Highly selective peroxisome proliferator-activated receptor δ (PPARδ) modulator demonstrates improved safety profile compared to GW501516.

Compound 1 regulates significantly fewer genes than the PPARδ modulator, GW501516. Both compounds are efficacious in a thermal injury model of muscle regeneration. The restricted gene profile of 1 relative to GW501516 suggests that 1 may be pharmacoequivalent to GW501516 with fewer PPAR-related safety concerns.

Novel highly selective peroxisome proliferator-activated receptor δ (PPARδ) modulators with pharmacokinetic properties suitable for once-daily oral dosing.

Optimization of benzamide PPARδ modulator 1 led to (E)-6-(2-((4-(furan-2-yl)-N-methylbenzamido)methyl)phenoxy)-4-methylhex-4-enoic acid (18), a potent selective PPARδ modulator with significantly improved exposure in multiple species following oral administration.

Mutant induced neurons and humanized mice enable identification of Niemann-Pick type C1 proteostatic therapies.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

Membrane-associated farnesylated UCH-L1 promotes alpha-synuclein neurotoxicity and is a therapeutic target for Parkinson's disease.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is linked to Parkinson's disease (PD) and memory and is selectively expressed in neurons at high levels. Its expression pattern suggests a function distinct from that of its widely expressed homolog UCH-L3. We report here that, in contrast to UCH-L3, UCH-L1 exists in a membrane-associated form (UCH-L1(M)) in addition to the commonly studied soluble form. C-terminal farnesylation promotes the association of UCH-L1 with cellular membranes, including the endoplasmic reticulum. The amount of UCH-L1(M) in transfected cells is shown to correlate with the intracellular level of alpha-synuclein, a protein whose accumulation is associated with neurotoxicity and the development of PD. Reduction of UCH-L1(M) in cell culture models of alpha-synuclein toxicity by treatment with a farnesyltransferase inhibitor (FTI-277) reduces alpha-synuclein levels and increases cell viability. Proteasome function is not affected by UCH-L1(M), suggesting that it may negatively regulate the lysosomal degradation of alpha-synuclein. Therefore, inhibition of UCH-L1 farnesylation may be a therapeutic strategy for slowing the progression of PD and related synucleinopathies.

Dopamine-modified alpha-synuclein blocks chaperone-mediated autophagy.

Altered degradation of alpha-synuclein (alpha-syn) has been implicated in the pathogenesis of Parkinson disease (PD). We have shown that alpha-syn can be degraded via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins. Pathogenic mutants of alpha-syn block lysosomal translocation, impairing their own degradation along with that of other CMA substrates. While pathogenic alpha-syn mutations are rare, alpha-syn undergoes posttranslational modifications, which may underlie its accumulation in cytosolic aggregates in most forms of PD. Using mouse ventral medial neuron cultures, SH-SY5Y cells in culture, and isolated mouse lysosomes, we have found that most of these posttranslational modifications of alpha-syn impair degradation of this protein by CMA but do not affect degradation of other substrates. Dopamine-modified alpha-syn, however, is not only poorly degraded by CMA but also blocks degradation of other substrates by this pathway. As blockage of CMA increases cellular vulnerability to stressors, we propose that dopamine-induced autophagic inhibition could explain the selective degeneration of PD dopaminergic neurons.

Botulinum neurotoxin serotype A inhibitors: small-molecule mercaptoacetamide analogs.

Botulinum neurotoxin elicits its paralytic activity through a zinc-dependant metalloprotease that cleaves proteins involved in neurotransmitter release. Currently, no drugs are available to reverse the effects of botulinum intoxication. Herein we report the design of a novel series of mercaptoacetamide small-molecule inhibitors active against botulinum neurotoxin serotype A. These analogs show low micromolar inhibitory activity against the isolated enzyme. Structure-activity relationship studies for a series of mercaptoacetamide analogs of 5-amino-3-phenylpyrazole reveal components essential for potent inhibitory activity.

PPARδ modulation rescues mitochondrial fatty acid oxidation defects in the mdx model of muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a recessive, fatal X-linked disease that is characterized by progressive skeletal muscle wasting due to the absence of dystrophin, which is an a essential protein that bridges the inner cytoskeleton and extra-cellular matrix. This study set out to characterize the mitochondria in primary muscle satellite cell derived myoblasts from mdx mice and wild type control mice. Compared to wild type derived cells the mdx derived cells have reduced mitochondrial bioenergetics and have fewer mitochondria. Here, we demonstrate that a novel PPARδ modulator improves mitochondrial function in the mdx mice, which supports that modulating PPARδ may be therapeutically beneficial in DMD patients.

Correction to "Selective PPARδ Modulators Improve Mitochondrial Function: Potential Treatment for Duchenne Muscular Dystrophy (DMD)".

[This corrects the article DOI: 10.1021/acsmedchemlett.8b00287.].

