenance of GSCs. Inhibition of this pathway impairs self-renewal as analyzed in tumorsphere formation and GBM expansion as analyzed in brain slice culture. Interestingly, both the canonical and non-canonical branches of the NF-κB pathway are shown to contribute to this phenotype. One source of NF-κB activation in GBM involves the TGF-ß/TAK1 signaling axis. Together, our results demonstrate a role for the NF-κB pathway in GSCs and provide a mechanistic basis for its potential as a therapeutic target in glioblastoma.

Dual activities of the anti-cancer drug candidate PBI-05204 provide neuroprotection in brain slice models for neurodegenerative diseases and stroke.

We previously reported neuroprotective activity of the botanical anti-cancer drug candidate PBI-05204, a supercritical CO2 extract of Nerium oleander, in brain slice and in vivo models of ischemic stroke. We showed that one component of this neuroprotective activity is mediated through its principal cardiac glycoside constituent, oleandrin, via induction of the potent neurotrophic factor brain-derived neurotrophic factor (BDNF). However, we also noted that the concentration-relation for PBI-05204 in the brain slice oxygen-glucose deprivation (OGD) model is considerably broader than that for oleandrin as a single agent. We thus surmised that PBI-05204 contains an additional neuroprotective component(s), distinct from oleandrin. We report here that neuroprotective activity is also provided by the triterpenoid constituents of PBI-05204, notably oleanolic acid. We demonstrate that a sub-fraction of PBI-05204 (Fraction 0-4) containing oleanolic and other triterpenoids, but without cardiac glycosides, induces the expression of cellular antioxidant gene transcription programs regulated through antioxidant transcriptional response elements (AREs). Finally, we show that Fraction 0-4 provides broad neuroprotection in organotypic brain slice models for neurodegeneration driven by amyloid precursor protein (APP) and tau implicated in Alzheimer's disease and frontotemporal dementias, respectively, in addition to ischemic injury modeled by OGD.

Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective.

Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI's role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease.

Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models.

Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of Huntington's disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species formation or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1, which guided the development of ferrostatins with improved properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid peroxidation mediates diverse disease phenotypes.

Experimental models for identifying modifiers of polyglutamine-induced aggregation and neurodegeneration.

Huntington's disease (HD) typifies a class of inherited neurodegenerative disorders in which a CAG expansion in a single gene leads to an extended polyglutamine tract and misfolding of the expressed protein, driving cumulative neural dysfunction and degeneration. HD is invariably fatal with symptoms that include progressive neuropsychiatric and cognitive impairments, and eventual motor disability. No curative therapies yet exist for HD and related polyglutamine diseases; therefore, substantial efforts have been made in the drug discovery field to identify potential drug and drug target candidates for disease-modifying treatment. In this context, we review here a range of early-stage screening approaches based in in vitro, cellular, and invertebrate models to identify pharmacological and genetic modifiers of polyglutamine aggregation and induced neurodegeneration. In addition, emerging technologies, including high-content analysis, three-dimensional culture models, and induced pluripotent stem cells are increasingly being incorporated into drug discovery screening pipelines for protein misfolding disorders. Together, these diverse screening strategies are generating novel and exciting new probes for understanding the disease process and for furthering development of therapeutic candidates for eventual testing in the clinical setting.

In vitro and in vivo neuroprotective activity of the cardiac glycoside oleandrin from Nerium oleander in brain slice-based stroke models.

