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Home , Acetylcholinesterase, Aldicarb, Chlorine, Chlorosarin, Chlorosoman, Chlorpyrifos

Review biomolecules : The “Hub” for ReviewNeurodegenerative Diseases and Chemical Weapons Acetylcholinesterase: The “Hub” for Convention Neurodegenerative Diseases and Chemical WeaponsSamir F. de A. Cavalcante Convention 1,2,3,*, Alessandro B. C. Simas 2,*, Marcos C. Barcellos 1, Victor G. M. de Oliveira 1, Roberto B. Sousa 1, Paulo A. de M. Cabral 1 and Kamil Kuča 3,*and Tanos C. C. França 3,4,* Samir F. de A. Cavalcante 1,2,3,* , Alessandro B. C. Simas 2,*, Marcos C. Barcellos 1, Victor1 Institute G. M. ofde Chemical, Oliveira Biological,1, Roberto Radiological B. Sousa and1, Paulo Nuclear A. Defense de M. Cabral (IDQBRN),1, Kamil Brazilian Kuˇca Army3,* and TanosTechnological C. C. França Center3,4,* (CTEx), Avenida das Américas 28705, Rio de Janeiro 23020-470, Brazil; [email protected] (M.C.B.); [email protected] (V.G.M.d.O.); [email protected] 1 Institute of Chemical, Biological, Radiological and Nuclear Defense (IDQBRN), Brazilian Army (R.B.S.); [email protected] (P.A.d.M.C.) Technological Center (CTEx), Avenida das Américas 28705, Rio de Janeiro 23020-470, Brazil; 2 [email protected] Mors Institute of Research (M.C.B.); on Natural [email protected] Products (IPPN), Federal (V.G.M.d.O.); University of Rio de Janeiro (UFRJ), CCS,[email protected] Bloco H, Rio de Janeiro (R.B.S.); 21941-902, [email protected] Brazil (P.A.d.M.C.) 32 DepartmentWalter Mors of Institute , of Research Faculty of on Science, Natural Un Productsiversity (IPPN), of Hradec Federal Králové, University Rokitanskeho of Rio de 62, Janeiro 50003 (UFRJ), HradecCCS, Bloco Králové, H, Rio Czech de JaneiroRepublic 21941-902, Brazil 43 LaboratoryDepartment of ofMolecular Chemistry, Modelling Faculty ofApplied Science, to UniversityChemical and of Hradec Biological Krá lovDefenseé, Rokitanskeho (LMACBD), 62, Military Institute50003 Hradec of Engineering Králové, (IME), Czech Praça Republic General Tibúrcio 80, Rio de Janeiro 22290-270, Brazil *4 Correspondence:Laboratory of Molecular [email protected] Modelling Applied (S to.F.d.A.C.); Chemical [email protected] and Biological Defense (A.B.C.S.); (LMACBD), [email protected] Institute of(K.K.); Engineering [email protected] (IME), Praça General (T.C.C.F.); Tibúrcio Tel. 80, +55-21-998-419-561 Rio de Janeiro 22290-270, (S.F.d.A.C.); Brazil +55-21- * 998-744-886Correspondence: (A.B.C.S.); [email protected] +420-603-289-166 (K.K.) (S.F.d.A.C.); [email protected] (A.B.C.S.); [email protected] (K.K.); [email protected] (T.C.C.F.); Tel.: +55-21-998-419-561 (S.F.d.A.C.); Received:+55-21-998-744-886 16 February 2020; (A.B.C.S.); Accepted:+420-603-289-166 3 March 2020; (K.K.) Published: 7 March 2020  Abstract:Received: 16This February article 2020; Accepted:describes 3 Marchacetylcholinesterase 2020; Published: 7 March(AChE), 2020 an involved in parasympathetic , its activity, and how its inhibition can be pharmacologically Abstract:useful for Thistreating article , describes caused acetylcholinesterase by Alzheimer’s (AChE), disease, an enzymeor as a involvedwarfare method in parasympathetic due to the actionneurotransmission, of nerve agents. its activity, The chemical and how concepts its inhibition related can to bethe pharmacologically irreversible inhibition useful of for AChE, treating its reactivation,dementia, caused and byaging Alzheimer’s are discussed, disease, along or as awith warfare a relationship method due to to the actioncurrent of international nerve agents. legislationThe chemical on conceptschemical relatedweapons. to the irreversible inhibition of AChE, its reactivation, and aging are discussed, along with a relationship to the current international legislation on chemical weapons. Keywords: acetylcholinesterase; Alzheimer’s disease; nerve agents; Chemical Weapons Convention Keywords: acetylcholinesterase; Alzheimer’s disease; nerve agents; Chemical Weapons Convention

1. Introduction 1. Introduction The use of chemical weapons (CW) is quite common throughout history. Over time, the use of incendiaries,The use darts, of chemical spears, weapons and arrows (CW) impregnated is quite common with throughout from history.different Over organisms, time, the animals, use of andincendiaries, plants, the darts, lethality spears, of andsubstances arrows impregnatedused for warfare with have poisons been from dramatically different organisms,improved. animals,Despite internationaland plants, the agreements lethality of already substances in existence used for by warfare the end have of 19th been century, dramatically these improved.did not inhibit Despite the large-scaleinternational use agreements of CW during already World in existence War I (WWI) by the (1914–1918), end of 19th when century, millions these didof casualties not inhibit were the provokedlarge-scale mainly use of by CW , during World , War and, I (WWI) particularly, (1914–1918), when mustard millions (HD of1) (Figure casualties 1). wereThis compound,provoked mainly due to byits phosgene,physicochemical chlorine, characteristic and, particularly,s, is known sulfur as “area mustard denial (HD weapon”,1) (Figure precluding1). This furthercompound, access due to tothe its place physicochemical where it has characteristics,been deployed is[1]. known as “area denial weapon”, precluding further access to the place where it has been deployed [1].

S Cl Cl

1

Biomolecules 2020, 10, x; doi: Figure 1. Sulfur mustard HD1. www.mdpi.com/journal/biomolecules

Biomolecules 2020, 10, 414; doi:10.3390/biom10030414 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, x 2 of 21 Biomolecules 2020, 10, 414 2 of 22

Figure 1. Sulfur mustard HD1. After WWI and more actively during WWII, while looking for new , pesticides, German scientists developed a a series series of of pentavalent pentavalent phosphorus compounds compounds that that contained contained a leaving a leaving group such suchas a .as a halogen. They Theydiscovered discovered that thatthese these compounds compounds were were very very toxic, toxic, and and capable capable of of inhibiting acetylcholinesterase (AChE). (AChE). These These findings findings resulted resulted in in the the weaponization weaponization of of the the first first nerve nerve agents, agents, 2 3 4 called also G-agents, which included 2 (GB), 3 (GD), and 4 (GA) (Figure2 2).). SuchSuch CW were produced and stockpiled, but never used during WWIIWWII. [ 1[1–11]–11].

Figure 2. G-Agents. Figure 2. G-Agents. Later, during the 1950s, aiming to produce more effective effective pesticides, groups in the former Soviet Union, Great Britain, Sweden, and the United United St Statesates developed developed other other organophosphorus organophosphorus compounds which were more toxic and persistentpersistent (less volatile)volatile) than G-agents. These new chemicals were then named V-agents, whichwhich areare phosphonothioatesphosphonothioates ( 5(5and and6 ,6 Figure, Figure3)[ 3)4 [4,12–15].,12–15]. Like Like sulfur sulfur mustard mustard HD HD1, 1these, these compounds compounds can can be be similarly similarly regarded regarded as as “area “area denial denial weapons” weapons” [2, 8[2,8].].

Figure 3. V-Agents.V-Agents.

