Design, synthesis and evaluation of novel cholinesterase inhibitors

Alzheimerjeva bolezen je kronična progresivna nevrodegenerativna bolezen možganov. Vzroki za nastanek te bolezni še niso znani, je pa znano, da kopičenje amiloidni proteina β, proteina tau in oksidativni stres v možganih povzročijo odmiranje nevronov in propadanje sinaps. Ta nevrodegeneracija je najizrazitejša v holinergičnem živčno-prenašalnem sistemu. Posledica tega je zmanjšana koncentracija živčnega prenašalca acetilholina v možganih, kar povzroči motnje spomina, ki so značilne za bolnike z Alzheimerjevo boleznijo. Trenutno zdravljenje ali preprečevanje te bolezni ni mogoče in bolnikom so na voljo le štiri učinkovine za lajšanje simptomov. Holin-esterazi acetilholin-esteraza in butirilholin-esteraza prekineta holinergični živčni prenos v možganih tako, da katalizirata hidrolizo acetilholina. V možganih zdravih odraslih je acetilholin-esteraza odgovorna za 80%, butirilholin-esteraza pa za 20% holin-esterazne encimske aktivnosti. Z napredovanjem bolezni se encimska aktivnost acetilholin-esteraze zmanjšuje, butirilholin-esteraze pa povečuje. Tri od štirih učinkovin za lajšanje simptomov Alzheimerjeve bolezni so zaviralci holin-esteraz: selektivna zaviralca acetilholin-esteraze donepezil in galantamin ter zaviralec acetilholin-esteraze in butirilholin-esteraze rivastigmin. Te učinkovine imajo, zaradi zaviranja acetilholin-esteraze, številne slabosti (neželeni učinki zaradi zaviranja acetilholin-esteraze v perifernem živčnem sistemu in s tem povezan omejen odmerek ter neučinkovitost v poznih stadijih bolezni), katerim se da izogniti s selektivnim zaviranjem butirilholin-esteraze. Načrtovali, sintetizirali in ovrednotili smo obsežno serijo novih selektivnih reverzibilnih zaviralcev butirilholin-esteraze: sulfonamidne in N-propargilpiperidinske derivate spojine zadetka 1 (IC50 = 21,3 nM) in določili odnos med njihovo strukturo in delovanjem. Za sintezo ključih intermediatov v sintezni poti do načrtovanih zaviralcev smo razvili splošni in enostavni sintezni postopek. Sulfonamidni derivat 2 (IC50 = 4,9 nM) je obetavna spojini za nadaljnji predklinični razvoj. Spojina ni citotoksična in ščiti živčne celice pred strupenimi amiloidnega proteina β. Zavira butirilholin-esterazo v rezinah podganjih možganov in prehaja v možgane podgan po intraperitonealni aplikaciji. Dokazali smo, da spojina 2 izboljša spomin, kognitivne funkcije in učne sposobnosti miši v bolezenskem modelu zmanjšane holinergične aktivnosti pri Alzheimerjevi bolezni. V možganih bolnikov je povišana encimska aktivnost monoaminske oksidaze B. Produkti, ki nastanejo pri reakciji, ki jo katalizira ta encim, prispevajo k oksidativnemu stresu in nevrodegeneraciji pri Alzheimerjevi bolezni. Trije N-propargilpiperidnski derivati spojine zadetka 1 poleg butirilholin-esteraze zavirajo tudi monoaminsko oksidazo B. Zaviralec obeh encimov z nevroprotektivnim delovanjem (N-propargilpiperidin 6) je spojina vodnica za načrtovanje novih spojin z delovanjem na več tarč. V doktorski disertaciji predstavljamo, da smo s sistematičnimi spremembami strukture spojine zadetka 1 prišli do (i) obetavne spojine za nadaljni predklinični razvoj, (ii) štirih kristalnih struktur kompleksov butirilholin-esteraz z zaviralci, (iii) ortogonalno zaščitenih intermediatov za sintezo novih zaviralcev in (iv) spojine vodnice kot izhodišča za načrtovanje novih spojin z delovanjem na več tarč.Alzheimer\u27s disease is a chronic progressive neurodegenerative brain disorder. The etiology of this disease is not entirely understood, although it is known that accumulation of amyloid β peptide, protein tau and oxidative stress in the brain cause neuronal death and synaptic loss. This neurodegeneration is most severe in the cholinergic neurotransmitter system and results in a decrease in the levels of the neurotransmitter acetylcholine, which in turn produces memory deficits, characteristic for patients with Alzheimer\u27s disease. Currently, there are no means to cure or prevent Alzheimer\u27s disease, and only four drugs are available to patients for alleviating its symptoms. Cholinesterases acetylcholinesterase and butyrycholinesterase terminate cholinergic neurotransmission in the brain by catalyzing the hydrolysis of acetylcholine. In the healthy brain, acetylcholinesterase accounts for 80% and butyrycholinesterase accounts for 20% of the cholinesterase enzymatic activity. As the disease progresses, enzymatic activity of acetylcholinesterase is reduced, while that of butyrycholinesterase increases. Three out of the four approved drugs for alleviating symptoms of patients with AD are cholinesterase inhibitors: the selective acetycholinesterase inhibitors donepezil and galantamine, and the pseudo-irreversible acetylcholinesterase and butyrycholinesterase inhibitor rivastigmine. These drugs have numerous limitations due to inhibition of acetycholinesterase (adverse side effects due to acetylcholinesterase inhibition in the peripheral nervous system, limited dosing and limited clinical efficacy in advanced stages of the disease) which can be overcome by selectively inhibiting butyrylcholinesterase. We have designed, synthesized and evaluated a comprehensive series of new selective reversible butyrycholinesterase inhibitors: sulfonamide and N-propargypiperidine derivatives of hit compound 1 (IC50 = 21.3 nM) and determined their structure–activity relationships. For preparing key intermediates in the synthesis of designed compounds, we developed a general and simple synthetic procedure. Sulfonamide derivative 2 (IC50 = 4.9 nM) is promising candidate for further preclinical development. This compound is noncytotoxic and protects neuronal cells from toxic amyloid β peptide species. Its inhibits butyrycholinesterase in rat brain slices and partitions into the brains of rats after intraperitoneal injection. Compound 2 and improves memory, cognitive functions, and learning abilities of mice in a disease model of the cholinergic hypofunction in Alzheimer\u27s disease. In the brains of patients with Alzheimer\u27s disease the enzymatic activity of monoamine oxidase B is increased and products formed in the reaction catalyzed by this enzyme contribute to the neurodegeneration. We developed three N-propargypiperidines derivatives of hit compound 1 that inhibit both butyrycholinesterase and monoamine oxidase B. N-propargylpiperidine 6 also has neuroprotective activity and therefore represents a lead compound for design of multitarget-directed ligands against Alzheimer’s disease. In this thesis, we describe the use of structural modifications of hit compound 1 to obtain (i) a promising candidate for further preclinical development, (ii) four crystal structures of complexes of butyrycholinesterase with its inhibitors, (iii) orthogonally protected intermediates for synthesis of new cholinesterase inhibitors, and (iv) lead compounds for design of new multitarget-directed ligands

