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.

High-Throughput Platform for Novel Reaction Discovery.

Detecting the formation of new chemical bonds in high-throughput synthesis is limited by the efficiency and scalability of reaction product detection, as conventional methods for isolating product from reaction mixtures are time consuming and labor intensive. Here, we report a miniaturizable purification method that enables the rapid, high-throughput isolation of quaternary ammonium-tagged products from reaction mixtures with excellent purity using inexpensive equipment that easily can be set up in a typical organic chemistry laboratory. This novel purification technique enabled us to establish a high-throughput reaction discovery platform. We validated this platform in a screen of 1536 reactions, and one previously unreported transformation was identified.

Viral Proteases as Targets for Coronavirus Disease 2019 Drug Development.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to be a global threat since its emergence. Although several COVID-19 vaccines have become available, the prospective timeframe for achieving effective levels of vaccination across global populations remains uncertain. Moreover, the emergence of SARS-CoV-2 variants presents continuing potential challenges for future vaccination planning. Therefore, development of effective antiviral therapies continues to be an urgent unmet need for COVID-19. Successful antiviral regimens for the treatment of human immunodeficiency virus and hepatitis C virus infections have established viral proteases as validated targets for antiviral drug development. In this context, we review protease targets in drug development, currently available antiviral protease inhibitors, and therapeutic development efforts on SARS-CoV-2 main protease and papain-like protease. SIGNIFICANCE STATEMENT: Coronavirus disease 2019 (COVID-19) continues to be a global threat since its emergence. The development of effective antiviral therapeutics for COVID-19 remains an urgent and long-term need. Because viral proteases are validated drug targets, specific severe acute respiratory syndrome coronavirus 2 protease inhibitors are critical therapeutics to be developed for treatment of COVID-19.

Application of niclosamide and analogs as small molecule inhibitors of Zika virus and SARS-CoV-2 infection.

Zika virus has emerged as a potential threat to human health globally. A previous drug repurposing screen identified the approved anthelminthic drug niclosamide as a small molecule inhibitor of Zika virus infection. However, as antihelminthic drugs are generally designed to have low absorption when dosed orally, the very limited bioavailability of niclosamide will likely hinder its potential direct repurposing as an antiviral medication. Here, we conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide to improve physicochemical properties such as microsomal metabolic stability, permeability and solubility. We found that the 5-bromo substitution in the salicylic acid region retains potency while providing better drug-like properties. Other modifications in the anilide region with 2'-OMe and 2'-H substitutions were also advantageous. We found that the 4'-NO2 substituent can be replaced with a 4'-CN or 4'-CF3 substituents. Together, these modifications provide a basis for optimizing the structure of niclosamide to improve systemic exposure for application of niclosamide analogs as drug lead candidates for treating Zika and other viral infections. Indeed, key analogs were also able to rescue cells from the cytopathic effect of SARS-CoV-2 infection, indicating relevance for therapeutic strategies targeting the COVID-19 pandemic.

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.

IKKß and mutant huntingtin interactions regulate the expression of IL-34: implications for microglial-mediated neurodegeneration in HD.

Neuronal interleukin-34 (IL-34) promotes the expansion of microglia in the central nervous system-microglial activation and expansion are in turn implicated in the pathogenesis of Huntington's disease (HD). We thus examined whether the accumulation of an amyloidogenic exon-1 fragment of mutant huntingtin (mHTTx1) modulates the expression of IL-34 in dopaminergic neurons derived from a human embryonic stem cell line. We found that mHTTx1 aggregates induce IL-34 production selectively in post-mitotic neurons. Exposure of neurons to DNA damaging agents or the excitotoxin NMDA elicited similar results suggesting that IL-34 induction may be a general response to neuronal stress including the accumulation of misfolded mHTTx1. We further determined that knockdown or blocking the activity of IκB kinase beta (IKKß) prevented the aggregation of mHTTx1 and subsequent IL-34 production. While elevated IL-34 itself had no effect on the aggregation or the toxicity of mHTTx1 in neuronal culture, IL-34 expression in a rodent brain slice model with intact neuron-microglial networks exacerbated mHTTx1-induced degeneration of striatal medium-sized spiny neurons. Conversely, an inhibitor of the IL-34 receptor reduced microglial numbers and ameliorated mHTTx1-mediated neurodegeneration. Together, these findings uncover a novel function for IKKß/mHTTx1 interactions in regulating IL-34 production, and implicate a role for IL-34 in non-cell-autonomous, microglial-dependent neurodegeneration in HD.