Although they were not used used in in the the World World Wars Wars,, the international community could soon testify testify their potential potential as Weapons Weapons of Mass Destruction (W (WMD)MD) when, in the 1980s, Saddam Hussein’s troops used chemical artillery containing G-agents, GB 2 and GA 4, along along with with sulfur sulfur mustard mustard HD 1, against civilian and military targets during the Iran– war. The tragic events in the Kurdish Kurdish city of Halabja Halabja show how abhorrent can be the use of these substanc substanceses as warfare agents [16–18]. [16–18]. In In 1994 1994 and and 1995, 1995, Japan waswas strickenstricken byby two two episodes episodes when when GB GB2 2was was employed employed in in the the terrorist terrorist actions actions carried carried out out by theby thesect sectAum Aum Shinrikyo Shinrikyo, provoking, provoking casualties casualties among among civilians civilians and and first first responders responders [4,19 [4,19–22].–22]. Although manymany papers papers have have dealt deal witht with chemical warfare over theover years, the theyears, use the of CW use is of currently CW is currentlya pressing a subject.pressing Recently,subject. Rece chemicalntly, chemical warfare agentswarfare have agents made have headlines made headlines due to theirdue to alleged their allegeddeployment deployment in the Civil in the War Civil ongoing War ongoing in Syria in (2011–) Syria and(2011–) as meansand as ofmeans murdering of murdering the North the Korean North Koreannational national Kim Jun Kim Nam Jun at theNam Kuala at the Lumpur Kuala InternationalLumpur International Airport in Airport 2017. A in report 2017. from A report Malaysian from Malaysianauthorities authorities confirmed thatconfirmed VX was that used VX by was the used perpetrators by the perpetrators [4,23–42]. [4,23–42]. In 2018, events events that that occurred occurred in in Salisbury Salisbury and and Amesbury, Amesbury, Great Britain, where a former Russian agent and other people were poisoned with an “unknown, high-purity grade military agent” with possible neurotoxic eeffects,ffects, prompted discussions about the so-called “Novichok” agents. These then elusive compounds compounds are are predicted predicted to to act act similarly similarly to toknown known nerve nerve agents agents,, but butwith with higher higher toxicity and differentand different physicochemical physicochemical properties, properties, for example, for example, being being at room solid temperature. at room temperature. After discussions After ondiscussions the actual on thestructures actual structures of the Novichok of the Novichok agents, agents, the most the most important important agreement agreement on on chemical chemical disarmament, thethe ChemicalChemical WeaponsWeapons Convention Convention (CWC), (CWC), has has acknowledged acknowledged four four novel novel sca scaffoldsffolds in itsin “Annexits “Annex on on Chemicals”, Chemicals”, which which are are to beto enteredbe entered into in forceto force in Junein June 2020. 2020. Figure Figure4 depicts 4 depicts examples examples of ofthese these compounds compounds (7– (107–)[104), 43[4,43–50].–50]. Biomolecules 2020, 10, 414 3 of 22 Biomolecules 2020, 10, x 3 of 21

Figure 4. SomeSome considered considered structures structures for for Novichok Novichok Agents Agents according according to to the Chemical Weapons Convention (CWC).

From pharmacological and toxicological points of view, the biological targets of classical (G- and V-) and “Novichok” nerve agents are the , a class of involved in a myriad of biological processes, such as respiration, cognition,cognition, and drug . Cholinesterases are found in twotwo isoforms,isoforms, AChEAChE (EC (EC 3.1.1.7) 3.1.1.7) and and butyrylcholinesterase (BChE), (BChE), EC EC 3.1.1.8. 3.1.1.8. Compounds Compounds that that can caninhibit inhibit cholinesterases cholinesterases may impactmay impact the body the positivelybody posi ortively negatively or negatively and, therefore, and, therefore, be either be explored either exploredas method as of method warfare of or warfar therapeuticallye or therapeutically [51–67]. [51–67]. Herein, we review the inhibition of cholinesterases and their outcome and how these chemical reactions can be subject of interestinterest in thethe treatmenttreatment ofof neurodegenerativeneurodegenerative diseases,diseases, likelike Alzheimer’sAlzheimer’s disease (AD) or as method of warfare,warfare, which is forbidden byby thethe CWC.CWC.

2. The The Chemical Chemical Weapons Convention After many international agreements have sought to forbid the use of toxic chemicals as a mode of warfare, the Chemical Weapons Convention (CWC)(CWC) entered into force in 1997, paving the way for the creation of the Organization for the Prohibit Prohibitionion of Chemical Weapons (OPCW), the international watchdog responsible for overseeing the implementationimplementation of the CWC. The headquartersheadquarters are located in The Hague, The Netherlands. As As of of January January 2020 2020,, there there are 193 signatories or State Parties to the CWC. In accordanceaccordance toto OPCW OPCW website, website, “98% “98% of of the th globale global population population lives lives under under the the protection protection of the of theConvention” Convention” and and “97% “97% of the of chemicalthe chemical weapons weapons stockpiles stockpiles declared declared by possessor by possessor States States have have been verifiablybeen verifiably destroyed” destroyed” [1,3]. A[1,3]. short A timeline short timeline of events of usingevents chemical using chemical warfare agentswarfare and agents the CWC and canthe CWCbe devised. can be Although devised. OPCWAlthough has OPCW made e ffhasorts made throughout efforts yearsthroughout to eliminate years CW,to eliminate which have CW, granted which haveit the granted Nobel Peace it the Prize Nobel in Peace 2013 [Prize1,2,6, 68in], 2013 the world[1,2,6,68], has the witnessed world has their witnessed recurrent their use inrecurrent conflicts use or interrorism conflicts (Chart or terrorism1). (Chart 1). Biomolecules 2020, 10, x 414 4 of 21 22 Biomolecules 2020, 10, x 4 of 21

Chart 1. Non-comprehensive timeline of the CWC and use of chemical warfare (CW). Chart 1. Non-comprehensiveNon-comprehensive timeline of the CWC and use of chemical warfare (CW). In the preamble of the CWC, the State Parties are called out to ensure “that the complete and effectiveIn the prohibition preamble of of the the development, CWC, the State production, Parties arear acquisition,e called out stockpiling, to ensure “that retention, the complete transfer and useeeffectiveffective of chemical prohibition weapons, of the and development, their destruction, production, represent acquisition, a necessary stockpiling, step towards retention, the achievement transfer and ofuse these of chemical common weapons,weapons, objectives” andand [1–3]. theirtheir destruction, destruction, represent represent a a necessary necessary step step towards towards the the achievement achievement of theseof theseThe common common current objectives” text objectives” of CWC [1– [1–3]. 3has]. the “Annex on Chemicals” section. There, compounds considered as chemicalThe current warfare texttext ofofagents CWCCWC hasorhas precursors the the “Annex “Annex are on on Chemicals”lis Chemted, icals”along section. section.with There,the There, compounds compounds considered consideredand . as Chemicalschemicalas chemical warfare are warfare divided agents agents in or Schedules precursors or precursors (1 are to listed,3), are accordingly alonglisted, with along to the the with toxins possibility the saxitoxin toxins of peaceful andsaxitoxin ricin. application Chemicalsand ricin. [1,3,4].areChemicals divided Schedules are in Schedules divided are subdivided in (1 toSchedules 3), accordingly in (1two to parts,3), to accordingly the A possibility and B, to being of the peaceful possibilityA for application toxic of chemicalspeaceful [1,3,4 ].application and Schedules B for precursors.are[1,3,4]. subdivided Schedules in twoare parts,subdivided A and in B, beingtwo parts, A for A toxic and chemicals B, being and A for B for toxic precursors. chemicals and B for precursors.Schedule 1 list toxic chemicals which which have have been been developed developed only only for for or or used used as as CW. CW. Given their toxicitytoxicitySchedule and and virtually virtually 1 list toxic no no peaceful peacefulchemicals applications, applications, which have they they been pose pose developed as as a a risk risk only to to the the for implementation implementation or used as CW. of ofGiven the the CWC. CWC. their Aftertoxicity the and terrorist virtually events no peaceful in Great applications, Britain in 2018,201 they8, Schedule pose as a 1 risk will to receive the implementation an amendment of includingthe CWC. thetheAfter “Novichok” the terrorist agents, agents, events which which in Great will will Britain be entered in 201 into 8, Scheduleforce in next 1 will June receive [[48].48]. Compounds an amendment described including in Figuresthe “Novichok” 11–4–4 areare Schedule Scheduleagents, which 1A1A chemicals.chemicals. will be entered ExamplesExamples into ofofforce ScheduleSchedule in next 1B1B June chemicalschemicals [48]. Compounds areare chlorosarinchlorosarin described 11 and in chlorosomanFigures 1–4 are 12 ,Schedule precursors 1A to chemicals. GB 2 and GDExamples 3, respectively of Schedule (Figure(Figure 1B 5 chemicals5))[ 3[3,4].,4]. are 11 and 12, precursors to GB 2 and GD 3, respectively (Figure 5) [3,4].

