Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor

ABSTRACTPreventing or reversing the pathological misfolding and self-association of alpha-synuclein (aSyn) can rescue a broad spectrum of pathological cellular insults that manifest in Parkinson’s Disease (PD), Dementia with Lewy bodies (DLB), and other alpha-synucleinopathies. We have developed a high-throughput, FRET-based drug discovery platform that combines high-resolution protein structural detection in living cells with an array of functional and biophysical assays to identify novel lead compounds that protect SH-SY5Y cells from aSyn induced cytotoxicity as well as inhibiting seeded aSyn aggregation, even at nanomolar concentrations.Our combination of cellular and cell-free assays allow us to distinguish between direct aSyn binding or indirect mechanisms of action (MOA). We focus on targeting oligomers with the requisite sensitivity to detect subtle protein structural changes that may lead to effective therapeutic discoveries for PD, DLB, and other alpha-synucleinopathies. Pilot high-throughput screens (HTS) using our aSyn cellular FRET biosensors has led to the discovery of the first nanomolar-affinity small molecules that disrupt toxic aSyn oligomers in cells and inhibit cell death. Primary neuron assays of aSyn pathology (e.g. phosphorylation of mouse aSyn PFF) show rescue of pathology for two of our tested compounds. Subsequent seeded thioflavin-t (ThioT) aSyn aggregation assays demonstrate these compounds deter or block aSyn fibril assembly. Other hit compounds identified in our HTS are known to modulate oxidative stress, autophagy, and ER stress, providing validation that our biosensor is sensitive to indirect MOA as well.

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High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211026245 ◽

2021 ◽

pp. 247255522110262

Author(s):

Jonathan Choy ◽

Yanqing Kan ◽

Steve Cifelli ◽

Josephine Johnson ◽

Michelle Chen ◽

...

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Screening Strategy ◽

Time Resolved ◽

Protein Levels ◽

Increased Risk ◽

Compound Screening ◽

High Throughput Screens ◽

Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

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Assay Optimization and Screening of RNA-Protein Interactions by AlphaScreen

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107306128 ◽

2007 ◽

Vol 12 (7) ◽

pp. 946-955 ◽

Cited By ~ 33

Author(s):

Nicholas L. Mills ◽

Anang A. Shelat ◽

R. Kiplin Guy

Keyword(s):

Drug Discovery ◽

Small Molecules ◽

High Throughput ◽

Protein Interactions ◽

Proof Of Concept ◽

Rna Molecules ◽

Lead Compounds ◽

Assay Optimization ◽

Rna Targets ◽

Hiv 1

The lack of lead compounds that specifically recognize and manipulate the function of RNA molecules limits our ability to consider RNA targets valid for drug discovery. Herein is reported a high-throughput biochemical screen for inhibitors of RNA-protein interactions based on AlphaScreen technology that incorporates several layers of specificity measurements into the primary screen. This screen was used to analyze approximately 5500 compounds from a collection of bioactive small molecules to detect inhibitors of the HIV-1 Rev-RRE and BIV Tat-TAR interactions. This proof-of-concept screen validates the assay as one that accurately identifies hit molecules and determines the selectivity of those hits. ( Journal of Biomolecular Screening 2007: 946-955)

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Multi-Omics Technologies Applied to Tuberculosis Drug Discovery

Applied Sciences ◽

10.3390/app10134629 ◽

2020 ◽

Vol 10 (13) ◽

pp. 4629 ◽

Cited By ~ 1

Author(s):

Aaron Goff ◽

Daire Cantillon ◽

Leticia Muraro Wildner ◽

Simon J Waddell

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Discovery ◽

High Throughput ◽

A Priori ◽

Drug Action ◽

Biological Molecules ◽

Lead Compound ◽

Omics Technologies ◽

Lead Compounds ◽

High Throughput Screens

Multi-omics strategies are indispensable tools in the search for new anti-tuberculosis drugs. Omics methodologies, where the ensemble of a class of biological molecules are measured and evaluated together, enable drug discovery programs to answer two fundamental questions. Firstly, in a discovery biology approach, to find new targets in druggable pathways for target-based investigation, advancing from target to lead compound. Secondly, in a discovery chemistry approach, to identify the mode of action of lead compounds derived from high-throughput screens, progressing from compound to target. The advantage of multi-omics methodologies in both of these settings is that omics approaches are unsupervised and unbiased to a priori hypotheses, making omics useful tools to confirm drug action, reveal new insights into compound activity, and discover new avenues for inquiry. This review summarizes the application of Mycobacterium tuberculosis omics technologies to the early stages of tuberculosis antimicrobial drug discovery.