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.

Protein aggregation can inhibit clathrin-mediated endocytosis by chaperone competition.

Protein conformational diseases exhibit complex pathologies linked to numerous molecular defects. Aggregation of a disease-associated protein causes the misfolding and aggregation of other proteins, but how this interferes with diverse cellular pathways is unclear. Here, we show that aggregation of neurodegenerative disease-related proteins (polyglutamine, huntingtin, ataxin-1, and superoxide dismutase-1) inhibits clathrin-mediated endocytosis (CME) in mammalian cells by aggregate-driven sequestration of the major molecular chaperone heat shock cognate protein 70 (HSC70), which is required to drive multiple steps of CME. CME suppression was also phenocopied by HSC70 RNAi depletion and could be restored by conditionally increasing HSC70 abundance. Aggregation caused dysregulated AMPA receptor internalization and also inhibited CME in primary neurons expressing mutant huntingtin, showing direct relevance of our findings to the pathology in neurodegenerative diseases. We propose that aggregate-associated chaperone competition leads to both gain-of-function and loss-of-function phenotypes as chaperones become functionally depleted from multiple clients, leading to the decline of multiple cellular processes. The inherent properties of chaperones place them at risk, contributing to the complex pathologies of protein conformational diseases.

Natural genetic variation of integrin alpha L (Itgal) modulates ischemic brain injury in stroke.

During ischemic stroke, occlusion of the cerebrovasculature causes neuronal cell death (infarction), but naturally occurring genetic factors modulating infarction have been difficult to identify in human populations. In a surgically induced mouse model of ischemic stroke, we have previously mapped Civq1 to distal chromosome 7 as a quantitative trait locus determining infarct volume. In this study, genome-wide association mapping using 32 inbred mouse strains and an additional linkage scan for infarct volume confirmed that the size of the infarct is determined by ancestral alleles of the causative gene(s). The genetically isolated Civq1 locus in reciprocal recombinant congenic mice refined the critical interval and demonstrated that infarct size is determined by both vascular (collateral vessel anatomy) and non-vascular (neuroprotection) effects. Through the use of interval-specific SNP haplotype analysis, we further refined the Civq1 locus and identified integrin alpha L (Itgal) as one of the causative genes for Civq1. Itgal is the only gene that exhibits both strain-specific amino acid substitutions and expression differences. Coding SNPs, a 5-bp insertion in exon 30b, and increased mRNA and protein expression of a splice variant of the gene (Itgal-003, ENSMUST00000120857), all segregate with infarct volume. Mice lacking Itgal show increased neuronal cell death in both ex vivo brain slice and in vivo focal cerebral ischemia. Our data demonstrate that sequence variation in Itgal modulates ischemic brain injury, and that infarct volume is determined by both vascular and non-vascular mechanisms.

BDNF mediates neuroprotection against oxygen-glucose deprivation by the cardiac glycoside oleandrin.

