ELX-02 Generates Protein via Premature Stop Codon Read-Through without Inducing Native Stop Codon Read-Through Proteins.

ELX-02 is a clinical stage, small-molecule eukaryotic ribosomal selective glycoside acting to induce read-through of premature stop codons (PSCs) that results in translation of full-length protein. However, improved read-through at PSCs has raised the question of whether native stop codon (NSC) fidelity would be impacted. Here, we compare read-through by ELX-02 in PSC and NSC contexts. DMS-114 cells containing a PSC in the TP53 gene were treated with ELX-02 and tested for increased nuclear p53 protein expression while also monitoring two other proteins for NSC read-through. Additionally, blood samples were taken from healthy subjects pre- and post-treatment with ELX-02 (0.3-7.5 mg/kg). These samples were processed to collect white blood cells and then analyzed by western blot to identify native and potentially elongated proteins from NSC read-through. In a separate experiment, lymphocytes cultivated with vehicle or ELX-02 (20 and 100 µg/ml) were subjected to proteomic analysis. We found that ELX-02 produced significant read-through of the PSC found in TP53 mRNA in DMS-114 cells, resulting in increased p53 protein expression and consistent with decreased nonsense-mediated mRNA degradation. NSC read-through protein products were not observed in either DMS-114 cells or in clinical samples from subjects dosed with ELX-02. The number of read-through proteins identified by using proteomic analysis was lower than estimated, and none of the NSC read-through products identified with >2 peptides showed dose-dependent responses to ELX-02. Our results demonstrate significant PSC read-through by ELX-02 with maintained NSC fidelity, thus supporting the therapeutic utility of ELX-02 in diseases resulting from nonsense alleles. SIGNIFICANCE STATEMENT: ELX-02 produces significant read-through of premature stop codons leading to full-length functional protein, demonstrated here by using the R213X mutation in the TP53 gene of DMS-114 cells. In addition, three complementary techniques suggest that ELX-02 does not promote read-through of native stop codons at concentrations that lead to premature stop codon read-through. Thus, ELX-02 may be a potential therapeutic option for nonsense mutation-mediated genetic diseases.

Intravitreal administration of small molecule read-through agents demonstrate functional activity in a nonsense mutation mouse model.

The prevalence of nonsense mutations as a class within genetic diseases such as inherited retinal disorders (IRDs) presents an opportunity to develop a singular, common therapeutic agent for patients whose treatment options are otherwise limited. We propose a novel approach to addressing IRDs utilizing Eukaryotic Ribosome Selective Glycosides, ELX-01 and ELX-06, delivered to the eye by intravitreal (IVT) injection. We assessed read-through activity in vitro using a plasmid-based dual luciferase assay and in vivo in a mouse model of oculocutaneous albinism type 2. These models interrogate a naturally occurring R262X nonsense mutation in the OCA2 gene. ELX-01 and ELX-06 both produced a concentration-dependent increase in read-through of the OCA2 R262X mutation in the dual luciferase assay, with an effect at the top concentration which is superior to both gentamicin and G418. When testing both compounds in vivo, a single IVT injection produced a dose-dependent increase in melanin, consistent with compound read-through activity and functional restoration of the Oca2 protein. These results establish that ELX-01 and ELX-06 produce read-through of a premature stop codon in the OCA2 gene both in vitro and in vivo. The in vivo results suggest that these compounds can be dosed IVT to achieve read-through at the back of the eye. These data also suggest that ELX-01 or ELX-06 could serve as a common therapeutic agent across nonsense mutation-mediated IRDs and help to establish a target exposure range for development of a sustained release IVT formulation.

Genetics of cystogenesis in base-edited human organoids reveal therapeutic strategies for polycystic kidney disease.

In polycystic kidney disease (PKD), microscopic tubules expand into macroscopic cysts. Among the world's most common genetic disorders, PKD is inherited via heterozygous loss-of-function mutations but is theorized to require additional loss of function. To test this, we establish human pluripotent stem cells in allelic series representing four common nonsense mutations, using CRISPR base editing. When differentiated into kidney organoids, homozygous mutants spontaneously form cysts, whereas heterozygous mutants (original or base corrected) express no phenotype. Using these, we identify eukaryotic ribosomal selective glycosides (ERSGs) as PKD therapeutics enabling ribosomal readthrough of these same nonsense mutations. Two different ERSGs not only prevent cyst initiation but also limit growth of pre-formed cysts by partially restoring polycystin expression. Furthermore, glycosides accumulate in cyst epithelia in organoids and mice. Our findings define the human polycystin threshold as a surmountable drug target for pharmacological or gene therapy interventions, with relevance for understanding disease mechanisms and future clinical trials.