Figure 5. Chlorosarin and chlorosoman. Figure 5. Chlorosarin and chlorosoman. Figure 5. Chlorosarin and chlorosoman. Schedule 2 contains toxic chemical chemicalss that that have have relevant relevant industrial industrial production, production, like the the precursors precursors forfor the Schedule synthesis 2 contains of pesticides toxic chemicalor mediciness that for have example. relevant Similar Similar industrial to to Schedule Schedule production, 1, 1, it it like is is divided the precursors in two parts.for the Amiton Amitonsynthesis (VG, (VG, of pesticides1313),), a a toxic toxic or chemical chemical related related for example. to to VX VX (5) (5) Similar is is an an example exampleto Schedule of of Schedule Schedule1, it is divided 2A 2A chemical. chemical. in two Diethylparts. Amiton methylphosphonate (VG, 13), a toxic (DEMP, chemical 1414),), isis related anan example to VX of (5) Schedule is an example 2B chemical, of Schedule as it can can 2A be be precursor precursorchemical. forforDiethyl toxic methylphosphonate nerve agents, but also (DEMP, flame-retardant flame-retardant 14), is an example materials of (Figure(FigureSchedule6 6))[ 2B 3[3,4].,4 chemical,]. as it can be precursor for toxic nerve agents, but also flame-retardant materials (Figure 6) [3,4]. Biomolecules 2020, 10, 414 5 of 22 Biomolecules 2020, 10, x 5 of 21

Figure 6. Amiton and Diethyl methylphosphonate (DEMP). Figure 6. Amiton and Diethyl methylphosphonate (DEMP). Schedule 33 lists lists chemicals chemicals that that are usually are usually manufactured manufactured in large amountsin large foramounts peaceful for applications. peaceful Notwithstanding,applications. Notwithstanding, they could be they employed could be either employed as CW either or precursors as CW or for precursors CW listed for in CW Schedules listed in 1 andSchedules 2. Schedule 1 and 32. is Schedule also divided 3 is inalso two divided parts. Partin two A listsparts. toxic Part compounds, A lists toxic such compounds, as phosgene, such and as Partphosgene, B refers and to precursors,Part B refers which to precursors, are manufactured which are in manufactured large scale for in peaceful large scale uses, for e.g., peaceful phosphorus uses, trichloridee.g., phosphorus15 and trichloride triethyl phosphite 15 and triethyl16 (Figure phosphite7)[3,4]. 16 (Figure 7) [3,4].

Figure 7. and triethyl phosphite. Figure 7. Phosphorus trichloride and triethyl phosphite. Although synthetic relationships are not the criteriacriteria for the Schedule Chemicals, Scheme 11 illustrates a a possible possible synthetic synthetic route route for for GB GB 22, ,a a Schedule Schedule 1A 1A chemical, chemical, from from triethyl triethyl phosphite phosphite 1616, a, aSchedule Schedule 3B 3B chemical. chemical. Michaelis-Arbuzov Michaelis-Arbuzov reaction reaction of of 1616 withwith iodomethane iodomethane 1717 affordsaffords DEMP DEMP ( 1414,, Schedule 2B chemical). DEMP DEMP may may be be then then chlorinated using thionyl 18, a ScheduleSchedule 3B3B chemical, yielding methylphosphonylmethylphosphonyl dichloride (MPDC, 19),), anotheranother ScheduleSchedule 2B2B chemical.chemical. MPDC is then reacted with with a a source source of of fluoride, fluoride, generating generating methylphosphonyl methylphosphonyl difluoride difluoride (MPDF, (MPDF, 2020), ),also also a aSchedule Schedule 2B 2B chemical. chemical. Reaction Reaction of MPDF of MPDF with with isopropanol isopropanol 21 in21 presencein presence of a , of a base,affords aff theords toxic the toxicSchedule Schedule 1 chemical. 1 chemical. There There is comprehensive is comprehensive literature literature on on the the synthesis synthesis of of organophosphorusorganophosphorus compounds and researchers must read carefully the CWC, in the the OPCW OPCW website website (www.opcw.org) (www.opcw.org) to learn more onon thethe internationalinternational legislationlegislation [[1–4,69–73].1–4,69–73].

Scheme 1. Synthetic relationship of scheduled chemicals. 3. Cholinesterases 3. Cholinesterases Chemistry is ubiquitous, its principles and applications underlie innumerous biological processes, Chemistry is ubiquitous, its principles and applications underlie innumerous biological as is the case with cholinesterases, key enzymes in the metabolism of different species. This class processes, as is the case with cholinesterases, key enzymes in the metabolism of different species. of enzyme in superior animals is present in two different isoforms, as aforementioned, AChE This class of enzyme in superior animals is present in two different isoforms, as aforementioned, and BChE. AChE is involved in the of neurotransmission processes (parasympathetic AChE and BChE. AChE is involved in the regulation of neurotransmission processes neurotransmission), being important for (but not limited to) cognition and respiration, for example. (parasympathetic neurotransmission), being important for (but not limited to) cognition and BChE is found in plasma, being responsible for of different esters, property that has been respiration, for example. BChE is found in plasma, being responsible for hydrolysis of different esters, studied to understand a series of metabolic events or for treatment of addiction, for example. property that has been studied to understand a series of metabolic events or for treatment of cocaine addiction, for example. Furthermore, it has been extensively studied due to its stoichiometric reaction with organophosphorus compounds, which indicates that it is not only a biomarker of exposure to Biomolecules 2020, 10, 414 6 of 22

Biomolecules 2020,, 10,, xx 6 of 21 Furthermore, it has been extensively studied due to its stoichiometric reaction with organophosphorus thesecompounds, toxic chemicals which indicates but alsothat a potential it is not treatment only a biomarker for intoxication, of exposure acting to theseas bioscavenger toxic chemicals of nerve but agentsalso a potential [61–67,74–83]. treatment for intoxication, acting as bioscavenger of nerve agents [61–67,74–83]. Cholinesterases are -estearases, which which means means that that their activity relies on a serine residue associated to a and a glutamateglutamate toto composecompose thethe so-called catalyticcatalytic triad.triad. The structures of cholinesterases have have been been extensively extensively studied studied in in or orderder to to better understand their their biochemical biochemical roles. roles. The selective, reversible inhibition of AChE is important in the treatment of AD, allowing research on novel drugs. On On other other hand, hand, their their irreversible irreversible inhibition inhibition caused caused by by nerve nerve agents agents (Figures (Figures 11–4),–4), organophosphorus and pesticides, like 2222 andand aldicarbaldicarb 23 (Figure(Figure 88)8)) may maymay be bebe fatal, depending on the the level level of of exposure exposure and and if if not not identified identified and and immedi immediatelyately addressed addressed [61–67,84]. [61–67,84].