The principal active constituent of the botanical drug candidate PBI-05204, a supercritical CO(2) extract of Nerium oleander, is the cardiac glycoside oleandrin. PBI-05204 shows potent anticancer activity and is currently in phase I clinical trial as a treatment for patients with solid tumors. We have previously shown that neriifolin, which is structurally related to oleandrin, provides robust neuroprotection in brain slice and whole animal models of ischemic injury. However, neriifolin itself is not a suitable drug development candidate and the FDA-approved cardiac glycoside digoxin does not cross the blood-brain barrier. We report here that both oleandrin as well as the full PBI-05204 extract can also provide significant neuroprotection to neural tissues damaged by oxygen and glucose deprivation as occurs in ischemic stroke. Critically, we show that the neuroprotective activity of PBI-05204 is maintained for several hours of delay of administration after oxygen and glucose deprivation treatment. We provide evidence that the neuroprotective activity of PBI-05204 is mediated through oleandrin and/or other cardiac glycoside constituents, but that additional, non-cardiac glycoside components of PBI-05204 may also contribute to the observed neuroprotective activity. Finally, we show directly that both oleandrin and the protective activity of PBI-05204 are blood brain barrier penetrant in a novel model for in vivo neuroprotection. Together, these findings suggest clinical potential for PBI-05204 in the treatment of ischemic stroke and prevention of associated neuronal death.

Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins.

A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction and oxidation of disulfides. Through a small molecule screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated ER membranes and trigger apoptotic cell death via mitochondrial outer-membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon 1 and Aß peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI represents a new mechanism linking protein misfolding and apoptotic cell death.

Identification and evaluation of small molecule pan-caspase inhibitors in Huntington's disease models.

Huntington's Disease (HD) is characterized by a mutation in the huntingtin (Htt) gene encoding an expansion of glutamine repeats on the N terminus of the Htt protein. Numerous studies have identified Htt proteolysis as a critical pathological event in HD postmortem human tissue and mouse HD models, and proteases known as caspases have emerged as attractive HD therapeutic targets. We report the use of the substrate activity screening method against caspase-3 and -6 to identify three novel, pan-caspase inhibitors that block proteolysis of Htt at caspase-3 and -6 cleavage sites. In HD models these irreversible inhibitors suppressed Hdh(111Q/111Q)-mediated toxicity and rescued rat striatal and cortical neurons from cell death. In this study, the identified nonpeptidic caspase inhibitors were used to confirm the role of caspase-mediated Htt proteolysis in HD. These results further implicate caspases as promising targets for HD therapeutic development.

Composite primary neuronal high-content screening assay for Huntington's disease incorporating non-cell-autonomous interactions.

Huntington's disease (HD) is a fatal neurodegenerative disease characterized by progressive cognitive, behavioral, and motor deficits and caused by expansion of a polyglutamine repeat in the Huntingtin protein (Htt). Despite its monogenic nature, HD pathogenesis includes obligatory non-cell-autonomous pathways involving both the cortex and the striatum, and therefore effective recapitulation of relevant HD disease pathways in cell lines and primary neuronal monocultures is intrinsically limited. To address this, the authors developed an automated high-content imaging screen in high-density primary cultures of cortical and striatal neurons together with supporting glial cells. Cortical and striatal neurons are transfected separately with different fluorescent protein markers such that image-based high-content analysis can be used to assay these neuronal populations separately but still supporting their intercellular interactions, including abundant synaptic interconnectivity. This assay was reduced to practice using transfection of a mutant N-terminal Htt domain and validated via a screen of ~400 selected small molecules. Both expected as well as novel candidate targets for HD emerged from this screen; of particular interest were target classes with close relative proximity to clinical testing. These findings suggest that composite primary cultures incorporating increased levels of biological complexity can be used for high-content imaging and "high-context" screening to represent molecular targets that otherwise may be operant only in the complex tissue environment found in vivo during disease pathogenesis.

Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity.

Although expanded polyglutamine (polyQ) repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected. The polyQ expansion that causes Huntington's disease (HD) is in the first exon (HDx-1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids and two proline (polyP) repeat domains, regulate the toxicity of mutant Htt. The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in HDx-1 toxicity, aggregation, localization, and turnover. Three domains in the P-rich region (PRR) of HDx-1 are defined by intrabodies: MW7 binds the two polyP domains, and Happ1 and Happ3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as V(L)12.3, which binds the N-terminal 17 aa, decrease the toxicity and aggregation of HDx-1, but they do so by different mechanisms. The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which V(L)12.3 does not change. In contrast, expression of V(L)12.3 increases nuclear Htt. We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD. We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells.