Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

Cherry, Jonathan J.; Kobayashi, Dione; Lynes, Maureen M.; Naryshkin, Nikolai; Tiziano, Francesco Danilo; Zaworski, Phillip G.; Rubin, Lee L.; Jarecki, Jill

doi:10.1089/adt.2014.587

Cited by 34 publications

(32 citation statements)

References 224 publications

(291 reference statements)

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“…12 As with other SMN quantification methods, lysis reagents used in the ECLIA protocol have to be chosen carefully to ensure extraction and dissociation of both cytoplasmic and nuclear SMN. 16 In the protocol described here, a commercially available lysis buffer known to work well in the ECLIA setting (MSD) is used, slightly modified for SMN extraction. Compared to other extraction buffers used in preliminary trials, it yielded higher concentrations of SMN in relation to whole protein (data not shown), suggesting a more efficient extraction of nuclear SMN.…”

Section: Discussionmentioning

confidence: 99%

“…Second, age is an important factor to consider when assessing SMN levels in clinical specimens, as there is evidence of a general decline of SMN levels across tissue types and species, 16 and several studies using ECLIA to measure SMN levels in PBMCs of SMA patients, carriers, and controls have stressed the necessity to control for age when performing any statistical analysis. 13,19 Third, even during a short period of time, SMN levels measured in PBMCs fluctuate substantially within individuals; when assessed at five time points over the course of 1 month, blood SMN levels were shown to vary from 2 to 10 times in six different healthy adults.…”

Section: Discussionmentioning

confidence: 99%

“…15,16 For any drug discovery program based on that mode of action, a key prerequisite is the reliable and accurate quantification of SMN protein levels in various fluids, cells, and tissues. Moreover, as some of the preclinical drug candidates eventually enter clinical testing, these quantification methods also need to perform rapidly and on a high-throughput basis.…”

Section: Introductionmentioning

confidence: 99%

“…To meet these criteria, different SMN-specific enzyme-linked immunosorbent assays (ELISAs) have been developed during the past years that allow for both SMA drug screening and SMN measurement in human and mouse tissue samples, including peripheral blood mononuclear cells (PBMCs). [16][17][18][19] Although ELISAs can detect much lower protein quantities than classical western blotting (dynamic range of 50-3,200 pg/mL), 19 in terms of sensitivity they are outperformed by a recently developed detection method called electrochemiluminescence immunoassay (ECLIA) whose principle of function is described elsewhere. 20,21 The aim of this study was the development and verification of this high-throughput assay for simple and accurate quantification of SMN protein levels that allows a highly sensitive detection limited to 30 pg/mL.…”

Section: Introductionmentioning

confidence: 99%

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Detection of Survival Motor Neuron Protein in Buccal Cells Through Electrochemiluminescence-Based Assay

Steinkellner

Etzler²,

Gmeiner³

et al. 2015

ASSAY and Drug Development Technologies

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Spinal muscular atrophy (SMA) is a severe autosomal recessive disorder affecting one in every 10,000 live births. The disease is characterized by loss of alpha-motor neurons in the spinal cord that leads to progressive atrophy and weakness of limb and trunk muscles. This neuromuscular disorder results from deletions and/or mutations within the survival motor neuron 1 (SMN1) gene, leading to a pathologically decreased expression of functional full-length SMN protein. Here we report on the investigation to measure SMN protein levels through electrochemiluminescence immunoassay (ECLIA). This simple assay is a highly quantitative method able to measure SMN protein levels in human, mouse, and rat samples throughout a wide working range with low intra- and interassay error. The sensitivity for human SMN is 30 pg/mL and provides a new tool for the set up of high-throughput screening for basic research. Moreover, we describe a novel tool for a noninvasive assessment of SMN in buccal cells derived from healthy donors, SMA carriers, and SMA patients. The availability of a validated quantitative ECLIA should improve the investigation of novel compounds for the treatment of SMA.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Detection of Survival Motor Neuron Protein in Buccal Cells Through Electrochemiluminescence-Based Assay

Steinkellner

Etzler²,

Gmeiner³

et al. 2015

ASSAY and Drug Development Technologies

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“…Proces opracowaniu koncepcji działania cząsteczki poprzez proces badań przedklinicznych i klinicznych czas do opracowania leku i wprowadzenia go na rynek jest długi, złożony i trwa zwykle dłużej niż dekadę [32]. Na rynek trafia zaledwie kilka (1-2%) opracowywanych substancji.…”