Estrogen receptor ß ligand therapy activates PI3K/Akt/mTOR signaling in oligodendrocytes and promotes remyelination in a mouse model of multiple sclerosis.

The identification of a drug that stimulates endogenous myelination and spares axon degeneration during multiple sclerosis (MS) could potentially reduce the rate of disease progression. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we have previously shown that prophylactic administration of the estrogen receptor (ER) ß ligand 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) decreases clinical disease, is neuroprotective, stimulates endogenous myelination, and improves axon conduction without altering peripheral cytokine production or reducing central nervous system (CNS) inflammation. Here, we assessed the effects of therapeutic DPN treatment during peak EAE disease, which represents a more clinically relevant treatment paradigm. In addition, we investigated the mechanism of action of DPN treatment-induced recovery during EAE. Given that prophylactic and therapeutic treatments with DPN during EAE improved remyelination-induced axon conduction, and that ER (α and ß) and membrane (m)ERs are present on oligodendrocyte lineage cells, a direct effect of treatment on oligodendrocytes is likely. DPN treatment of EAE animals resulted in phosphorylated ERß and activated the phosphatidylinositol 3-kinase (PI3K)/serine-threonine-specific protein kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, a pathway required for oligodendrocyte survival and axon myelination. These results, along with our previous studies of prophylactic DPN treatment, make DPN and similar ERß ligands immediate and favorable therapeutic candidates for demyelinating disease.

Oestrogen receptor beta ligand: a novel treatment to enhance endogenous functional remyelination.

Demyelinating diseases, such as multiple sclerosis, are characterized by inflammatory demyelination and neurodegeneration of the central nervous system. Therapeutic strategies that induce effective neuroprotection and enhance intrinsic repair mechanisms are central goals for future therapy of multiple sclerosis. Oestrogens and oestrogen receptor ligands are promising treatments to prevent multiple sclerosis-induced neurodegeneration. In the present study we investigated the capacity of oestrogen receptor ß ligand treatment to affect callosal axon demyelination and stimulate endogenous myelination in chronic experimental autoimmune encephalomyelitis using electrophysiology, electron microscopy, immunohistochemistry and tract-tracing methods. Oestrogen receptor ß ligand treatment of experimental autoimmune encephalomyelitis mice prevented both histopathological and functional abnormalities of callosal axons despite the presence of inflammation. Specifically, there were fewer demyelinated, damaged axons and more myelinated axons with intact nodes of Ranvier in oestrogen receptor ß ligand-treated mice. In addition, oestrogen receptor ß ligand treatment caused an increase in mature oligodendrocyte numbers, a significant increase in myelin sheath thickness and axon transport. Functional analysis of callosal axon conduction showed a significant improvement in compound action potential amplitudes, latency and in axon refractoriness. These findings show a direct neuroprotective effect of oestrogen receptor ß ligand treatment on oligodendrocyte differentiation, myelination and axon conduction during experimental autoimmune encephalomyelitis.

Targeting G542X CFTR nonsense alleles with ELX-02 restores CFTR function in human-derived intestinal organoids.

BACKGROUND: Promoting full-length protein production is a requisite step to address some of the remaining unmet medical need for those with Cystic Fibrosis (CF) nonsense alleles. ELX-02 promotes read-through of mRNA transcripts bearing nonsense mutations, including the most common CF nonsense allele G542X, in several different preclinical models including human bronchial epithelial cells. Here we evaluate ELX-02 mediated read-through using the CFTR-dependent Forskolin-induced swelling (FIS) assay across a selection of G542X genotype patient derived organoids (PDOs). METHODS: CFTR functional restoration was evaluated in ELX-02 treated G542X homozygous and heterozygous PDOs in the CFTR-dependent FIS assay. CFTR mRNA abundance and integrity were evaluated by qPCR and Nanostring analysis while PDO protein was detected by capillary based size-exclusion chromatography. RESULTS: PDOs homozygous for G542X or heterozygous with a second minimally functional allele had significantly increased CFTR activity with ELX-02 in a dose-dependent fashion across a variety of forskolin induction concentrations. The functional increases are similar to those obtained with tezacaftor/ivacaftor in F508del homozygous PDOs. Increased CFTR C- and B-band protein was observed in accordance with increased function. In addition, ELX-02 treatment of a G542X/G542X PDO results in a 5-fold increase in CFTR mRNA compared with vehicle treated, resulting in normalization of CFTR mRNA as measured via Nanostring. CONCLUSIONS: These data with ELX-02 in PDOs are consistent with previous G542X model evaluations. These results also support the on-going clinical evaluation of ELX-02 as a read-through agent for CF caused by the G542X allele.