We have previously shown that the botanical drug candidate PBI-05204, a supercritical CO2 extract of Nerium oleander, provides neuroprotection in both in vitro and in vivo brain slice-based models for focal ischemia (Dunn et al., 2011). Intriguingly, plasma levels of the neurotrophin BDNF were increased in patients treated with PBI-05204 in a phase I clinical trial (Bidyasar et al., 2009). We thus tested the hypothesis that neuroprotection provided by PBI-05204 to rat brain slices damaged by oxygen-glucose deprivation (OGD) is mediated by BDNF. We found, in fact, that exogenous BDNF protein itself is sufficient to protect brain slices against OGD, whereas downstream activation of TrkB receptors for BDNF is necessary for neuroprotection provided by PBI-05204, using three independent methods. Finally, we provide evidence that oleandrin, the principal cardiac glycoside component of PBI-05204, can quantitatively account for regulation of BDNF at both the protein and transcriptional levels. Together, these findings support further investigation of cardiac glycosides in providing neuroprotection in the context of ischemic stroke.

Differential roles of Aß processing in hypoxia-induced axonal damage.

Axonopathy is a common and early phase in neurodegenerative and traumatic CNS diseases. Recent work suggests that amyloid ß (Aß) produced from amyloid precursor protein (APP) may be a critical downstream mediator of CNS axonopathy in CNS diseases, particularly those associated with hypoxia. We critically tested this hypothesis in an adult retinal explant system that preserves the three-dimensional organization of the retina while permitting direct imaging of two cardinal features of early-stage axonopathy: axonal structural integrity and axonal transport capacity. Using this system, we found via pharmacological inhibition and genetic deletion of APP that production of Aß is a necessary step in structural compromise of retinal ganglion cell (RGC) axons induced by the disease-relevant stressor hypoxia. However, identical blockade of Aß production was not sufficient to protect axons from associated hypoxia-induced reduction in axonal transport. Thus, Aß mediates distinct facets of hypoxia-induced axonopathy and may represent a functionally selective pharmacological target for therapies directed against early-stage axonopathy in CNS diseases.

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.

An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics.

This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced in microgram quantities with consistent purity and potency in less than 24 h. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo. The proposed production process is efficient and can be readily scaled up and deployed wherever a viral pathogen might emerge. The current emergence of viral variants of SARS-CoV-2 has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.

A new kinetic framework for synaptic vesicle trafficking tested in synapsin knock-outs.

At least two rate-limiting mechanisms in vesicle trafficking operate at mouse Schaffer collateral synapses, but their molecular/physical identities are unknown. The first mechanism determines the baseline rate at which reserve vesicles are supplied to a readily releasable pool. The second causes the supply rate to depress threefold when synaptic transmission is driven hard for extended periods. Previous models invoked depletion of a reserve vesicle pool to explain the reductions in the supply rate, but the mass-action assumption at their core is not compatible with kinetic measurements of neurotransmission under maximal-use conditions. Here we develop a new theoretical model of rate-limiting steps in vesicle trafficking that is compatible with previous and new measurements. A physical interpretation is proposed where local reserve pools consisting of four vesicles are tethered to individual release sites and are replenished stochastically in an all-or-none fashion. We then show that the supply rate depresses more rapidly in synapsin knock-outs and that the phenotype can be fully explained by changing the value of the single parameter in the model that would specify the size of the local reserve pools. Vesicle-trafficking rates between pools were not affected. Finally, optical imaging experiments argue against alternative interpretations of the theoretical model where vesicle trafficking is inhibited without reserve pool depletion. This new conceptual framework will be useful for distinguishing which of the multiple molecular and cell biological mechanisms involved in vesicle trafficking are rate limiting at different levels of synaptic throughput and are thus candidates for physiological and pharmacological modulation.

CalDAG-GEFI down-regulation in the striatum as a neuroprotective change in Huntington's disease.