Figure 8. Chlorpyrifos and . Figure 8. Chlorpyrifos and aldicarb. AChE 2424catalyzes catalyzescatalyzes the thethe breakdown breakdownbreakdown of acetylcholineofof acetylcholineacetylcholine (ACh, (ACh,(ACh,25), into 25), acetate into acetate26 and 26 andand 27cholinecholinethrough 27 througha tetrahedral a tetrahedral transition transition state 28 at state the post-synaptic28 atat thethe post-synapticpost-synaptic cleft, ending cleft,cleft, the endingending ,thethe actionaction in potential,potential, accordance inin withaccordance the proposal with the below proposal (Scheme below2). (Scheme 2).

Scheme 2. Hydrolysis of ACh through AChE. Scheme 2. Hydrolysis of ACh through AChE. A schematic schematic representation representation of of different different sites sites of ofAChEAChE AChE isis shownshown is shown inin FigureFigure in Figure 99 [4,8,61–67].[4,8,61–67].9[ 4,8,61 –TheThe67]. esteraticThe esteratic site (catalytic site (catalytic site, ES), site, isis ES), responsibleresponsible is responsible forfor thethe for hydrolysishydrolysis the hydrolysis reactionreaction reaction throughthrough through thethe triadtriad the serine-serine- triad histidine-glutamateserine-histidine-glutamate (whose (whose position position in the in enzyme the enzyme primary primary structure structure may may slightly slightly vary vary across species, as as cholinesterases cholinesterases are are much much conserved conserved enzyme enzymes).s). Also Also relevant relevant is the is theperipheral peripheral anionic anionic site (PAS)site (PAS) which which is formed is formed by aromatic by aromatic amino amino acid residues. residues. There There are studies are studies correlating correlating PAS PAS and andthe occurrencethe occurrence of β-amyloid of β-amyloid proteins (Aβ 1–42).1–42). (Aβ 1–42). TheseThese Theseinsolubleinsoluble insoluble oligopeptioligopepti oligopeptidesdes together together with tau with tangles tau aretangles related are to related the onset to the of AD. onset PAS of AD.and its PAS aromatic and its re aromaticgion assist region cationic assist (or cationic electrophilic) (or electrophilic) substrates tosubstrates take the to right take direction the right towards direction the towards catalytic the gorge. catalytic The gorge. catalytic The anio catalyticnic site anionic (CAS) site contains (CAS) aromaticcontains aromaticamino acid amino residues, acid residues,like like tryptophan and phenylalanine, and phenylalanine, which interacts which interactswith the withcationic the motifcationic of motifthe substrates, of the substrates, and helps and to helpsbring tothe bring ester the motif ester of motifsubstrates of substrates close to the close catalytic to the catalytic ES. The oxyanionES. The oxyanion pocket (OAS) pocket helps (OAS) to stabilize helps to the stabilize tetrahedral the tetrahedral transition state transition (28) during state (ACh28) during hydrolysis. ACh Finally,hydrolysis. the acyl Finally, pocket the site acyl (APS) pocket is the site point (APS) where is the the point difference where the between difference AChE between and BChE AChE may and be assessed,BChE may with be assessed,AChE having with a AChE smaller having APS than a smaller BChE, APS determining than BChE, the determining size of the the size ofto thebe hydrolyzedsubstrate to [61–67,85–93]. be hydrolyzed [61–67,85–93]. Biomolecules 2020, 10, 414 7 of 22 Biomolecules 2020, 10, x 7 of 21

Figure 9.9. ActiveActive site of AChE [4] [4] (ES: Estearic site, PAS: PeripheralPeripheral AnionicAnionic Site,Site, CAS:CAS: CatalyticCatalytic AnionicAnionic Site,Site, OAS:OAS: OxianionOxianion Pocket,Pocket, APS:APS: AcylAcyl PocketPocket Site,Site, Ser:Ser: Serine, His: Histidine, Glu: Glutamate, Glutamate, Phe: Phenylalanine, Phenylalanine, Asp: Asp: Aspartat Aspartate,e, Ala: Ala: Alanine, Alanine, Trp: Trp: Tryptophan Tryptophan,, Tyr: Tyr:) Tyrosine) Reproduced Reproduced with withpermission. permission.

4. The Inhibition of Acetylcholinesterase As discussed, AChE is the enzyme involved in the breakdown of ACh 2525,, thethe parasympatheticparasympathetic , into choline 26 and acetate 27,, whichwhich areare reuptakenreuptaken forfor dede novonovo synthesissynthesis ofof thethe neurotransmitter. Therefore,Therefore, AChE AChE is is a a pharmaceutical pharmaceutical target, target, as as its its inhibition inhibition provokes provokes an an increase increase of neurotransmitterof neurotransmitter concentration at the at post-synaptic the post-syn cleft,aptic leading cleft, leading to exacerbated to exacerbated cholinergic response overresponse the nerve over the structures nerve structures that require that parasympathetic require parasympathetic stimulation, stimulation, such as neuromuscular such as neuromuscular junctions. Thisjunctions. outcome This may outcome be positive, may be as positive, in the case as in of the treatment case of oftreatment diseases of where diseases the where transitory, the transitory, reversible inhibitionreversible ofinhibition AChE located of AChE in located the central in the nervous central system nervous (CNS) system delivers (CNS) valuable delivers response,valuable response, e.g., AD, ore.g., negative, AD, or negative, when toxic when chemicals, toxic chemicals, such as such nerve as agentsnerve agents (Figures (Figures1–4) or 1-4) organophosphorus or organophosphorus and carbamateand carbamate pesticides pesticides (Figure (Figure8), cause 8), cause irreversible irreversible inhibition inhibition at neuromuscular at neuromuscular junctions, junctions, which which may bemay life-threatening be life-threatening [53,54 [53,54,94–103].,94–103]. Proposed reactionsreactions between between AChE AChE and and inhibitors inhibitors are are represented represented in the in Schemethe Scheme3. Inhibitors 3. Inhibitors have anhave electrophilic an electrophilic site that site reacts that with reacts the nucleophilicwith the nucleophilic hydroxyl of hydroxyl the serine of residue the serine at the residue catalytic at triad. the Thecatalytic bond triad. is then The formed bond byis then displacement formed by of displacement a . of a In leaving case of group. irreversible In case AChE of irreversible inhibitors, likeAChE classic inhibitors, nerve agentslike classic (G- andnerve V-Series), agents (G- organophosphorus and V-Series), organophosphorus compounds 29, the compounds leaving group 29, the is eitherleaving a group halogen is (fluorideeither a halogen in GB 2 (fluorideand GD in3) GB or other 2 and suitable GD 3) or entity other ( suitable entity in GA (cyanide4, Scheme in3 A).GA In4, theScheme case of3A). In the case of clinicalof carbamates use 30, usuallyof clinical reversible use 30 AChE, usually inhibitors reversible (exemption AChE inhibitors given to the(exemption toxic pesticides given to and the “Novichok” toxic pesticides carbamates and “Novichok” described carbamates previously indescribed this text), previously an in31 thisis displacedtext), an alcohol (Scheme 313 isB). displaced (Scheme 3B). Biomolecules 2020, 10, x 8 of 21

Biomolecules 2020, 10, 414 8 of 22 Biomolecules 2020, 10, x 8 of 21

Scheme 3. Reactions of AChE with different inhibitors.