Section: Próby Terapeutyczneunclassified

Spinal muscular atrophy – novel therapies, new challenges

Jędrzejowska¹,

Kostera‐Pruszczyk²

2017

Child Neurology

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STRESZCZENIERdzeniowy zanik mięśni jest postępującą chorobą neurodegeneracyjną, w przebiegu której dochodzi do utraty motoneuronów rdzenia kręgowego, a w konsekwencji osłabienia i zaniku mięśni. Choroba charakteryzuje się bardzo dużą zmiennością przebiegu klinicznego -od letalnej formy wrodzonej po postać dorosłą, o średniej przeżycia jak w populacji ogólnej. Choroba związana jest z mutacjami genu SMN1. Liczba kopii bliźniaczo podobnego genu SMN2 warunkuje nasilenie objawów klinicznych. Analizy ostatnich lat zaowocowały weryfikacją aktualnej klasyfikacji, poprawą standardów opieki, poznaniem przebiegu historii naturalnej choroby. W zderzeniu z nowymi badaniami nad podłożem molekularnym i patogenezą choroby możliwe stało się podjęcie prób terapeutycznych. Badania kliniczne wykazały skuteczność nusinersenu (anysensowny oligonukleotyd), i doprowadziły do rejestracji leku do leczenia SMA. Najlepsze efekty uzyskano u pacjentów, u których włączono leczenie w okresie przedobjawowym. Trwają badania nad kolejnymi substancjami, których działanie oparte jest o różnorodne strategie-począwszy od terapii genowej (AVXS101), przez mikromolekuły modyfikujące składanie pre-mRNA SMN2 (LMI070, RG7916), po neuroprotektory (Olesoxime) i wiele innych. W pracy dokonujemy przeglądu najnowszych danych dotyczą-cych klasyfikacji, historii naturalnej, standardów opieki i prób terapeutycznych. Wczesne rozpoznanie i leczenie może zmienić historię naturalną choroby. Dlatego tak ważne wydaje się wczesne rozpoznanie, a w przyszłości być może wprowadzenie skriningu noworodkowego. Słowa kluczowe: SMA 5q, białko SMN, terapia. ABSTRACTSpinal muscle atrophy (SMA) is a progressive neurodegenerative disease that results in the loss of motoneurons in the spinal cord and, consequently, in muscle weakness and atrophy. The disease is characterized by high variability of the clinical course -from the early neonatal form to the adult form, with the average survival as in the general population. The disease is caused by mutations in the SMN1 gene. The number of copies of a similar SMN2 gene determines the severity of clinical symptoms. Recent analyzes allowed to verify current classification, improve care standards, learn the natural history of SMA. Clinical studies showed the efficacy of nusinersen (an antysense oligonucleotide) and led to the registration of the drug for SMA treatment. The best results were seen in presymptomatic patients. Research is underway on substances that are based on a variety of strategies, from gene therapy (AVXS101) to SMN2 pre-mRNA modulation micronucleus (LMI070, RG7916) to neuroprotectants (Olesoxime) and many others. We review the latest data on classification, natural history, care standards and therapeutic trials. Early diagnosis and treatment can change the natural history of the disease. That is why it is so important to have an early diagnosis and, perhaps, a neonatal screening in the future.

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Fluorescent Turn‐On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap‐Recognizing Proteins

Kasprzyk

Starek

Ciechanowicz

et al. 2019

Chemistry A European J

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The m 7 Gc ap is au nique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specificallyinteracts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecularp robest os tudy important cap-recognizing proteins, we synthesized m 7 Gn ucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymaticc leavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. Ap yrene-labeled m 7 GTP analogue showed up to eightfold enhanced fluorescencee mission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-folde nhancementu pon cleavageb yd ecapping scavenger (DcpS) enzyme. These observations serveda st he basis for developing binding-and hydrolytic-activity assays. The assay utility was validated with previously characterizedl ibraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also appliedtostudy hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.

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Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

Cited by 34 publications

References 224 publications

Detection of Survival Motor Neuron Protein in Buccal Cells Through Electrochemiluminescence-Based Assay

Detection of Survival Motor Neuron Protein in Buccal Cells Through Electrochemiluminescence-Based Assay

Spinal muscular atrophy – novel therapies, new challenges

Fluorescent Turn‐On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap‐Recognizing Proteins

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