Selective PPARδ Modulators Improve Mitochondrial Function: Potential Treatment for Duchenne Muscular Dystrophy (DMD).

The X-ray structure of the previously reported PPARδ modulator 1 bound to the ligand binding domain (LBD) revealed that the amide moiety in 1 exists in the thermodynamically disfavored cis-amide orientation. Isosteric replacement of the cis-amide with five-membered heterocycles led to the identification of imidazole 17 (MA-0204), a potent, selective PPARδ modulator with good pharmacokinetic properties. MA-0204 was tested in vivo in mice and in vitro in patient-derived muscle myoblasts (from Duchenne Muscular Dystrophy (DMD) patients); 17 altered the expression of PPARδ target genes and improved fatty acid oxidation, which supports the therapeutic hypothesis for the study of MA-0204 in DMD patients.

Small molecule metalloprotease inhibitor with in vitro, ex vivo and in vivo efficacy against botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to mankind and are the causative agents of the neuroparalytic disease botulism. Their ease of production and extreme toxicity have caused these neurotoxins to be classified as Tier 1 bioterrorist threat agents and have led to a sustained effort to develop countermeasures to treat intoxication in case of a bioterrorist attack. While timely administration of an approved antitoxin is effective in reducing the severity of botulism, reversing intoxication requires different strategies. In the present study, we evaluated ABS 252 and other mercaptoacetamide small molecule active-site inhibitors of BoNT/A light chain using an integrated multi-assay approach. ABS 252 showed inhibitory activity in enzymatic, cell-based and muscle activity assays, and importantly, produced a marked delay in time-to-death in mice. The results suggest that a multi-assay approach is an effective strategy for discovery of potential BoNT therapeutic candidates.

Development and advanced validation of an optimized method for the quantitation of Aß42 in human cerebrospinal fluid.

Cerebrospinal fluid (CSF) biomarkers have been extensively utilized in the diagnosis of Alzheimer's disease (AD) and characterization of progression. One important CSF biomarker is the amyloid beta 42 (Aß(42)) peptide, a key player in AD pathogenesis. The INNOTEST® Aß(42) ELISA kit has been widely used but an advanced level of method development and validation has not been reported. To support a clinical trial in AD, we successfully completed a Good Laboratory Practices (GLP)-level validation of the method to establish the parameters of precision, accuracy, parallelism, selectivity, specificity, and linearity of dilution of the assay in CSF matrix, as well as CSF storage stability. Several modifications were required to optimize the assay and ensure consistent results in a clinical-trial setting. These included the use of additional calibrators, an adjusted standard curve range, a minimum required dilution (MRD) of CSF by 6-fold to avoid matrix interference and mitigation of analyte adsorption to labware by the addition of Tween-20. The optimized method displayed a quantitative range of 375-4,500 pg/mL. The inter-assay precision was ≤12.1 % CV and the inter-assay relative accuracy was ≤10.9 % absolute bias, bringing the total error of the assay to ≤23 %. The intra-assay precision of the assay at the high validation standard and below was ≤5.5 % CV; this enables sensitive detection of biomarker changes across a therapeutic regime. The INNOTEST® Aß(42) ELISA kit, modified as reported here, may be appropriate for many applications, including regulatory agency acceptable clinical diagnosis and pharmacodynamic assessment.

Phosphorylation at Ser-129 but not the phosphomimics S129E/D inhibits the fibrillation of alpha-synuclein.

alpha-Synuclein (alpha-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related alpha-synulceinopathies. However, whether phosphorylation promotes or inhibits alpha-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of alpha-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type alpha-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated alpha-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of alpha-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of alpha-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern alpha-syn aggregation and toxicity in PD and related alpha-synulceinopathies.

The impact of the E46K mutation on the properties of alpha-synuclein in its monomeric and oligomeric states.

The third and most recently identified Parkinson's disease-linked variant of the neuronal protein alpha-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164-173). Herein, we present biochemical and biophysical characterization of E46K alpha-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K alpha-synuclein formed insoluble fibrils in vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K alpha-synuclein fibrils possess a typical amyloid ultrastructure. E46K alpha-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of alpha-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type alpha-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of alpha-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT alpha-synuclein that are observed in vitro.

Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy.

Aberrant alpha-synuclein degradation is implicated in Parkinson's disease pathogenesis because the protein accumulates in the Lewy inclusion bodies associated with the disease. Little is known, however, about the pathways by which wild-type alpha-synuclein is normally degraded. We found that wild-type alpha-synuclein was selectively translocated into lysosomes for degradation by the chaperone-mediated autophagy pathway. The pathogenic A53T and A30P alpha-synuclein mutants bound to the receptor for this pathway on the lysosomal membrane, but appeared to act as uptake blockers, inhibiting both their own degradation and that of other substrates. These findings may underlie the toxic gain-of-function by the mutants.