5. Reversible Inhibition of AChE: A Tool for Treatment of AD It is reckoned that ACh is involved in the cognition mechanism. Therefore, increased levels of this neurotransmitter may contribute to improvement. Based on this knowledge, the selective inhibition of AChE at the CNS has been used for treatment of AD, a multifactorial disease that causes affected people to suffer memory impairment and progressive neurodegeneration, which is not only fatal, but also a burden to the health system and people involved in patient care [100–107]. Currently, there are only four drugs available to ameliorate the memory loss and other symptoms related to AD. Nonetheless, they can only act in a certain level of impairment and are unable to halt the progression of the pathology. Consequently, knowledge on the different Scheme 3. Reactions of AChE with different inhibitors. mechanisms that can contributeScheme 3. Reactionsto the onset of AChE of ADwith differentand discover inhibitors. of the early neurological, 5.biochemical, Reversible and Inhibition behavioral of AChE:changes, A useful Tool for for Treatment the development of AD of a more efficient treatments. Three 5.of Reversible these compounds Inhibition act of AChE:as AChE A Tool inhi forbitors, Treatment of AD (a carbamate, 33), (a It is reckoned that ACh is involved in the cognition mechanism. Therefore, increased levels of this benzylpiperidine derivative, 34), and (an of natural occurrence, 35). The fourth neurotransmitterIt is reckoned may that contribute ACh is involved to memory in the improvement. cognition mechanism. Based on Therefore, this knowledge, increased the selectivelevels of compound is (an adamantane derivative, 36), which affects other receptors inhibitionthis neurotransmitter of AChE at the may CNS contribute has been usedto memory for treatment improvement. of AD, a multifactorialBased on this disease knowledge, that causes the (glutamatergic, serotoninergic, nicotinic receptors) [107–127]. These compounds are aselectiveffected people inhibition to su offf erAChE memory at the impairment CNS has been and used progressive for treatment neurodegeneration, of AD, a multifactorial which is not disease only shown in Figure 10. fatal,that causes but also affected a burden people to the to healthsuffer memory system and impairment people involved and progressive in patient neurodegeneration, care [100–107]. which is notRivastigmine only fatal, but is also regarded a burden to react to the with health the systemserine residueand people of the involved ES present in patient in AChE care and [100–107]. BChE (as shownCurrently, in Scheme there are 3), only whereas four drugsdonepezil available can act to amelioratein both CAS the and memory PAS of loss AChE. and other These symptoms actions Currently, there are only four drugs available to ameliorate the memory loss and other relatedincrease to the AD. synaptic Nonetheless, levels of they ACh, can rendering only act in positive a certain cognitive level of response impairment of the and patient. are unable In case to haltof symptoms related to AD. Nonetheless, they can only act in a certain level of impairment and are thedonepezil, progression action of on the PAS pathology. is further Consequently, regarded to knowledgereduce the onbiosynthesis the different of insoluble mechanisms β-amyloid that can unable to halt the progression of the pathology. Consequently, knowledge on the different contributeoligopeptides to thethat onset bring of about AD and inflammatory discover of resp the earlyonse that neurological, contribute biochemical, to the neurodegeneration and behavioral mechanisms that can contribute to the onset of AD and discover of the early neurological, changes,characteristic useful of forAD. the Galantamine development acts ofsimultaneous a more efficiently as an treatments. AChE inhibitor Three and of theseallosteric compounds modulator act biochemical, and behavioral changes, useful for the development of a more efficient treatments. Three asof AChEnicotinic inhibitors, receptors, rivastigmine increasing the (a carbamate,affinity for ACh33), donepezil[114–128]. (aRe benzylpiperidinesearch in progress derivative,on novel AD34 ), of these compounds act as AChE inhibitors, rivastigmine (a carbamate, 33), donepezil (a anddrugs galantamine has covered (an different alkaloid disciplin of naturales and occurrence, been focused35 on). Thefurther fourth development compound of AChE is memantine inhibitors, (an benzylpiperidine derivative, 34), and galantamine (an alkaloid of natural occurrence, 35). The fourth adamantanebeta-secretase derivative, 1 (BACE-1)36 ),inhibitors, which aff ectsserotonin other receptors5-HT4 receptor (glutamatergic, inhibitors serotoninergic,and PROTAC (PRO acetylcholineteolysis compound is memantine (an adamantane derivative, 36), which affects other receptors nicotinicTargeting receptors) Chimeras) [compounds,107–127]. These among compounds other approaches are shown [129–146]. in Figure 10. (glutamatergic, serotoninergic, acetylcholine nicotinic receptors) [107–127]. These compounds are shown in Figure 10. Rivastigmine is regarded to react with the serine residue of the ES present in AChE and BChE (as shown in Scheme 3), whereas donepezil can act in both CAS and PAS of AChE. These actions increase the synaptic levels of ACh, rendering positive cognitive response of the patient. In case of donepezil, action on PAS is further regarded to reduce the biosynthesis of insoluble β-amyloid oligopeptides that bring about inflammatory response that contribute to the neurodegeneration characteristic of AD. Galantamine acts simultaneously as an AChE inhibitor and of nicotinic receptors, increasing the affinity for ACh [114–128]. Research in progress on novel AD Figure 10. Currently approved drugs for treatment of Alzheimer’s disease (AD). drugs has covered different disciplines and been focused on further development of AChE inhibitors, beta-secretaseRivastigmine 1 (BACE-1) is regarded inhibitors, to react with the serine 5-HT4 residue receptor of inhibitors the ES present and inPROTAC AChE and (PRO BChEteolysis (as shownTargeting in SchemeChimeras3),) compounds, whereas donepezil among can othe actr approaches in both CAS [129–146]. and PAS of AChE. These actions increase the synaptic levels of ACh, rendering positive cognitive response of the patient. In case of donepezil, action on PAS is further regarded to reduce the biosynthesis of insoluble β-amyloid oligopeptides that bring about inflammatory response that contribute to the neurodegeneration characteristic of AD. Galantamine acts simultaneously as an AChE inhibitor and allosteric modulator of nicotinic receptors, increasing the affinity for ACh [114–128]. Research in progress on novel AD drugs has covered

Biomolecules 2020, 10, 414 9 of 22 Biomolecules 2020, 10, x 9 of 21 Biomolecules 2020, 10, x 9 of 21 different disciplinesFigure 10. and Currently been focused approved on drugs further for development treatment of Alzheimer’s of AChE inhibitors,disease (AD). beta-secretase 1 Figure 10. Currently approved drugs for treatment of Alzheimer’s disease (AD). (BACE-1) inhibitors, serotonin 5-HT4 receptor inhibitors and PROTAC (PROteolysis Targeting Chimeras) The use of carbamates as drugs is based on the comparison of hydrolysis rates of complexes compounds,The use amongof carbamates other approaches as drugs is [129 based–146 ].on the comparison of hydrolysis rates of complexes between AChE and its natural substrate ACh and those yielded by reaction with carbamates (which betweenThe AChE use of and carbamates its natural as substrate drugs is basedACh and on thethose comparison yielded by ofreaction hydrolysis with ratescarbamates of complexes (which reversibly carbamoylate the ES; Scheme 3B) which usually show lower rates [108,118,119]. Other betweenreversibly AChE carbamoylate and its natural the ES; substrate Scheme ACh 3B) andwhich those usually yielded show by reactionlower rates with [108,118,119]. carbamates (which Other important uses for AChE reversible inhibitors are the treatment of dementia related to Parkinson’s reversiblyimportant uses carbamoylate for AChE the reversible ES; Scheme inhibitors3B) which are the usually treatment show of lower dementia rates related [ 108,118 to, 119Parkinson’s]. Other disease, and and . importantdisease, and uses glaucoma for AChE and reversible myasthenia inhibitors gravis. are the treatment of dementia related to Parkinson’s Other carbamate that acts as AChE reversible inhibitor is 37 (Figure 11), a drug disease,Other and carbamate glaucoma that and acts myasthenia as AChE gravis. reversible inhibitor is pyridostigmine 37 (Figure 11), a drug used for treatment of myasthenia gravis, which has been used as protective measure for those usedOther for treatment carbamate of that myasthenia acts as AChE gravis, reversible which inhibitorhas been isused pyridostigmine as protective37 measure(Figure 11 for), a those drug working in places where nerve agents might have been deployed, as during Gulf War (1991). usedworking for treatmentin places of where myasthenia nerve gravis,agents which might has have been been used deployed, as protective as measureduring Gulf for those War working (1991). Overdose of pyridostigmine has been studied as a possible cause of the “Gulf War Syndrome” inOverdose places whereof pyridostigmine nerve agents has might been have studied been deployed,as a possible as during cause Gulfof the War “Gulf (1991). War OverdoseSyndrome” of [109,110,147–149]. pyridostigmine[109,110,147–149]. has been studied as a possible cause of the “Gulf War Syndrome” [109,110,147–149].

