Imaging Butyrylcholinesterase in Multiple Sclerosis

Thorne, Michael; Cash, Meghan K.; Reid, G. Andrew; Burley, Darcy E.; Luke, David Fasholé; Pottie, Ian R.; Darvesh, Sultan

doi:10.1007/s11307-020-01540-6

Cited by 15 publications

(11 citation statements)

References 52 publications

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“…30 Moreover, AChE and BChE can be used as biomarkers for the detection of Alzheimer's disease 1,31,32 , Hirschsprung's disease 33,34 , and multiple sclerosis. 35,36 To date, only a few AChE [37][38][39][40][41][42] and BChE [43][44][45][45][46][47] fluorescent probes and histochemical methods 48 enable tissue ChEs location and activity determination. Accordingly, the development of new chemical probes for investigating the roles of ChEs in physiological and pathophysiological processes in vitro and ex vivo is required by biologists and neurobiologists.…”

Section: Introductionmentioning

confidence: 99%

Pseudo-irreversible butyrylcholinesterase inhibitors: SAR, kinetic, computational, and crystallographic study of the N-dialkyl O-arylcarbamate warhead

Meden

Knez

Brazzolotto³

et al. 2022

Preprint

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Beside reversible butyrylcholinesterse inhibitors (BChEIs), a plethora of covalent ones, typically pseudo-irreversible carbamates, have been reported in literature. For the latter, however, in most cases the proper confirmation of their covalent mode of action is lacking. Additionally, the available reports on the structure-activity relationships of the O-arylcarbamate warhead are incomplete. Therefore, a follow-up on a series of pseudo-irreversible covalent carbamate human butyrylcholinesterase inhibitors (hBChEIs) and the structure-activity relationships of the N-dialkyl O-arylcarbamate warhead is presented. The covalent mechanism of binding was tested by IC50 time-dependency profiles, and sequentially and increasingly confirmed by kinetic analysis, whole protein LC-MS, and crystallographic evidence. The computational studies provided valuable insights into the steric constraints and identified problematic, bulky carbamate warheads that could not reach and carbamoylate the catalytic Ser198. QM calculations lent further evidence that the steric effects seemed to be a key factor in determining the covalent binding behaviour of these carbamate ChEIs and their duration of action. Furthermore, the introduction of a clickable terminal alkyne moiety into one of the carbamate N-substituents and in situ derivatization with an azide-containing fluorophore enabled fluorescent labelling of plasma hBChE. This proof-of-concept study highlighted the potential of this novel approach and these compounds to be further developed as clickable molecular probes for investigating tissue localization and activity of ChEs.

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Section: Introductionmentioning

confidence: 99%

Pseudo-irreversible butyrylcholinesterase inhibitors: SAR, kinetic, computational, and crystallographic study of the N-dialkyl O-arylcarbamate warhead

Meden

Knez

Brazzolotto³

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…30 Moreover, AChE and BChE can be used as biomarkers for detection of Alzheimer's disease 1,31,32 , Hirschsprung's disease 33,34 and multiple sclerosis. 35,36 To date, only a few AChE [37][38][39][40][41][42] and BChE [43][44][45][45][46][47] fluorescent probes and histochemical methods 48 have enabled tissue ChE location and activity determination. Accordingly, the development of new chemical probes for investigating the roles of ChEs in physiological and pathophysiological processes in vitro and ex vivo is required by biologists and neurobiologists.…”

Section: Introductionmentioning

confidence: 99%

Pseudo-irreversible butyrylcholinesterase inhibitors: structure–activity relationships, and kinetic, computational, and crystallographic study of the N-dialkyl O-arylcarbamate warhead

