Synthesis, molecular modelling and biological evaluation of novel heterodimeric, multiple ligands targeting cholinesterases and amyloid beta

Cholinesterases and amyloid beta are one of the major biological targets in the search for a new and efficacious treatment of Alzheimer’s disease. The study describes synthesis and pharmacological evaluation of new compounds designed as dual binding site acetylcholinesterase inhibitors. Among the synthesized compounds, two deserve special attention—compounds 42 and 13. The former is a saccharin derivative and the most potent and selective acetylcholinesterase inhibitor (EeAChE IC50 = 70 nM). Isoindoline-1,3-dione derivative 13 displays balanced inhibitory potency against acetyl- and butyrylcholinesterase (BuChE) (EeAChE IC50 = 0.76 μM, EqBuChE IC50 = 0.618 μM), and it inhibits amyloid beta aggregation (35.8% at 10 μM). Kinetic studies show that the developed compounds act as mixed or non-competitive acetylcholinesterase inhibitors. According to molecular modelling studies, they are able to interact with both catalytic and peripheral active sites of the acetylcholinesterase. Their ability to cross the blood-brain barrier (BBB) was confirmed in vitro in the parallel artificial membrane permeability BBB assay. These compounds can be used as a solid starting point for further development of novel multifunctional ligands as potential anti-Alzheimer’s agents

Synthesis and biological evaluation of benzochromenopyrimidinones as cholinesterase inhibitors and potent antioxidant, non-hepatotoxic agents for Alzheimer’s disease

We report herein the straightforward two-step synthesis and biological assessment of novel racemic benzochromenopyrimidinones as non-hepatotoxic, acetylcholinesterase inhibitors with antioxidative properties. Among them, compound 3Bb displayed a mixed-type inhibition of human acetylcholinesterase (IC50 = 1.28 ± 0.03 μM), good antioxidant activity, and also proved to be non-hepatotoxic on human HepG2 cell line.JMC thanks Government of Spain for support (SAF2016-65586-R), JJ and OS thank MH CZ- DRO (UHHK 00179906).We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Longitudinal evaluation of a novel BChE PET tracer as an early in vivo biomarker in the brain of a mouse model for Alzheimer disease

Purpose: The increase in butyrylcholinesterase (BChE) activity in the brain of Alzheimer disease (AD) patients and animal models of AD position this enzyme as a potential biomarker of the disease. However, the information on the ability of BChE to serve as AD biomarker is contradicting, also due to scarce longitudinal studies of BChE activity abundance. Here, we report 11C-labeling, in vivo stability, biodistribution, and longitudinal study on BChE abundance in the brains of control and 5xFAD (AD model) animals, using a potent BChE selective inhibitor, [11C]4, and positron emission tomography (PET) in combination with computerised tomography (CT). We correlate the results with in vivo amyloid beta (Aβ) deposition, longitudinally assessed by [18F]florbetaben-PET imaging. Methods: [11C]4 was radiolabelled through 11C-methylation. Metabolism studies were performed on blood and brain samples of female wild type (WT) mice. Biodistribution studies were performed in female WT mice using dynamic PET-CT imaging. Specific binding was demonstrated by ex vivo and in vivo PET imaging blocking studies in female WT and 5xFAD mice at the age of 7 months. Longitudinal PET imaging of BChE was conducted in female 5xFAD mice at 4, 6, 8, 10 and 12 months of age and compared to age-matched control animals. Additionally, Aβ plaque distribution was assessed in the same mice using [18F]florbetaben at the ages of 2, 5, 7 and 11 months. The results were validated by ex vivo staining of BChE at 4, 8, and 12 months and Aβ at 12 months on brain samples. Results: [11C]4 was produced in sufficient radiochemical yield and molar activity for the use in PET imaging. Metabolism and biodistribution studies confirmed sufficient stability in vivo, the ability of [11C]4 to cross the blood brain barrier (BBB) and rapid washout from the brain. Blocking studies confirmed specificity of the binding. Longitudinal PET studies showed increased levels