Figure 11. Pyridostigmine bromide. Figure 11. Pyridostigmine bromide. 6. Irreversible Irreversible Inhibition of AChE: The Chemistry of Nerve Agents 6. Irreversible Inhibition of AChE: The Chemistry of Nerve Agents As expected, the the irreversible irreversible AChE AChE inhibition inhibition may may result result in inlife-threatening life-threatening effects, effects, according according to As expected, the irreversible AChE inhibition may result in life-threatening effects, according to theto the level level of ofexposure exposure and and resources resources available available to to treat treat affected affected victims. The The overstimulation may the level of exposure and resources available to treat affected victims. The overstimulation may trigger a series of symptoms represented by the acronym SLUDGEM (Salivation, Lachrimation, trigger a series of symptoms represented by the acronym SLUDGEM (Salivation, Lachrimation, Urination, DDefecation,efecation,G astrintestinalGastrintestinal disturbs, disturbs,Emesis, Emesis,Myosis, Myosis, and M uscleand spasms)Muscle whichspasms) illustrates which Urination, Defecation, Gastrintestinal disturbs, Emesis, Myosis, and Muscle spasms) which illustratesthe clinical the eff clinicalects.Acute effects. exposure Acute exposure may be may fatal be due fatal to due the to respiratory the respiratory failure failure resulting resulting from illustrates the clinical effects. Acute exposure may be fatal due to the respiratory failure resulting fromthe continuous the continuous stimulation stimulation of neuromuscular of neuromuscular junctions junctions at diaphragm, at diaphragm, as described as described earlier earlier in this in from the continuous stimulation of neuromuscular junctions at diaphragm, as described earlier in paperthis paper [6,14 [6,14,19,20,53,54,150].,19,20,53,54,150]. Classic Classic (G- and (G- V-Series) and V-Series) and “Novichok” and “Novichok” nerve agentsnerve (Figuresagents (Figures1–4) listed 1– this paper [6,14,19,20,53,54,150]. Classic (G- and V-Series) and “Novichok” nerve agents (Figures 1– 4)in listed the CWC in the in CWC its Schedule in its Schedule 1, Part A, 1, arePart example A, are example compounds. compounds. Structurally Structurally related related to nerve to agents, nerve 4) listed in the CWC in its Schedule 1, Part A, are example compounds. Structurally related to nerve agents,some pesticides, some pesticides, such as such chlorpyrifos as chlorpyrifos22 and 22 aldicarb and aldicarb23 (Figure 23 (Figure6), 6), paraoxon38 (Figure 38 (Figure 12) (used 12) agents, some pesticides, such as chlorpyrifos 22 and aldicarb 23 (Figure 6), paraoxon 38 (Figure 12) (usedin many in studiesmany forstudies development for development of of towardsantidotes towards cholinesterase inhibitors), andinhibitors), and39 (used in many studies for development of antidotes towards cholinesterase inhibitors), and (Figuremalathion 12), 39 a compound(Figure 12), still a usedcompound as aerosol still to used combat as arbovirusesaerosol to combat vectors inarboviruses developing vectors countries in malathion 39 (Figure 12), a compound still used as aerosol to combat arboviruses vectors in (Figuredeveloping 12)[ countries3–6,8,19,45 (Figure,46,50,53 12),54 [3–6,8,19,45,46,50,53,54,151–154].,151–154]. developing countries (Figure 12) [3–6,8,19,45,46,50,53,54,151–154].

Figure 12. Organophosphorus pesticides paraoxon and malathion. Figure 12. Organophosphorus pesticides paraoxon and malathion. The toxicity related to organophosphorus compoundscompounds may be explained through the high The toxicity related to organophosphorus compounds may be explained through the high affinityaffinity of of phosphorus phosphorus and and , oxygen, with with higher higher energy energy bonds, bonds, 335 335 and and 544 544kJ/mol, kJ/mol, for P-O for and P-O P=O and affinity of phosphorus and oxygen, with higher energy bonds, 335 and 544 kJ/mol, for P-O and P=O bonds,P=O bonds, respectively respectively [155]. [155 Poisoning]. Poisoning and and environmental environmental contamination contamination by by organophosphorus bonds, respectively [155]. Poisoning and environmental contamination by organophosphorus pesticides poses a serious public health challenge in countries countries with with lack lack of of control control of of these these substances. substances. pesticides poses a serious public health challenge in countries with lack of control of these substances. Nonetheless, occupationaloccupational poisoningpoisoning is is not not the the sole sole problem. problem. Cases Cases of pesticideof use use in suicides in suicides are alsoare Nonetheless, occupational poisoning is not the sole problem. Cases of pesticide use in suicides are alsoknown. known. The The World World Health Health Organization Organization (WHO) (WHO) estimated estimated more more than than 200,000 200,000 casualties casualties per per year year by also known. The World Health Organization (WHO) estimated more than 200,000 casualties per year by poisoning with organophosphorus pesticides in developing countries. Recently in India, more by poisoning with organophosphorus pesticides in developing countries. Recently in India, more than 20 children died after eating a meal prepared with oil stored in -contaminated than 20 children died after eating a meal prepared with oil stored in monocrotophos-contaminated Biomolecules 2020, 10, 414 10 of 22

Biomolecules 2020,, 10,, xx 10 of 21 poisoning with organophosphorus pesticides in developing countries. Recently in India, more than 20 Biomolecules 2020, 10, x 10 of 21 bottleschildren (40 died,, FigureFigure after eating13)13) [156,157].[156,157]. a meal preparedAsAs anan alternative,alternative, with oil stored manymanyin countriescountries monocrotophos-contaminated havehave adoptedadopted neonicotinoidsneonicotinoids bottles ( 40asas, ,Figureinsecticides,bottles 13(40)[, 156Figure whichwhich,157 ]. 13)areare As [156,157].lessless an toxictoxic alternative, totoAs mammaliansmammalians an manyalternative, countries andand many birds.birds. have countries However,However, adopted have theythey adopted areare underunder neonicotinoids scrutinyscrutiny as insecticides, duedue toasto toxicitywhichtoxicityinsecticides, are forfor less pollinatorpollinator which toxic are to insectsinsects mammaliansless toxic [158,159].[158,159]. to mammalians and birds. However, and birds. they However, are under they scrutiny are under due scrutiny to toxicity due for to pollinatortoxicity for insects pollinator [158 ,insects159]. [158,159].

Figure 13. Monocrotophos. Figure 13. Monocrotophos. Figure 13. Monocrotophos. Depending on the AChE adduct formed withwith organophosphorusorganophosphorus compoundscompounds ((31,, SchemeScheme 3A),3A), thethe displacementdisplacementDepending on ofof the thethe AChE O-alkylO- adduct moietymoiety formed cancan lead with to to organophosphorus enzymaticenzymatic aging.aging. compounds TheThe generatedgenerated ( 31 ,, phosphonateScheme 33A),A), anionthe displacementdisplacement 41 isis stabilizedstabilized of of the the O-alkylbyby O-alkyl thethe protonated moietyprotonated moiety can can lead histidinehistidine lead to enzymatic to imidazoleimidazoleenzymatic aging. moietymoietyaging. The generated Thelocatedlocated generated atat phosphonate thethe phosphonatecatalyticcatalytic anion ES,ES, affecting41anionis stabilized 41 theis antidotestabilized by the efficacy. protonated by the Adductprotonated histidine of GD imidazolehistidine with AChE imidazole moiety 42 quicklyquickly located moiety undergoesundergoes at thelocated catalytic suchsuch at theprocessprocess ES, catalytic affecting (Scheme(Scheme theES, 4)antidoteaffecting [160–167]. ethefficacy. Adduct efficacy. of GD Adduct with AChE of GD42 withquickly AChE undergoes 42 quickly such undergoes process such (Scheme process4)[160 (Scheme–167]. 4) [160–167].