Bacterial adhesion on prosthetic and orthotic material surfaces

Prosthetic and orthotic parts, such as prosthetic socket and inner sides of orthoses, are often in contact with human skin, giving bacteria the capability to adhere and form biofilms on the materials of those parts which can further cause infections. The purpose of this study was to determine the extent of bacterial adhesion of Staphylococcus aureus and Staphylococcus epidermidis on twelve different prosthetic and orthotic material surfaces and how roughness, hydrophobicity, and surface charge of this materials affect the adhesion. The roughness, contact angle, zeta potential of material surfaces, and adhesion rate of Staphylococcus aureus and Staphylococcus epidermidis were measured on all twelve prosthetic and orthotic materials, i.e., poly(methyl methacrylate), thermoplastic elastomer, three types of ethylene polyvinyl acetates (pure, with low-density polyethylene and with silver nanoparticles), silicone, closed-cell polyethylene foams with and without nanoparticles, thermo and natural cork, and artificial and natural leather. The greatest degree of adhesion was measured on both closed-cell polyethylene foams, followed by artificial thermo cork and leather. The lowest adhesion extent was observed on ethylene-vinyl acetate. The bacterial adhesion extent increases with the increasing surface roughness. Smaller deviations of this rule are the result of the surface’s hydrophobicity and charge

Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer\u27s disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds

Synthesis and initial characterization of a reversible, selective 18F-labeled radiotracer for human butyrylcholinesterase

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 A(β) 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[(18)F]. IC(50) 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 IC(50) 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 (K(2) = 68.0 nM) in comparison to the free enzyme (K(1) = 32.9 nM). CONCLUSIONS: The reversibly acting, selective radiotracer is synthetically easily accessible and retains promising activity and binding potential on hBChE. Radiosynthesis with (18)F labeling of tosylates was feasible in a reasonable time frame and good radiochemical yield

Discovery, biological evaluation, and crystal structure of a novel nanomolar selective butyrylcholinesterase inhibitor.