of BChE in the cerebral cortex, hippocampus, striatum, thalamus, cerebellum and brain stem in aged AD mice compared to WT littermates. [18F]Florbetaben-PET imaging showed similar trend of Aβ plaques accumulation in the cerebral cortex and the hippocampus of AD animals as the one observed for BChE at ages 4 to 8 months. Contrarily to the results obtained by ex vivo staining, lower abundance of BChE was observed in vivo at 10 and 12 months than at 8 months of age. Conclusions: The BChE inhibitor [11C]4 crosses the BBB and is quickly washed out of the brain of WT mice. Comparison between AD and WT mice shows accumulation of the radiotracer in the AD-affected areas of the brain over time during the early disease progression. The results correspond well with Aβ accumulation, suggesting that BChE is a promising early biomarker for incipient AD

Design, synthesis and evaluation of potential drugs for treatment of Alzheimer\u27s disease with multiple mechanisms of action

Nevrodegenerativna obolenja zaradi staranja prebivalstva in posledično večjega števila bolnikov predstavljajo pereč zdravstveni problem. Med najbolj prepoznanimi je Alzheimerjeva bolezen, napredujoča motnja centralnega živčnega sistema, ki vodi v propad predelov možganov, posledično izgubo spomina ter motenj ostalih kognitivnih sposobnosti. Točen mehanizem nastanka in poteka bolezni še vedno ni pojasnjen, znano pa je, da do klinične slike vodi več patoloških procesov na molekularnem in celičnem nivoju. Izrazite poškodbe holinergičnega sistema in upad koncentracije živčnega prenašalca acetil holina privedejo do tipičnih motenj v spominu, ki se lajšajo z zaviralci holin esteraz. Encim butirilholin esteraza (BChE) opravlja v nekaterih predelih možganov podporno vlogo pri zaključevanju holinergičnega prenosa, z napredovanjem bolezni pa lahko ta vloga celo prevlada, saj encim opravi glavnino hidrolazne aktivnosti. Zaradi povečane aktivnosti in izražanja v poznih stadijih boleznih predstavlja BChE pomembno tarčo za lajšanje simptomov v teh stopnjah Alzheimerjeve bolezni. Poleg opisanega igrajo pri razvoju bolezni pomembno vlogo tudi številni drugi procesi. Nastanek in agregacija amiloida beta do amiloidnih plakov je ena od najbolj izrazitih patoloških sprememb, opaženih pri bolnikih. Različne zvrsti amiloida beta povezujejo z nevrotoksičnostjo, h kateri pa pomembno prispevajo tudi povečan oksidativni stres in motnje v porazdelitvi biološko pomembnih dvovalentnih kovinskih ionov. Trenutna simptomatska terapija Alzheimerjeve bolezni nima velikega vpliva na potek bolezni. Za učinkovito zdravljenje in izboljšanje simptomov bi bilo zato potrebno delovati na več tarč hkrati. Razvoj potencialnih učinkovin, ki bi lahko vzročno vplivale na nastanek in potek bolezni, se je tako usmeril v načrtovanje učinkovin z multiplim mehanizmom delovanja (multifunkcionalne spojine). Selektiven zaviralec BChE 1 (IC50 = 21,3 nM), ki je bil rezultat uspešnega strukturno podprtega virtualnega rešetanja, in njegovo kristalno strukturo v kompleksu s človeško BChE, smo uporabili kot osnovo za načrtovanje multifunkcionalnih ligandov predstavljenih v tem delu. Spojina 1 zavira spontano agregacijo amiloida beta do toksičnih oligomerov in fibrilov, zaradi česar deluje nevroprotektivno. Ob upoštevanju pomembne vloge kovinskih ionov v nastanku Alzheimerjeve bolezni, smo naftalen spojine 1 zamenjali z nitroksolinom, ki deluje kot kelator dvovaletnih kovinskih ionov. Spojina 8g z nanomolarno zaviralno aktivnostjo na človeški BChE (IC50 = 215 nM) selektivno kompleksira bakrove(II) ione in zavira spontano agregacijo amiloida beta, hkrati pa ima tudi šibek antioksidativni učinek. Kristalna struktura te spojine v kompleksu s človeško BChE je razkrila vezavo nitroksolina v acil vezavni žepek in s tem pokazala, da je ta del vezavnega mesta sposoben vezati tudi od naftalena večje fragmente. Derivate nitroksolina tako smatramo kot dobro izhodišče za nadaljnjo optimizacijo učinkovin za terapijo Alzheimerjeve bolezni z multiplim mehanizmom delovanja. Vzporedno z razvojem multifunkcionalnih ligandov je potekala tudi optimizacija zaviralne aktivnosti spojine zadetka 1 ter raziskovanje odnosa med strukturo in zaviralnim delovanjem analogov. Velika