Scheme 4. Aging of AChE-GD adduct. Scheme 4. Aging of AChE-GD adduct. Scheme 4. Aging of AChE-GD adduct. The knowledge on the reaction between nerve ag agentsents and cholinesterases may be useful to identifyidentifyThe exposure exposure knowledge to to such suchon the toxic toxic reaction chemicals. chemicals. between InIn blbl blood oodnerve and ag plasmaents and samples, cholinesterases nerve agents may may be beuseful found to ininidentify their their hydrolyzedhydrolyzedexposure to forms, forms,such toxic mainly mainly chemicals. alkylphosp alkylphosphonicalkylphosp In blhonicood and , plasma but alsosamples, as adducts nerve with agents BChE may 44 be.. Iffound If this this adductin their ishydrolyzed aged 45,, itsits forms, digestiondigestion mainly yieldsyields alkylphosp aa phosphylatedphosphylated honic acids, nonapeptitdenonapeptitde nonapeptitde but also 46as46 (FGESAGAAS,adducts(FGESAGAAS,(FGESAGAAS, with BChE A:A: A: Alanine,Alanine, Alanine, 44. If this E:E: E: Glutamate,adduct is aged F: Phenylalanine, 45, its digestion G: G: yields Glycine, Glycine, a phosphylated S: S: Serine Serine))) thatthat nonapeptitde cancan bebe usedused 46 asas (FGESAGAAS, proof proof of of exposure exposure A: Alanine, to to nerve nerve E: agents.Glutamate, Nevertheless, Nevertheless, F: Phenylalanine, if if the the enzyme enzyme G: Glycine, is is not not S:aged aged Serine 4444,),, treatmenttreatment that can be withwith used fluoridefluoride fluoride as proof ionsions of enables enablesexposure aa de tode nervenovo novo synthesisagents. Nevertheless, of of the the organophosphorus organophosphorus if the enzyme compound compoundis not aged 47 4744.. TheseThese., Thesetreatment compoundscompounds compounds with fluoride cancan can bebe besuccessfullysuccessfully successfully enables detecteddetected a detectedde novo byby chromatographicbysynthesis chromatographic of the organophosphorus and and spectrometric spectrometric compound techniques techniques 47. These(Scheme(Scheme (Scheme compounds 5).5).5). ThisThis This canreactionreaction reaction be successfully between between detected BChEBChEBChE andandand by neurotoxicchromatographic CWC Schedule and spectrometric 1A not only techniqueshighlights the (Scheme importance 5). This of this reaction enzyme between as as a a biomarker, biomarker, BChE andbut alsoneurotoxic illustratesillustrates CWC its its Schedule potential potential use1A use not as aas only bioscavenger. a bioscavenger.highlights Therefore, the Therefore,importance it can it beof can athis potential be enzyme a potential prophylactic as a biomarker, prophylactic measure but measurebyalso reacting illustrates by inreacting stoichiometric its potential in stoichiometric manneruse as a with manner bioscavenger. nerve with agents nerve Therefore, [168 agents–181 ].[168–181].it can be a potential prophylactic measure by reacting in stoichiometric manner with nerve agents [168–181].

Scheme 5. Digestion and fluoride reactivation products from biological matrices. Scheme 5. Digestion and fluoride reactivation products from biological matrices. Scheme 5. Digestion and fluoride reactivation products from biological matrices. 7. Rescuing Cholinesterases: Antidotes towards Nerve Agents 7. Rescuing Cholinesterases: Antidotes towards Nerve Agents Biomolecules 2020, 10, 414 11 of 22

7. Rescuing Cholinesterases: Antidotes towards Nerve Agents BiomoleculesBiomolecules 2020 ,2020 10, x, 10, x 11 of 2111 of 21 In order to rescue organophosphorus-inhibited AChE and BChE, appropriate antidote therapy mustIn orderIn be order promptly to rescue to rescue employed. organophos organophos Rapidphorus-inhibitedphorus-inhibited response is required AChE AChE and to lessen BChE,and BChE, the appropriate risk appropriate of neurological antidote antidote therapy damage therapy and must be promptly employed. Rapid response is required to lessen the risk of neurological damage musteven be promptly death. Antidotes employed. usually Rapid are response a mixture is ofrequired three di toff erentlessen compounds, the risk of neurological an enzyme reactivatordamage (to and even death. Antidotes usually are a mixture of three different compounds, an enzyme reactivator andremove even death. the organophosphorus Antidotes usually are from a mixture catalytic of ES), three an different compounds, agent an (to enzyme counteract reactivator the effects of (to remove the organophosphorus from catalytic ES), an anticholinergic agent (to counteract the (to theremove higher the concentration organophosphorus of the neurotransmitter),from catalytic ES), and an ananticholinergic agent (to control(to counteract ). the Up to effects of the higher concentration of the neurotransmitter), and an anticonvulsant (to control effectsdate, of pyridinium the higher concentration have been of clinically the neur usedotransmitter), as AChE reactivators, and an anticonvulsant 48(to, obidoximecontrol 49, seizures). Up to date, pyridinium oximes have been clinically used as AChE reactivators, pralidoxime seizures).trimedoxime Up to date,50, HI-6 pyridinium51, HLö-7 oximes52 and have K027 been53 clinicallyare representative used as AChE compounds reactivators, (Figure pralidoxime 14)[4,182– 186]. 48, 49, trimedoxime 50, HI-6 51, HLö-7 52 and K027 53 are representative compounds 48, Atobidoxime the physiological 49, trimedoxime pH oximes 50, aHI-6fford 51 oximates., HLö-7 These52 and K027 53 are reactivate representative cholinesterases compounds through (Figure 14) [4,182–186]. At the physiological pH oximes afford oximates. These nucleophiles (Figurea nucleophilic 14) [4,182–186]. attack At on the phosphorusphysiological atom, pH oximes releasing afford the hydroxyloximates. serine These residue. nucleophiles Scheme 6 reactivate cholinesterases through a nucleophilic attack on the phosphorus atom, releasing the reactivatedepicts cholinesterases the reaction of athrough pyridinium a nucleophilic oximate 54 attackand an on organophosphorus-inhibited the phosphorus atom, releasing cholinesterase the hydroxylhydroxyl serine serine residue. residue. Scheme Scheme 6 depicts 6 depicts the reactionthe reaction of a ofpyridinium a pyridinium oximate oximate 54 and 54 anand an 56 [4,99,160,161,185–193]. organophosphorus-inhiborganophosphorus-inhibited itedcholinesterase cholinesterase 56 [4,99,160,161,185–193]. 56 [4,99,160,161,185–193].

Figure 14. Oximes clinically used as antidotes for organophosphorus poisoning. X = chloride, FigureFigure 14.14.Oximes Oximes clinically clinically used used asantidotes as antidotes for organophosphorus for organophosphorus poisoning. poisoning. X = chloride, X = mesylatechloride, mesylate or for 48, chloride for 49, bromide for 50 and 53, chloride or mesylate for 51 and 52. ormesylate iodine foror iodine 48, chloride for 48, for chlori 49, bromidede for 49, for bromide 50 and for 53, 50 chloride and 53, or ch mesylateloride or formesylate 51 and for 52. 51 and 52.