International audienceButyrylcholinesterase (BChE) is regarded as a promising drug target as its levels and activity significantly increase in the late stages of Alzheimer's disease. To discover novel BChE inhibitors, we used a hierarchical virtual screening protocol followed by biochemical evaluation of 40 highest scoring hit compounds. Three of the compounds identified showed significant inhibitory activities against BChE. The most potent, compound 1 (IC50 = 21.3 nM), was resynthesized and resolved into its pure enantiomers. A high degree of stereoselective activity was revealed, and a dissociation constant of 2.7 nM was determined for the most potent stereoisomer (+)-1. The crystal structure of human BChE in complex with compound (+)-1 was solved, revealing the binding mode and providing clues for potential optimization. Additionally, compound 1 inhibited amyloid β(1-42) peptide self-induced aggregation into fibrils (by 61.7% at 10 μM) and protected cultured SH-SY5Y cells against amyloid-β-induced toxicity. These data suggest that compound 1 represents a promising candidate for hit-to-lead follow-up in the drug-discovery process against Alzheimer's disease

N-Propargylpiperidines with naphthalene-2-carboxamide or naphthalene-2-sulfonamide moieties: Potential multifunctional anti-Alzheimer's agents.

International audienceIn the brains of patients with Alzheimer's disease, the enzymatic activities of butyrylcholinesterase (BChE) and monoamine oxidase B (MAO-B) are increased. While BChE is a viable therapeutic target for alleviation of symptoms caused by cholinergic hypofunction, MAO-B is a potential therapeutic target for prevention of neurodegeneration in Alzheimer's disease. Starting with piperidine-based selective human (h)BChE inhibitors and propargylamine-based MAO inhibitors, we have designed, synthesized and biochemically evaluated a series of N-propargylpiperidines. All of these compounds inhibited hBChE with good selectivity over the related enzyme, acetylcholinesterase, and crossed the blood-brain barrier in a parallel artificial membrane permeation assay. The crystal structure of one of the inhibitors (compound 3) in complex with hBChE revealed its binding mode. Three compounds (4, 5, 6) showed concomitant inhibition of MAO-B. Additionally, the most potent hBChE inhibitor 7 and dual BChE and MAO-B inhibitor 6 were non-cytotoxic and protected neuronal SH-SY5Y cells from toxic amyloid β-peptide species

Treatment of canine cognitive dysfunction with novel butyrylcholinesterase inhibitor

Canine cognitive dysfunction (CCD) is common in aged dogs and has many similarities with Alzheimer’s disease. Unfortunately, like Alzheimer’s disease, CCD cannot be cured. In the present study, we treated dogs with CCD with our newly developed and characterized butyrylcholinesterase inhibitor (BChEi). Seventeen dogs were randomized into two groups (treated with BChEi and untreated) and followed for 6 months at regular check-ups. The dogs’ cognitive status was determined by a Canine Dementia Scale (CADES) questionnaire and two cognitive tests. In dogs with moderate cognitive impairment, treatment caused significant improvement in the clinical rating of cognitive abilities and the performance-based tests of cognitive functioning when compared to the untreated group (p < 0.001). Dogs treated with BChEi showed markedly improved cognitive function with enhanced quality of life. No side effects were observed in the treated dogs with moderate cognitive impairment. According to the results of this preliminary study, there is an indication that novel BChEi may be a promising drug for the treatment of CCD in dogs and may be an interesting candidate for the treatment of Alzheimer\u27s disease in humans. However, further clinical studies are needed to confirm this

The Magic of Crystal Structure-Based Inhibitor Optimization: Development of a Butyrylcholinesterase Inhibitor with Picomolar Affinity and in Vivo Activity

International audienceThe enzymatic activity of butyrylcholinesterase (BChE) in the brain increases with the progression of Alzheimer's disease, thus classifying BChE as a promising drug target in advanced Alzheimer's disease. We used structure-based drug discovery approaches to develop potent, selective, and reversible human BChE inhibitors. The most potent, compound 3, had a picomolar inhibition constant versus BChE due to strong cation-π interactions, as revealed by the solved crystal structure of its complex with human BChE. Additionally, compound 3 inhibits BChE ex vivo and is noncytotoxic. In vitro pharmacokinetic experiments show that compound 3 is highly protein bound, highly permeable, and metabolically stable. Finally, compound 3 crosses the blood-brain barrier, and it improves memory, cognitive functions, and learning abilities of mice in a scopolamine model of dementia. Compound 3 is thus a promising advanced lead compound for the development of drugs for alleviating symptoms of cholinergic hypofunction in patients with advanced Alzheimer's disease