Huntingtin protein (Htt) is ubiquitously expressed, yet Huntington's disease (HD), a fatal neurologic disorder produced by expansion of an Htt polyglutamine tract, is characterized by neurodegeneration that occurs primarily in the striatum and cerebral cortex. Such discrepancies between sites of expression and pathology occur in multiple neurodegenerative disorders associated with expanded polyglutamine tracts. One possible reason is that disease-modifying factors are tissue-specific. Here, we show that the striatum-enriched protein, CalDAG-GEFI, is severely down-regulated in the striatum of mouse HD models and is down-regulated in HD individuals. In the R6/2 transgenic mouse model of HD, striatal neurons with the largest aggregates of mutant Htt have the lowest levels of CalDAG-GEFI. In a brain-slice explant model of HD, knock-down of CalDAG-GEFI expression rescues striatal neurons from pathology induced by transfection of polyglutamine-expanded Htt exon 1. These findings suggest that the striking down-regulation of CalDAG-GEFI in HD could be a protective mechanism that mitigates Htt-induced degeneration.

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.

Preclinical target validation for non-addictive therapeutics development for pain.

INTRODUCTION: The Helping to End Addiction Long-termSM Initiative supports a wide range of programs to develop new or improved prevention and opioid addiction treatment strategies. An essential component of this effort is to accelerate development of non-opioid pain therapeutics. In all fields of medicine, therapeutics development is an arduous process and late-stage translational efforts such as clinical trials to validate targets are particularly complex and costly. While there are plentiful novel targets for pain treatment, successful clinical validation is rare. It is therefore crucial to develop processes whereby therapeutic targets can be reasonably 'de-risked' prior to substantial late-stage validation efforts. Such rigorous validation of novel therapeutic targets in the preclinical space will give potential private sector partners the confidence to pursue clinical validation of promising therapeutic concepts and compounds. AREAS COVERED: In 2020, the National Institutes of Health (NIH) held the Target Validation for Non-Addictive Therapeutics Development for Pain workshop to gather insights from key opinion leaders in academia, industry, and venture-financing. EXPERT OPINION: The result was a roadmap for pain target validation focusing on three modalities: 1) human evidence; 2) assay development in vitro; 3) assay development in vivo.

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.

Identification of anti-inflammatory targets for Huntington's disease using a brain slice-based screening assay.

Huntington's disease (HD) is a late-onset, neurodegenerative disease for which there are currently no cures nor disease-modifying treatments. Here we report the identification of several potential anti-inflammatory targets for HD using an ex vivo model of HD that involves the acute transfection of human mutant huntingtin-based constructs into rat brain slices. This model recapitulates key components of the human disease, including the formation of intracellular huntingtin protein (HTT)-containing inclusions and the progressive neurodegeneration of striatal neurons-both occurring within the native tissue context of these neurons. Using this "high-throughput biology" screening platform, we conducted a hypothesis-neutral screen of a collection of drug-like compounds which identified several anti-inflammatory targets that provided neuroprotection against HTT fragment-induced neurodegeneration. The nature of these targets provide further support for non-cell autonomous mechanisms mediating significant aspects of neuropathogenesis induced by mutant HTT fragment proteins.

Development of a low-pressure microtargeting biolistic device for transfection of retinal explants.

Biolistic transfection offers a key experimental method for molecular perturbation of bona fide, postmitotic neurons within their native local environment in explanted tissues. However, current, commercially available biolistic devices unavoidably deliver traumatic injury to surface layers of explanted tissues because of helium co-emission with DNA-coated gold particles during the shooting process. This makes such methods unsuitable for use with the delicate tissue layers of the mammalian retina. Here, we report the development of a novel and inexpensive microtargeting biolistic device that avoids the trauma associated with conventional entrainment biolistic methods, permitting rapid and efficient transfection of retinal ganglion cells in the adult mammalian retina without significant damage to their local microenvironment. By using low helium inflow pressures and vacuum diversion to eliminate helium emission during the transfection process, we found that the current method allowed efficient transfection as well as morphological and functional preservation of retinal ganglion cells and their local glial microenvironment in transfected retinal explants from adult rats. The use of an ethanol-gold suspension further supported rapid and extended shooting sequences and reduced shot-to-shot variation during transfection compared to existing tubing-based devices. This new biolistic device should be useful not only in the retina, but also in other tissue explant settings in which preservation of local cellular and tissue integrity is a priority.