. . Scheme 6. Reactivation of AChE through pyridinium oximes. SchemeScheme 6.6. Reactivation of AChE throughthrough pyridinium oximes.oximes. MarketedMarketed kits kitsalso alsocontain contain atropine 59 as59 anticholinergicas anticholinergic agent, agent, and an and anticonvulsant, an anticonvulsant, such suchas as Marketed kits also contain atropine 59 as anticholinergic agent, and an anticonvulsant, such as diazepamdiazepam 60 60(Figure(Figure 15). 15 ).Seizures Seizures may may bebe relatedrelated to to the the excitotoxicity excitotoxicity that that takes takes place place due to due unbalanced to 60 (Figure 15). Seizures may be related to the excitotoxicity that takes place due to unbalancedlevels of glutamatelevels of glutamate and γ-aminobutyric and γ-aminobutyric acid (GABA) acid in(GABA) brain in in the brain course in the of AChEcourse inhibition.of AChE The unbalanced levels of glutamate and γ-aminobutyric acid (GABA) in brain in the course of AChE inhibition.use of glutamate The use of antagonists glutamate and antagonists GABA and GABA may beagonists a valuable may therapeutic be a valuable approach therapeutic [4,194– 197]. approachinhibition. [4,194–197]. The use of glutamate antagonists and GABA agonists may be a valuable therapeutic approach [4,194–197]. Biomolecules 2020, 10, 414 12 of 22 Biomolecules 2020, 10, x 12 of 21

Figure 15. Atropine and diazepam. Figure 15. Atropine and diazepam. Due toto thethe cationiccationic profile profile of of all all available available antidotes, antidotes, permeation permeation into into the blood-brainthe blood-brain barrier barrier (BBB) (BBB)hinders hinders effective effective treatment. treatment. Additionally, Additionally, an efficient an “universal efficient antidote’“universal towards antidote’ all cholinesterase towards all cholinesteraseinhibitors is not inhibitors yet available is not yet [198 available]. To tackle [198]. these To tackle limitations, these limitation developments, development of more lipophilic of more lipophiliccompounds compounds and novel routesand novel of administration routes of administra of antidotestion of have antidotes beenextensively have been extensively studied [4,199 studied–205]. [4,199–205]. 8. Final Remarks 8. FinalCholinesterases Remarks are constitutive enzymes, essential to the correct performance of the parasympatheticCholinesterases nervous are systemconstitutive (PNS). enzymes, The chemistry essential of AChE to inhibitionthe correct and performance aging in the contextof the parasympatheticof the contemporary nervous issue system of terrorism (PNS). The by chemical chemistry warfare of AChE is aninhibition attractive and conduit aging in for the chemical context ofeducation the contemporary and for dual issue use, of military terrorism and by civilian chemical research warfare work. is AChEan attractive has a pivotal conduit role for in chemical memory educationand learning, and thereforefor dual use, increased military knowledge and civilian on research AChE function work. AChE may facilitatehas a pivotal the developmentrole in memory of andfurther, learning, more therefore effective therapiesincreased towardsknowledge neurodegenerative on AChE function diseases, may facilitate like AD. the Besides development the varied of further,conceptual more background effective therapies in Chemistry towards that itneurodegenerative demands, the subject diseases, is also like multidisciplinary, AD. Besides the since varied it is conceptualrelated to pharmacology, background in toxicology, Chemistry medicinal that it demands, chemistry, the international subject is also politics, multidisciplinary, health, environmental since it is relatedand forensics to pharmacology, sciences, as well ethicaltoxicology, issues. medicinal chemistry, international politics, health, Authorenvironmental Contributions: and forensicsConceptualization, sciences, as well S.F.d.A.C., ethical A.B.C.S., issues. K.K. and T.C.C.F.; writing—original draft preparation,Author Contributions: S.F.d.A.C., A.B.C.S.,Conceptualization, and T.C.C.F.; S.F.d.A.C., writing—review A.B.C.S., and K. editing,K. and S.F.d.A.C.,T.C.C.F.; writing—original A.B.C.S., M.C.B., R.B.S.,draft V.G.M.d.O., P.A.d.M.C., K.K. and T.C.C.F.; project administration, S.F.d.A.C., A.B.C.S., K.K., T.C.C.F.; funding preparation,acquisition, S.F.d.A.C.,S.F.d.A.C., A.B.C.S.,A.B.C.S., M.C.B.,and T.C.C.F.; V.G.M.d.O., writing—review P.A.d.M.C., and K.K. editing, and T.C.C.F.S.F.d.A.C., All A.B.C.S., authors haveM.C.B., read R.B.S., and V.G.M.d.O.,agreed to the P.A.d.M.C., published versionK.K. and of T.C.C.F.; the manuscript. project administration, S.F.d.A.C., A.B.C.S., K.K., T.C.C.F.; funding acquisition, S.F.d.A.C., A.B.C.S., M.C.B., V.G.M.d.O., P.A.d.M.C., K.K. and T.C.C.F. All authors have read and Funding: This research project was funded by Organisation for the Prohibition of Chemical Weapons—OPCW, agreedgrant number to the published L/ICA/ICB version/201062 /of15 the (2016-2018) manuscript. and Brazilian Army. It was also supported by This work was also Funding:supported This by the research Czech project Science was Foundation funded (no.by Organisation 18-01734S) and for Universitythe Prohibition of Hradec of Chemical Kralove Weapons—OPCW, (Faculty of Science, no. VT2019-2021). grant number L/ICA/ICB/201062/15 (2016-2018) and Brazilian Army. It was also supported by This work was Acknowledgments:also supported by theAuthors Czech Science would Foundation like to thank (no. Brazilian 18-01734S) Army and and Universi Walterty Morsof Hradec Institute Kralove of Research (Faculty onof Science,Natural Products—Federalno. VT2019-2021). University of Rio de Janeiro, Brazil, for infrastructure, Brazilian National Authority and Brazilian Ministry of Defense for support, University of Hradec Králové for technical and financial cooperation. Acknowledgments:S.F.d.A.C. also acknowledges Authors would OPCW like and to Organic thank Brazilian Synthesis Army Group an atd Spiez Walter Laboratory—Switzerland Mors Institute of Research for theon NaturalOrganic Products—Federal Synthesis Fellowship University in 2012. Theof Rio Authors de Janeiro, also acknowledgesBrazil, for infrastructure, Bentham Science Brazilian Publishers National for Authority granting the use of the Figure9, published originally in the Ref. 4, and Angelo A. T. Silva (Federal Institute of Rio de Janeiro andat Nil Brazilianópolis, Rio Ministry de Janeiro, of Brazil)Defense for for discussions support, onUniver Graphicalsity of Abstract. Hradec Králové for technical and financial cooperation. S.F.d.A.C. also acknowledges OPCW and Organic Synthesis Group at Spiez Laboratory— SwitzerlandConflicts of Interest:for the OrganicThe funders Synthesis had no Fellowship role in the design in 2012. of theThe study; Authors in the also collection, acknowledges analyses, Bentham or interpretation Science of data; in the writing of the manuscript, or in the decision to publish the results. Publishers for granting the use of the Figure 9, published originally in the Ref. 4, and Angelo A. T. Silva (Federal InstituteDisclaimer: of RioDue de to Janeiro information at Nilópolis, presented Rio in de this Jane manuscript,iro, Brazil) the for Authors discussions and Publisheron Graphical should Abstract. not be responsible or held accountable for misuse or incorrect handling of toxic organophosphorus compounds. Readers must Conflictsaccess the websiteof Interest: of the The Organization funders had for theno Prohibitionrole in the of design Chemical of Weaponsthe study; (OPCW, in the www.opcw.org collection, analyses,) and read or interpretationcarefully Chemical of data; Weapons in the writ Conventioning of the to manuscript, obtain further or in sources the decision of information to publish about the results. legal framework when working with the organophosphorus chemistry. Disclaimer: Due to information presented in this manuscript, the Authors and Publisher should not be responsible or held accountable for misuse or incorrect handling of toxic organophosphorus compounds. Readers must access the website of the Organization for the Prohibition of Chemical Weapons (OPCW, www.opcw.org) and read carefully Chemical Weapons Convention to obtain further sources of information about legal framework when working with the organophosphorus chemistry.

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