Loop 2 structure in glycine and GABA(A) receptors plays a key role in determining ethanol sensitivity.

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and gamma-aminobutyric acid type A receptors (GABA(A)Rs). To test this, we mutated Loop 2 in the alpha1 subunit of GlyRs and in the gamma subunit of alpha1beta2gamma2GABA(A)Rs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of alpha1GlyR subunits with Loop 2 from the deltaGABA(A)R (deltaL2), but not the gammaGABA(A)R subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the gamma subunit of GABA(A)Rs with deltaL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA(A) gamma-deltaL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA(A)Rs. The deltaL2 mutations did not affect GlyR or GABA(A)R sensitivity, respectively, to Zn(2+) or diazepam, which suggests that these deltaL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and deltaL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.

Targets for ethanol action and antagonism in loop 2 of the extracellular domain of glycine receptors.

The present studies used increased atmospheric pressure in place of a traditional pharmacological antagonist to probe the molecular sites and mechanisms of ethanol action in glycine receptors (GlyRs). Based on previous studies, we tested the hypothesis that physical-chemical properties at position 52 in extracellular domain Loop 2 of alpha1GlyRs, or the homologous alpha2GlyR position 59, determine sensitivity to ethanol and pressure antagonism of ethanol. Pressure antagonized ethanol in alpha1GlyRs that contain a non-polar residue at position 52, but did not antagonize ethanol in receptors with a polar residue at this position. Ethanol sensitivity in receptors with polar substitutions at position 52 was significantly lower than GlyRs with non-polar residues at this position. The alpha2T59A mutation switched sensitivity to ethanol and pressure antagonism in the WTalpha2GlyR, thereby making it alpha1-like. Collectively, these findings indicate that (i) polarity at position 52 plays a key role in determining sensitivity to ethanol and pressure antagonism of ethanol; (ii) the extracellular domain in alpha1- and alpha2GlyRs is a target for ethanol action and antagonism and (iii) there is structural-functional homology across subunits in Loop 2 of GlyRs with respect to their roles in determining sensitivity to ethanol and pressure antagonism of ethanol. These findings should help in the development of pharmacological agents that antagonize ethanol.

Attenuation of corpus callosum axon myelination and remyelination in the absence of circulating sex hormones.

Sex differences in the structure and organization of the corpus callosum (CC) can be attributed to genetic, hormonal or environmental effects, or a combination of these factors. To address the role of gonadal hormones on axon myelination, functional axon conduction and immunohistochemistry analysis of the CC in intact, gonadectomized and hormone-replaced gonadectomized animals were used. These groups were subjected to cuprizone diet-induced demyelination followed by remyelination. The myelinated component of callosal compound action potential was significantly decreased in ovariectomized and castrated animals under normal myelinating condition. Compared to gonadally intact cohorts, both gonadectomized groups displayed more severe demyelination and inhibited remyelination. Castration in males was more deleterious than ovariectomy in females. Callosal conduction in estradiol-supplemented ovariectomized females was significantly increased during normal myelination, less attenuated during demyelination, and increased beyond placebo-treated ovariectomized or intact female levels during remyelination. In castrated males, the non-aromatizing steroid dihydrotestosterone was less efficient than testosterone and estradiol in restoring normal myelination/axon conduction and remyelination to levels of intact males. Furthermore, in both sexes, estradiol supplementation in gonadectomized groups increased the number of oligodendrocytes. These studies suggest an essential role of estradiol to promote efficient CC myelination and axon conduction in both sexes.

An animal model of cortical and callosal pathology in multiple sclerosis.

The pathological and radiological hallmarks of multiple sclerosis (MS) include multiple demyelinated lesions disseminated throughout the white matter of the central nervous system (CNS). More recently, the cerebral cortex has been shown to be affected in MS, but the elucidation of events causing cortical demyelination has been hampered by the lack of animal models reflecting such human cortical pathology. In this report, we have described the presence of cortical gray matter and callosal white matter demyelinating lesions in the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Similar to the pathological lesions of MS patients, EAE lesions have been classified as type I-leukocortical, type II-intracortical and type III-subpial. All of these lesions had varying degrees of demyelination, inflammatory cells and reactive astrocytes. Similar to MS, cortical layers during EAE showed demyelination, microglia activation, synaptic protein alterations and apoptotic cells. In addition, the callosal white matter during EAE had many inflammatory demyelinating lesions and axon degeneration. Functional electrophysiological conduction analysis showed deficits in both myelinated and unmyelinated callosal axons during early and late EAE. The chronic EAE mouse model has features that mimic cortical and callosal pathology of MS, and can be potentially used to screen agents to prevent these features of disease.