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Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors

npj Parkinson s Disease ◽

10.1038/s41531-021-00195-6 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Anthony R. Braun ◽

Elly E. Liao ◽

Mian Horvath ◽

Prakriti Kalra ◽

Karen Acosta ◽

...

Keyword(s):

Drug Discovery ◽

Single Molecule ◽

Conformational Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Alpha Synuclein ◽

Time Resolved ◽

Primary Mouse ◽

Fret Biosensors

AbstractWe have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson’s disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.

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Time-resolved FRET screening identifies small molecular modifiers of mutant Huntingtin conformational inflexibility in patient-derived cells

10.1101/2021.01.18.427095 ◽

2021 ◽

Author(s):

Johannes H. Wilbertz ◽

Julia Frappier ◽

Sandra Muller ◽

Sabine Gratzer ◽

Walter Englaro ◽

...

Keyword(s):

Small Molecules ◽

High Throughput ◽

High Throughput Screening ◽

Age Of Onset ◽

Conformational Flexibility ◽

Cag Repeat ◽

Mutant Huntingtin ◽

Loss Of Function ◽

Human Patient ◽

Time Resolved

AbstractHuntington’s disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1 encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTT’s conformational flexibility in human patient fibroblasts. We applied an antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay, measuring endogenous HTT flexibility using two validated HTT-specific antibodies. The ratio of TR-FRET signal at 4°C and 20°C differs between wtHTT and mHTT and allowed to perform a high-throughput screening using HTT flexibility as a read-out. We identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. This novel screening approach has the potential to identify previously unknown HD drugs and drug targets.

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Use of a Novel Homogeneous Fluorescent Technology in High Throughput Screening

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719600100407 ◽

1996 ◽

Vol 1 (4) ◽

pp. 203-210 ◽

Cited By ~ 50

Author(s):

Janet M. Kolb ◽

Gregory Yamanaka ◽

Susan P. Manly

Keyword(s):

Time Delay ◽

High Throughput ◽

High Throughput Screening ◽

Time Resolved Fluorescence ◽

Reaction Conditions ◽

Time Resolved ◽

Attractive Candidate ◽

Endogenous Fluorescence ◽

High Throughput Screens ◽

Fluorescent Compounds

A new fluorescent technology called homogeneous time-resolved fluorescence (HTRF) is sensitive, homogeneous, and quite tolerant to extremes in reaction conditions. These characteristics make this technique an attractive candidate for use in high throughput screens. The assay system uses a pair of fluorescent compounds to label biomolecules. The long-lived nature of the fluorescence of one of them, europium cryptate, facilitates the homogeneous nature of the assay. Furthermore, the introduction of a time delay in reading the signal eliminates the principal difficulty in applying fluorescence to screening formats, that of endogenous fluorescence of samples tested (especially natural products). This technique is robust and sensitive, and we report here its utility in a high throughput screening format.

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Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening

Biosensors ◽

10.3390/bios8040099 ◽

2018 ◽

Vol 8 (4) ◽

pp. 99 ◽

Cited By ~ 12

Author(s):

Tory Schaaf ◽

Ang Li ◽

Benjamin Grant ◽

Kurt Peterson ◽

Samantha Yuen ◽

...

Keyword(s):

High Precision ◽

Small Molecule ◽

High Throughput ◽

Fluorescence Lifetime ◽

High Throughput Screening ◽

Structural Changes ◽

Dynamic Range ◽

Resonance Energy ◽

Fluorescence Measurements ◽

Fret Biosensors

We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.

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UbFluor: a mechanism-based probe for HECT E3 ligases

Chemical Science ◽

10.1039/c6sc01167e ◽

2016 ◽

Vol 7 (8) ◽

pp. 5587-5595 ◽

Cited By ~ 13

Author(s):

David T. Krist ◽

Sungjin Park ◽

Galyah H. Boneh ◽

Sarah E. Rice ◽

Alexander V. Statsyuk

Keyword(s):

Small Molecules ◽

High Throughput ◽

E3 Ligase ◽

E3 Ligases ◽

Catalytic Cysteine ◽

High Throughput Screens

UbFluor is a mechanism-based probe that undergoes a direct transthiolation reaction with the catalytic cysteine of the model HECT E3 ligase Rsp5. We show that UbFluor can be utilized to conduct high-throughput screens (HTS) of small molecules against HECT ligases.

