Small molecules that bind with high affinity and specificity to fibrils of the α-synuclein (αS) protein have the potential to serve as positron emission tomography (PET) imaging probes to aid in the diagnosis of Parkinson's disease and related synucleinopathies. To identify such molecules, we employed an ultra-high throughput in silico screening strategy using idealized pseudo-ligands termed exemplars to identify compounds for experimental binding studies. For the top hit from this screen, we used photo-crosslinking to confirm its binding site and studied the structure–activity relationship of its analogs to develop multiple molecules with nanomolar affinity for αS fibrils and moderate specificity for αS over Aβ fibrils. Lastly, we demonstrated the potential of the lead analog as an imaging probe by measuring binding to αS-enriched homogenates from mouse brain tissue using a radiolabeled analog of the identified molecule. This study demonstrates the validity of our powerful new approach to the discovery of PET probes for challenging molecular targets.
more »
« less
Synthesis and characterization of high affinity fluorogenic α-synuclein probes
Fluorescent small molecules are powerful tools for imaging α-synuclein pathology in vitro and in vivo . In this work, we explore benzofuranone as a potential scaffold for the design of fluorescent α-synuclein probes. These compounds have high affinity for α-synuclein, show fluorescent turn-on upon binding to fibrils, and display different binding to Lewy bodies, Lewy neurites and glial cytoplasmic inclusion pathologies in post-mortem brain tissue. These studies not only reveal the potential of benzofuranone compounds as α-synuclein specific fluorescent probes, but also have implications for the ways in which α-synucleinopathies are conformationally different and display distinct small molecule binding sites.
more »
« less
- Award ID(s):
- 1827457
- NSF-PAR ID:
- 10146570
- Date Published:
- Journal Name:
- Chemical Communications
- Volume:
- 56
- Issue:
- 24
- ISSN:
- 1359-7345
- Page Range / eLocation ID:
- 3567 to 3570
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Abstract Background Mutations in leucine-rich repeat kinase 2 (
LRRK2 ) are the most common cause of familial Parkinson’s disease (PD). These mutations elevate the LRRK2 kinase activity, making LRRK2 kinase inhibitors an attractive therapeutic. LRRK2 kinase activity has been consistently linked to specific cell signaling pathways, mostly related to organelle trafficking and homeostasis, but its relationship to PD pathogenesis has been more difficult to define.LRRK2 -PD patients consistently present with loss of dopaminergic neurons in the substantia nigra but show variable development of Lewy body or tau tangle pathology. Animal models carryingLRRK2 mutations do not develop robust PD-related phenotypes spontaneously, hampering the assessment of the efficacy of LRRK2 inhibitors against disease processes. We hypothesized that mutations inLRRK2 may not be directly related to a single disease pathway, but instead may elevate the susceptibility to multiple disease processes, depending on the disease trigger. To test this hypothesis, we have previously evaluated progression of α-synuclein and tau pathologies following injection of proteopathic seeds. We demonstrated that transgenic mice overexpressing mutant LRRK2 show alterations in the brain-wide progression of pathology, especially at older ages.Methods Here, we assess tau pathology progression in relation to long-term LRRK2 kinase inhibition. Wild-type or LRRK2G2019Sknock-in mice were injected with tau fibrils and treated with control diet or diet containing LRRK2 kinase inhibitor MLi-2 targeting the IC50 or IC90 of LRRK2 for 3–6 months. Mice were evaluated for tau pathology by brain-wide quantitative pathology in 844 brain regions and subsequent linear diffusion modeling of progression.
Results Consistent with our previous work, we found systemic alterations in the progression of tau pathology in LRRK2G2019Smice, which were most pronounced at 6 months. Importantly, LRRK2 kinase inhibition reversed these effects in LRRK2G2019Smice, but had minimal effect in wild-type mice, suggesting that LRRK2 kinase inhibition is likely to reverse specific disease processes in G2019S mutation carriers. Additional work may be necessary to determine the potential effect in non-carriers.