Meden

Knez

Brazzolotto³

et al. 2022

Preprint

View full text Add to dashboard Cite

Alongside reversible butyrylcholinesterase inhibitors, a plethora of covalent butyrylcholinesterase inhibitors have been reported in the literature, typically pseudo-irreversible carbamates. For these latter, however, most cases lack full confirmation of their covalent mode of action. Additionally, the available reports regarding the structure–activity relationships of the O-arylcarbamate warhead are incomplete. Therefore, a follow-up on a series of pseudo-irreversible covalent carbamate human butyrylcholinesterase inhibitors and the structure–activity relationships of the N-dialkyl O-arylcarbamate warhead are presented in this study. The covalent mechanism of binding was tested by IC50 time-dependency profiles, and sequentially and increasingly confirmed by kinetic analysis, whole protein LC-MS, and crystallographic analysis. Computational studies provided valuable insights into steric constraints and identified problematic, bulky carbamate warheads that cannot reach and carbamoylate the catalytic Ser198. Quantum mechanical calculations provided further evidence that steric effects appear to be a key factor in determining the covalent binding behaviour of these carbamate cholinesterase inhibitors and their duration of action. Additionally, the introduction of a clickable terminal alkyne moiety into one of the carbamate N-substituents and in situ derivatisation with azide-containing fluorophore enabled fluorescent labelling of plasma human butyrylcholinesterase. This proof-of-concept study highlights the potential of this novel approach and for these compounds to be further developed as clickable molecular probes for investigating tissue localisation and activity of cholinesterases.

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“…This could also be shown for the pseudoirreversible BChE inhibitor phenyl 4- 123 I-iodophenylcarbamate ( 123 I-PIP), which accumulated specifically in Aβ plaques with ChE activity in human AD brain tissue [ 29 ]. Additionally, this tracer has been investigated very recently as a potential diagnostic and treatment monitoring tool for BChE activity changes in multiple sclerosis showing promising preliminary results [ 30 ]. Regarding irreversible and highly selective inhibitors of BChE as radiotracers, carbamate-based inhibitors were investigated that transfer the radiolabeled moiety onto the enzyme, where it is covalently bound.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Initial Characterization of a Reversible, Selective 18F-Labeled Radiotracer for Human Butyrylcholinesterase

et al. 2021

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Purpose A neuropathological hallmark of Alzheimer’s disease (AD) is the presence of amyloid-β (Aβ) plaques in the brain, which are observed in a significant number of cognitively normal, older adults as well. In AD, butyrylcholinesterase (BChE) becomes associated with Aβ aggregates, making it a promising target for imaging probes to support diagnosis of AD. In this study, we present the synthesis, radiochemistry, in vitro and preliminary ex and in vivo investigations of a selective, reversible BChE inhibitor as PET-tracer for evaluation as an AD diagnostic. Procedures Radiolabeling of the inhibitor was achieved by fluorination of a respective tosylated precursor using K[18F]. IC50 values of the fluorinated compound were obtained in a colorimetric assay using recombinant, human (h) BChE. Dissociation constants were determined by measuring hBChE activity in the presence of different concentrations of inhibitor. Results Radiofluorination of the tosylate precursor gave the desired radiotracer in an average radiochemical yield of 20 ± 3 %. Identity and > 95.5 % radiochemical purity were confirmed by HPLC and TLC autoradiography. The inhibitory potency determined in Ellman’s assay gave an IC50 value of 118.3 ± 19.6 nM. Dissociation constants measured in kinetic experiments revealed lower affinity of the inhibitor for binding to the acylated enzyme (K2 = 68.0 nM) in comparison to the free enzyme (K1 = 32.9 nM). Conclusions The reversibly acting, selective radiotracer is synthetically easily accessible and retains promising activity and binding potential on hBChE. Radiosynthesis with 18F labeling of tosylates was feasible in a reasonable time frame and good radiochemical yield.

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Imaging Butyrylcholinesterase in Multiple Sclerosis

Cited by 15 publications

References 52 publications

Pseudo-irreversible butyrylcholinesterase inhibitors: SAR, kinetic, computational, and crystallographic study of the N-dialkyl O-arylcarbamate warhead

Pseudo-irreversible butyrylcholinesterase inhibitors: SAR, kinetic, computational, and crystallographic study of the N-dialkyl O-arylcarbamate warhead

Pseudo-irreversible butyrylcholinesterase inhibitors: structure–activity relationships, and kinetic, computational, and crystallographic study of the N-dialkyl O-arylcarbamate warhead

Synthesis and Initial Characterization of a Reversible, Selective 18F-Labeled Radiotracer for Human Butyrylcholinesterase

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