Molecular targets and mechanisms for ethanol action in glycine receptors.

Glycine receptors (GlyRs) are recognized as the primary mediators of neuronal inhibition in the spinal cord, brain stem and higher brain regions known to be sensitive to ethanol. Building evidence supports the notion that ethanol acting on GlyRs causes at least a subset of its behavioral effects and may be involved in modulating ethanol intake. For over two decades, GlyRs have been studied at the molecular level as targets for ethanol action. Despite the advances in understanding the effects of ethanol in vivo and in vitro, the precise molecular sites and mechanisms of action for ethanol in ligand-gated ion channels in general, and in GlyRs specifically, are just now starting to become understood. The present review focuses on advances in our knowledge produced by using molecular biology, pressure antagonism, electrophysiology and molecular modeling strategies over the last two decades to probe, identify and model the initial molecular sites and mechanisms of ethanol action in GlyRs. The molecular targets on the GlyR are covered on a global perspective, which includes the intracellular, transmembrane and extracellular domains. The latter has received increasing attention in recent years. Recent molecular models of the sites of ethanol action in GlyRs and their implications to our understanding of possible mechanism of ethanol action and novel targets for drug development in GlyRs are discussed.

Assaying the functional effects of demyelination and remyelination: revisiting field potential recordings.

The occurrence and histopathological characteristics of demyelination and neurodegeneration have been well described in different demyelinating mouse models. However, histopathological analysis is limiting in that it is unable to describe the functional consequences of demyelination and recovery after remyelination. Establishing the functional correlates of axon demyelination and remyelination is an important goal and can be used to measure axon function and develop neuroprotective therapies. This report describes a previously established, simple, easily applied method of electrophysiological measurement that can characterize white matter axonal dysfunction following demyelination and potential recovery after remyelination. It is designed to study in vitro stimulated compound action potentials in the corpus callosum of superfused brain slices at various time points and can be similarly used on white matter tracts in the optic nerve, spinal cord and cerebellum. Since behavioral testing can be performed prior to the brain slice electrophysiology, and the recorded slices can be post-fixed and subjected to histological analysis, correlates between behavior, axon function, and pathology can be determined. A temporal pattern of white matter functional deterioration and recovery can also be established to study mechanisms of demyelination-induced white matter injury and repair.

Roles for loop 2 residues of alpha1 glycine receptors in agonist activation.

The present study tested the hypothesis that several residues in Loop 2 of alpha1 glycine receptors (GlyRs) play important roles in mediating the transduction of agonist activation to channel gating. This was accomplished by investigating the effect of cysteine point mutations at positions 50-60 on glycine responses in alpha1GlyRs using two-electrode voltage clamp of Xenopus oocytes. Cysteine substitutions produced position-specific changes in glycine sensitivity that were consistent with a beta-turn structure of Loop 2, with odd-numbered residues in the beta-turn interacting with other agonist-activation elements at the interface between extracellular and transmembrane domains. We also tested the hypothesis that the charge at position 53 is important for agonist activation by measuring the glycine response of wild type (WT) and E53C GlyRs exposed to methanethiosulfonate reagents. As earlier, E53C GlyRs have a significantly higher EC(50) than WT GlyRs. Exposing E53C GlyRs to the negatively charged 2-sulfonatoethyl methanethiosulfonate, but not neutral 2-hydroxyethyl methanethiosulfonate, positively charged 2-aminoethyl methanethiosulfonate, or 2-trimethylammonioethyl methanethiosulfonate, decreased the glycine EC(50) to resemble WT GlyR responses. Exposure to these reagents did not significantly alter the glycine EC(50) for WT GlyRs. The latter findings suggest that the negative charge at position 53 is important for activation of GlyRs through its interaction with positive charge(s) in other neighboring agonist activation elements. Collectively, the findings provide the basis for a refined molecular model of alpha1GlyRs based on the recent x-ray structure of a prokaryotic pentameric ligand-gated ion channel and offer insight into the structure-function relationships in GlyRs and possibly other ligand-gated ion channels.

Evidence that ethanol acts on a target in Loop 2 of the extracellular domain of alpha1 glycine receptors.