večina sprememb v strukturi zaviralca je poslabšala zaviralno aktivnost. Z nadomestitvijo metoksietilne verige spojine 1 z 2-(dimetilamino)etilno verigo pa smo uspeli izkoristiti še dodatno interakcijo s Trp82 človeške BChE, kar je povečalo moč zaviranja, ki se pri spojini 3 giblje v pikomolarnem območju. Spojina je ohranila način vezave v aktivno mesto BChE in šibek nevroprotektiven učinek. Glede na pomembno vlogo oksidativnega stresa in motenj v porazdelitvi kovinskih ionov, smo v molekulo najboljšega reverzibilnega zaviralca BChE s piperidinsko strukturo 3 uvajali različne fragmente s sposobnostjo kompleksacije kovinskih ionov in antioksidativnimi lastnostmi. Spojini 8 in 11 z IC50 vrednostima za človeško BChE 13,8 ± 0,6 oziroma 11,1 ± 0,6 nM, spadata med najboljše zaviralce iz te serije. Kristalna struktura kompleksa spojine 11 in človeške BChE je, podobno kot za nitroksolinski derivat, pokazala vezavo 8-hidroksikinolinskega fragmenta v acil vezavni žep encima. Spojina je v kokristalu vezana tudi na površini med dvema monomeroma encima, kjer vezavno mesto tvorita dva plitva žepa. Alternativna vezava po vsej verjetnosti nima vpliva na aktivnost encima, spojina pa bi lahko bila potencialno uporabna kot spodbujevalec kristalizacije človeške BChE. 8-hidroksikinolinska derivata 8 in 11 kažeta tudi dobre antioksidativne lastnosti in kompleksacijo bakrovih(II) ionov, ki po vezavi v kompleks niso več redoks aktivni. Kljub temu, da spojini ne zavirata procesa spontane agregacije amiloida beta, spojina 11 ščiti nevroblastomske SH-SY5Y celice pred toksičnostjo fibrilov amiloida beta. Poleg navedenega oba analoga znižata znotrajcelične koncentracije reaktivnih kisikovih zvrsti, s pasivno difuzijo prehajata membrane v Caco2 celičnem modelu ter nista substrata za prenašalne proteine (primer P-glikoprotein). Derivata 8 in 11 zaradi dobrega profila dodatnih aktivnosti v primerjavi z nitroksolinskimi analogi in spodbudnih rezultatov na celičnih testih predstavljata naslednjo generacijo izboljšanih multifunkcionalnih ligandov za terapijo Alzheimerjeve bolezni. Poleg že vseh naštetih faktorjev je pri Alzheimerjevi bolezni opazna tudi povečana aktivnost monoamin oksidaze v določenih predelih možganov. Povečana oksidativna deaminacija zniža koncentracijo živčnih prenašalcev, kar privede do motenj v vedenju. Pri metabolizmu nastane tudi več toksičnih produktov, ki še dodatno prispevajo k oksidativnemu stresu in nevrodegeneraciji, zato predstavlja monoamin oksidaza dobro tarčo, preko katere lahko vplivamo tudi na vzroke nastanka Alzheimerjeve bolezni. Zelo pogosto se zaviralna aktivnost na monoamin oksidazi združi z zaviranjem ene ali obeh holin esteraz. V tem primeru govorimo o t.i. dualnih zaviralcih, ki jih je v literaturi opisanih kar nekaj, in jih lahko razdelimo na tri razrede: derivati propargilamina, kumarini in derivati naravnih spojin. Najbolj raziskana je zagotovo prva skupina, kjer spojina ASS234 in ladostigil kažeta dobre rezultate v predkliničnih testiranjih, slednji pa tudi v kliničnih fazah testiranj. Preko uvedbe propargilne skupine na piperidinski dušik analoga spojine zadetka 1 smo pri spojini 6 dosegli dualno mikromolarno zaviranje dveh encimov, BChE (IC50 = 2,600 ± 0,348 μM) in monoamin oksidaze B (IC50 = 53,904 ± 4,781 μM). Zaviranje monoamin oksidaze B je ireverzibilno in časovno odvisno, kar sovpada z literaturno znanimi podatki o zaviralcih propargilaminskega tipa. Spojina 6 v testu prehajanja s sistemom vzporednih umetnih membran kaže velik potencial za prehajanje krvno možganske pregrade, za nevroblastomske celice ni citotoksična, sočasno pa jih ščiti tudi pred toksičnostjo amiloida beta. N-propargilpiperidinske dualne zaviralce BChE in monoamin oksidaze B lahko tako smatramo kot dobro izhodišče za nadaljnjo optimizacijo in razvoj multiplih ligandov za terapijo Alzheimerjeve bolezni.Due to the aging of the population and the concomitant increase in the number of people with neurodegenerative diseases, these disorders are becoming an enormous health care problem. Among the most renowned is the Alzheimer\u27s disease, a progressive disorder of central nervous system leading to dysfunctions in certain areas of the brain, and subsequent loss of