Miroslav Pohanka

Cholinesterases, a target of pharmacology and toxicology.

BACKGROUND Cholinesterases are a group of serine hydrolases that split the neurotransmitter acetylcholine (ACh) and terminate its action. Of the two types, butyrylcholinesterase and acetylcholinesterase (AChE), AChE plays the key role in ending cholinergic neurotransmission. Cholinesterase inhibitors are substances, either natural or man-made that interfere with the break-down of ACh and prolong its action. Hence their relevance to toxicology and pharmacology. METHODS AND RESULTS The present review summarizes current knowledge of the cholinesterases and their inhibition. Particular attention is paid to the toxicology and pharmacology of cholinesterase-related inhibitors such as nerve agents (e.g. sarin, soman, tabun, VX), pesticides (e.g. paraoxon, parathion, malathion, malaoxon, carbofuran), selected plants and fungal secondary metabolites (e.g. aflatoxins), drugs for Alzheimers disease (e.g. huperzine, metrifonate, tacrine, donepezil) and Myasthenia gravis (e.g. pyridostigmine) treatment and other compounds (propidium, ethidium, decamethonium). CONCLUSIONS The crucial role of the cholinesterases in neural transmission makes them a primary target of a large number of cholinesterase-inhibiting drugs and toxins. In pharmacology, this has relevance to the treatment of neurodegenerative disorders.

Alzheimer´s disease and oxidative stress: a review.

Alzheimer´s disease (AD) is a neurodegenerative disorder with no known cure and rapid rise in incidence. The predominant cognitive impairment is currently treated using cognitive enhancers like cholinesterase inhibitors. The two molecular hallmarks of AD are amyloid plaques created from an amyloid precursor protein and hyperphosphorylated tau protein that is deposited as neurofibrillary tangles inside neurons. A number of pathological mechanisms follow or precede these formations. Alteration in mitochondrial function and deposition of heavy metals are reported. The disease progression is enhanced by oxidative stress. However, the role of oxidative stress is not universally accepted. The current review covers and discusses the basic evidence and role of oxidative stress in AD development.

Alpha7 Nicotinic Acetylcholine Receptor Is a Target in Pharmacology and Toxicology

Alpha7 nicotinic acetylcholine receptor (α7 nAChR) is an important part of the cholinergic nerve system in the brain. Moreover, it is associated with a cholinergic anti-inflammatory pathway in the termination of the parasympathetic nervous system. Antagonists of α7 nAChR are a wide group represented by conotoxin and bungarotoxin. Even Alzheimer’s disease drug memantine acting as an antagonist in its side pathway belongs in this group. Agonists of α7 nAChR are suitable for treatment of multiple cognitive dysfunctions such as Alzheimer’s disease or schizophrenia. Inflammation or even sepsis can be ameliorated by the agonistic acting compounds. Preparations RG3487, SEN34625/WYE-103914, SEN12333, ABT-107, Clozapine, GTS-21, CNI-1493, and AR-R17779 are representative examples of the novel compounds with affinity toward the α7 nAChR. Pharmacological, toxicological, and medicinal significance of α7 nAChR are discussed throughout this paper.

Acetylcholinesterase inhibitors: a patent review (2008 – present)

Introduction: Both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are present in the body in large amounts. AChE is an important part of the cholinergic nervous system taking place in the central and peripheral nervous system. AChE is a target of several toxins such as insecticide carbofuran, nerve agents, sarin, soman, tabun and VX. Beside toxins, drugs for treatment of Alzheimers disease and myasthenia gravis, such as galantamine, donepezil, rivastigmine, tacrine, huperzine, pyridostigmine and neostigmine, are known. Areas covered: The review gives an overview of the importance of the cholinergic nervous system, the biochemistry of AChE and the role of AChE inhibitors. Current efforts to introduce potent drugs for Alzheimers disease therapy and reduce toxicity, while keeping the maximal pharmacological effect, are also discussed. Expert opinion: The current research effort into AChE inhibitors can be divided into two categories. First, new toxins useful for agricultural purposes and second, novel drugs that need to be prepared, although there is less interest in the new toxins. The research for drugs for Alzheimers disease needs to focus on inhibitors that reduce the deposition of amyloid plaques, but do not initiate AChE expression.