Considerable evidence indicates that ethanol acts on specific residues in the transmembrane domains of glycine receptors (GlyRs). In this study, we tested the hypothesis that the extracellular domain is also a target for ethanol action by investigating the effect of cysteine substitutions at positions 52 (extracellular domain) and 267 (transmembrane domain) on responses to n-alcohols and propyl methanethiosulfonate (PMTS) in alpha1GlyRs expressed in Xenopus oocytes. In support of the hypothesis: (i) The A52C mutation changed ethanol sensitivity compared to WT GlyRs; (ii) PMTS produced irreversible alcohol-like potentiation in A52C GlyRs; and (iii) PMTS binding reduced the n-chain alcohol cutoff in A52C GlyRs. Further studies used PMTS binding to cysteines at positions 52 or 267 to block ethanol action at one site in order to determine its effect at other site(s). In these situations, ethanol caused negative modulation when acting at position 52 and positive modulation when acting at position 267. Collectively, these findings parallel the evidence that established the TM domain as a target for ethanol, suggest that positions 52 and 267 are part of the same alcohol pocket and indicate that the net effect of ethanol on GlyR function reflects the summation of its positive and negative modulatory effects on different targets.

Multiple sites of ethanol action in alpha1 and alpha2 glycine receptors suggested by sensitivity to pressure antagonism.

The current study used an ethanol antagonist, increased atmospheric pressure, to test the hypothesis that ethanol acts on multiple sites in glycine receptors (GlyRs). The effects of 12 times normal atmospheric pressure of helium-oxygen gas (pressure) on ethanol-induced potentiation of GlyR function in Xenopus oocytes expressing human alpha1, alpha2 or the mutant alpha1(A52S) GlyRs were measured using two-electrode voltage clamp. Pressure reversibly antagonized potentiation of glycine in alpha1 GlyR by 40-200 mm ethanol, but did not antagonize 10 and 25 mm ethanol in the same oocytes. In contrast, pressure did not significantly affect potentiation of glycine by 25-100 mm ethanol in alpha2 GlyRs, nor did pressure alter ethanol response in the A52S mutant. Pressure did not affect baseline receptor function or response to glycine in the absence of ethanol. These findings provide the first direct evidence for multiple sites of ethanol action in GlyRs. The sites can be differentiated on the basis of ethanol concentration, subunit and structural composition and sensitivities to pressure antagonism of ethanol. Parallel studies with butanol support this conclusion. The mutant alpha1(A52S) GlyR findings suggest that increased attention should be focused on the amino terminus as a potential target for ethanol action.

Ethanol potentiation of glycine receptors expressed in Xenopus oocytes antagonized by increased atmospheric pressure.

BACKGROUND: Behavioral and biochemical studies indicate that exposure to 12 times normal atmospheric pressure (12 ATA) of helium-oxygen gas (heliox) is a direct, selective ethanol antagonist. The current study begins to test the hypothesis that ethanol acts by a common mechanism on ligand-gated ion channels by expanding previous hyperbaric investigations on gamma-aminobutyric acid type A (GABA(A)) receptors (GABA(A)Rs) at the biochemical level to alpha(1)glycine (GlyRs) expressed in Xenopus oocytes. METHODS: Oocytes expressing wild-type alpha(1) homomeric GlyRs were voltage-clamped (-70 mV) and tested in the presence of glycine (EC(2)) +/- ethanol (50-200 mM) under 1 ATA control and 3 to 12 ATA heliox conditions. Glycine concentration response curves, strychnine/glycine interactions, and zinc (Zn2+) modulation of GlyR function was also tested. RESULTS: Pressure reversibly antagonized the action of ethanol. The degree of antagonism increased as pressure increased. Pressure did not significantly alter the effects of glycine, strychnine, or Zn2+, indicating that ethanol antagonism by pressure cannot be attributed to alterations by pressure of normal GlyR function. The antagonism did not reflect tolerance to ethanol, receptor desensitization, or receptor rundown. CONCLUSION: This is the first use of hyperbarics to investigate the mechanism of action of ethanol in recombinant receptors. The findings indicate that pressure directly and selectively antagonizes ethanol potentiation of alpha(1)GlyR function in a reversible and concentration- and pressure-dependent manner. The sensitivity of ethanol potentiation of GlyR function to pressure antagonism indicates that ethanol acts by a common, pressure-antagonism-sensitive mechanism in GlyRs and GABA(A)Rs. The findings also support the hypothesis that ethanol potentiation of GlyR function plays a role in mediating the sedative-hypnotic effects of ethanol.