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Alpha-Synuclein in Alcohol Use Disorder, Connections with Parkinson’s Disease and Potential Therapeutic Role of 5’ Untranslated Region-Directed Small Molecules

Biomolecules ◽

10.3390/biom10101465 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1465

Author(s):

Catherine M. Cahill ◽

Rozaleen Aleyadeh ◽

Jin Gao ◽

Changning Wang ◽

Jack T. Rogers

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Alcohol Use ◽

Small Molecules ◽

Untranslated Region ◽

Alcohol Use Disorder ◽

Lewy Bodies ◽

Neuronal Cell ◽

Alpha Synuclein ◽

Divalent Metal Ions

Alpha-synuclein (α-Syn) is a 140-amino acid (aa) protein encoded by the Synuclein alpha SNCA gene. It is the synaptic protein associated with Parkinson’s disease (PD) and is the most highly expressed protein in the Lewy bodies associated with PD and other alpha synucleopathies, including Lewy body dementia (LBD) and multiple system atrophy (MSA). Iron deposits are present in the core of Lewy bodies, and there are reports suggesting that divalent metal ions including Cu2+ and Fe2+ enhance the aggregation of α-Syn. Differential expression of α-Syn is associated with alcohol use disorder (AUD), and specific genetic variants contribute to the risk for alcoholism, including alcohol craving. Spliced variants of α-Syn, leading to the expression of several shorter forms which are more prone to aggregation, are associated with both PD and AUD, and common transcript variants may be able to predict at-risk populations for some movement disorders or subtypes of PD, including secondary Parkinsonism. Both PD and AUD are associated with liver and brain iron dyshomeostasis. Research over the past decade has shown that α-Syn has iron import functions with an ability to oxidize the Fe3+ form of iron to Fe2+ to facilitate its entry into cells. Our prior research has identified an iron-responsive element (IRE) in the 5’ untranslated region (5’UTR) of α-Syn mRNA, and we have used the α-Syn 5’UTR to screen for small molecules that modulate its expression in the H4 neuronal cell line. These screens have led us to identify several interesting small molecules capable of both decreasing and increasing α-Syn expression and that may have the potential, together with the recently described mesenchymal stem cell therapies, to normalize α-Syn expression in different regions of the alcoholic and PD brain.

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Small Molecule Drugs Targeting Non-Coding RNAs as Treatments for Alzheimer’s Disease and Related Dementias

Genes ◽

10.3390/genes12122005 ◽

2021 ◽

Vol 12 (12) ◽

pp. 2005

Author(s):

Lien D. Nguyen ◽

Rachel K. Chau ◽

Anna M. Krichevsky

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Small Molecules ◽

Circular Rnas ◽

Cellular Functions ◽

Protein Coding ◽

Lead Compounds ◽

Associated Proteins ◽

The Central Nervous System ◽

High Throughput Screens

Despite the enormous burden of Alzheimer’s disease and related dementias (ADRD) on patients, caregivers, and society, only a few treatments with limited efficacy are currently available. While drug development conventionally focuses on disease-associated proteins, RNA has recently been shown to be druggable for therapeutic purposes as well. Approximately 70% of the human genome is transcribed into non-protein-coding RNAs (ncRNAs) such as microRNAs, long ncRNAs, and circular RNAs, which can adopt diverse structures and cellular functions. Many ncRNAs are specifically enriched in the central nervous system, and their dysregulation is implicated in ADRD pathogenesis, making them attractive therapeutic targets. In this review, we first detail why targeting ncRNAs with small molecules is a promising therapeutic strategy for ADRD. We then outline the process from discovery to validation of small molecules targeting ncRNAs in preclinical studies, with special emphasis on primary high-throughput screens for identifying lead compounds. Screening strategies for specific ncRNAs will also be included as examples. Key challenges—including selecting appropriate ncRNA targets, lack of specificity of small molecules, and general low success rate of neurological drugs and how they may be overcome—will be discussed throughout the review.

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