Conclusions This work supports a protective role of LRRK2 kinase inhibition in G2019S carriers and provides a rational workflow for systematic evaluation of brain-wide phenotypes in therapeutic development.
-
more » « less
Abstract Many neurodegenerative diseases including frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD), multiple system atrophy (MSA), etc., show colocalized deposits of TDP-43 and α-synuclein (αS) aggregates. To understand whether these colocalizations are driven by specific molecular interactions between the two proteins, we previously showed that the prion-like C-terminal domain of TDP-43 (TDP-43PrLD) and αS synergistically interact to form neurotoxic heterotypic amyloids in homogeneous buffer conditions. However, it remains unclear if αS can modulate TDP-43 present within liquid droplets and biomolecular condensates called stress granules (SGs). Here, using cell culture and in vitro TDP-43PrLD – RNA liquid droplets as models along with microscopy, nanoscale AFM-IR spectroscopy, and biophysical analyses, we uncover the interactions of αS with phase-separated droplets. We learn that αS acts as a Pickering agent by forming clusters on the surface of TDP-43PrLD – RNA droplets. The aggregates of αS on these clusters emulsify the droplets by nucleating the formation of heterotypic TDP-43PrLD amyloid fibrils, structures of which are distinct from those derived from homogenous solutions. Together, these results reveal an intriguing property of αS to act as a Pickering agent while interacting with SGs and unmask the hitherto unknown role of αS in modulating TDP-43 proteinopathies.
-
more » « less
Abstract Repulsive electrostatic forces between prion‐like proteins are a barrier against aggregation. In neuropharmacology, however, a prion's net charge (
Z ) is not a targeted parameter. Compounds that selectively boost prionZ remain unreported. Here, we synthesized compounds that amplified the negative charge of misfolded superoxide dismutase‐1 (SOD1) by acetylating lysine‐NH3+in amyloid‐SOD1, without acetylating native‐SOD1. Compounds resembled a “ball and chain” mace: a rigid amyloid‐binding “handle” (benzothiazole, stilbene, or styrylpyridine); an aryl ester “ball”; and a triethylene glycol chain connecting ball to handle. At stoichiometric excess, compounds acetylated up to 9 of 11 lysine per misfolded subunit (ΔZ fibril=−8100 per 103subunits). Acetylated amyloid‐SOD1 seeded aggregation more slowly than unacetylated amyloid‐SOD1 in vitro and organotypic spinal cord (these effects were partially due to compound binding). Compounds exhibited reactivity with other amyloid and non‐amyloid proteins (e.g., fibrillar α‐synuclein was peracetylated; serum albumin was partially acetylated; carbonic anhydrase was largely unacetylated). -
more » « less
Abstract Repulsive electrostatic forces between prion‐like proteins are a barrier against aggregation. In neuropharmacology, however, a prion's net charge (
Z ) is not a targeted parameter. Compounds that selectively boost prionZ remain unreported. Here, we synthesized compounds that amplified the negative charge of misfolded superoxide dismutase‐1 (SOD1) by acetylating lysine‐NH3+in amyloid‐SOD1, without acetylating native‐SOD1. Compounds resembled a “ball and chain” mace: a rigid amyloid‐binding “handle” (benzothiazole, stilbene, or styrylpyridine); an aryl ester “ball”; and a triethylene glycol chain connecting ball to handle. At stoichiometric excess, compounds acetylated up to 9 of 11 lysine per misfolded subunit (ΔZ fibril=−8100 per 103subunits). Acetylated amyloid‐SOD1 seeded aggregation more slowly than unacetylated amyloid‐SOD1 in vitro and organotypic spinal cord (these effects were partially due to compound binding). Compounds exhibited reactivity with other amyloid and non‐amyloid proteins (e.g., fibrillar α‐synuclein was peracetylated; serum albumin was partially acetylated; carbonic anhydrase was largely unacetylated).
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/c9cc09849f