Progress of Biosensors Based on Cholinesterase Inhibition

Biosensors are available and applicable for detection and characterization of specific inhibitors of many enzymes. In this review, biosensors based on fixed acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) are presented. Inhibition of selected enzymes by various compounds, such as organophosphorus and carbamate pesticides, nerve agents (e.g. sarin or VX), and other natural toxins (e.g. aflatoxins), was employed to develop specific assays using biosensors only. Biosensor technology brings potential miniaturization and portability, when it is compared to standard methods. Construction of biosensors based on cholinesterases became a more important issue within the last decades. Novel approach with recombinant proteins, microelectrodes and immobilization protocol related to nanotechnologies opened new insight to the cholinesterase based biosensor construction and its perspective via routine assays. This review is focused on novel trends within such biosensors as a result of the known platform.

Synthesis and in vitro evaluation of N-alkyl-7-methoxytacrine hydrochlorides as potential cholinesterase inhibitors in Alzheimer disease

All approved drugs for Alzheimer disease (AD) in clinical practice ameliorate the symptoms of the disease. Among them, acetylcholinesterase inhibitors (AChEIs) are used to increase the cholinergic activity. Among new AChEI, tacrine compounds were found to be more toxic compared to 7-MEOTA (9-amino-7-methoxy-1,2,3,4-tetrahydroacridine). In this Letter, series of 7-MEOTA analogues (N-alkyl-7-methoxytacrine) were synthesized. Their inhibitory ability was evaluated on recombinant human acetylcholinesterase (AChE) and plasmatic human butyrylcholinesterase (BChE). Three novel compounds showed promising results towards hAChE better to THA or 7-MEOTA. Three compounds resulted as potent inhibitors of hBChE. The SAR findings highlighted the C(6)-C(7)N-alkyl chains for cholinesterase inhibition.

Improvement of acetylcholinesterase-based assay for organophosphates in way of identification by reactivators

Organophosphates present serious fulmination in several aspects of human life. Detection of organophosphates is frequently based on following acetylcholinesterase (AChE) inhibition. Although limit of detection and sensitivity for AChE-based assays seem to be intriguing, the identification of organophosphates is not currently efficient in this way. We introduce an improvement of AChE-based assay by reactivators using a selective come-back of AChE activity after previous inhibition. We have chosen four organophosphates: paraoxon-ethyl, paraoxon-methyl, trichlorfon, methamidophos as representative pesticides and the three most available reactivators: HI-6, obidoxime, pralidoxime. Reactivation was realized in the 96-wells photometric microplates and activity of human recombinant AChE was followed by reaction of Ellmans reagent with one of enzyme digestion product: thiocholine. Distinguishing of reactivation efficacy was judged by the independent two population t-test. The most significant identification was based on methamidophos inhibited AChE reactivation by HI-6 or pralidoxime and paraoxon-ethyl inhibited AChE by obidoxime; moreover, identification of trichlorfon and paraoxon-methyl was possible, too. Practical impact of described method is discussed.

Inhibitors of Acetylcholinesterase and Butyrylcholinesterase Meet Immunity

Acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of Alzheimer’s disease and other dementias. More recent use is for myasthenia gravis. Many of these inhibitors interact with the second known cholinesterase, butyrylcholinesterase (BChE). Further, evidence shows that acetylcholine plays a role in suppression of cytokine release through a “cholinergic anti-inflammatory pathway” which raises questions about the role of these inhibitors in the immune system. This review covers research and discussion of the role of the inhibitors in modulating the immune response using as examples the commonly available drugs, donepezil, galantamine, huperzine, neostigmine and pyridostigmine. Major attention is given to the cholinergic anti-inflammatory pathway, a well-described link between the central nervous system and terminal effector cells in the immune system.

Mycotoxin Assays Using Biosensor Technology: A Review

Toxic fungal metabolites—mycotoxins—cause poisonings after consumption of contaminated food commodities. The most probable intoxications are connected with eating poorly stored food or inhaling of moldy dust. One of the effective ways to protect people against mycotoxins is timely detection. Several methods such as affinity chromatography and enzyme-linked immunosorbent assay are commercially available for this purpose. Nevertheless, fast, sensitive, simple, portable, and low-cost devices are difficult to find. Application of biosensors appears to be a possible method to meet this need for mycotoxins assay.

Role of oxidative stress in infectious diseases. A review.

Oxidative stress plays a dual role in infections. Free radicals protect against invading microorganisms, and they can also cause tissue damage during the resulting inflammation. In the process of infection, there is generation of reactive species by myeloperoxidase, NADPH oxidase, and nitric oxide synthase. On the other hand, reactive species can be generated among others, by cytochrome P450, some metals, and xanthine oxidase. Some pathologies arising during infection can be attributed to oxidative stress and generation of reactive species in infection can even have fatal consequences. This article reviews the basic pathways in which reactive species can accumulate during infectious diseases and discusses the related health consequences.