memory and disturbances in other cognitive functions. The exact cause of the disease is still unknown, yet several pathological processes on molecular and cellular level lead to the observed clinical picture. Alterations in the cholinergic system and reduced levels of neurotransmitter acetylcholine lead to typical memory loss, which can be alleviated by cholinesterase inhibitors. Butyrylcholinesterase (BChE) helps with the termination of the cholinergic neurotransmission in some parts of the brain. With the progression of the disease its hydrolytic function becomes more important and can even take over the cholinesterase activity. Due to the increased activity and expression in latter stages of Alzheimer\u27s disease, BChE represents an important target for the alleviation of symptoms in the late stages of Alzheimer\u27s disease. Several other factors significantly contribute to the development of Alzheimer\u27s disease. Aggregation of amyloid beta peptide and the formation of senile plaques are one of the hallmark pathological changes. Apart from various amyloid beta species, increased oxidative stress and disturbances in bivalent metal ion distribution are also related to neurotoxicity. Current symptomatic management of Alzheimer\u27s disease does not have any significant effect on the disease progression, thus several pathological changes should be targeted simultaneously to address the underlying cause. The development of new drugs has therefore shifted towards multi-target-directed ligands (multifunctional ligands) capable of interacting with the factors related to the disease development and progression. Selective BChE inhibitor 1 (IC50 = 21.3 nM), a result of a successful structure based virtual screening, and its crystal structure in complex with human BChE was used as the basis for the design of multifunctional ligands presented herein. This compound also inhibits self-induced aggregation of amyloid beta to oligomers and fibrils and has a neuroprotective activity. Taking into the account the important role of metal ions in the disease pathogenesis, the naphthalene moiety of 1 was replaced with nitroxoline, a known chelating agent of bivalent metal ions, to yield the multifunctional ligand 8g. Compound 8g, with nanomolar inhibitory potency against human BChE (IC50 = 250 nM), also selectively chelates copper(II) ions, inhibits amyloid beta aggregation, and has weak antioxidant activity. The co-crystal structure of 8g with human BChE reveals the binding of the nitroxoline into the acyl binding pocket and proves that this part of the active site gorge can indeed accommodate fragments, bigger than naphthalene. This nitroxoline derivative can therefore be considered as a good starting point for further optimization of multi-target-directed ligands against Alzheimer\u27s disease. In parallel to the development of multifunctional ligands, the optimization of compound 1 was performed in order to increase the inhibitory potency and to elucidate the structure-activity relationships. The majority of structural modifications led to diminished inhibitory potency. On the other hand, the replacement of methoxyethyl side chain of 1 with 2-(dimethylamino)ethyl moiety sequestered an additional interaction with Trp82 of human BChE, which led to increased inhibitory potency. Compound 3 is a picomolar BChE inhibitor with the same binding mode into the BChE active site gorge as observed for 1. Derivative 3 also shows weak neuroprotective activity. The structure of the most potent reversible piperidine-based inhibitor 3 was used to design another series of multifunctional ligands with metal chelating and antioxidant properties. Compounds 8 and 11 with IC50 values for human BChE 13.8 ± 0.6 nM and 11.1 ± 0.6 nM, respectively, are among the best inhibitors in the series. Resolved crystal structure of compound 11 in human BChE has once again confirmed the binding of 8-hydroxyquinoline moiety in the acyl binding pocket as observed before for the nitroxoline analog. The compound is also bound to the interface of monomeric units of BChE where the binding site is composed of shallow pockets, one on each monomer. This alternative binding probably does not affect the enzyme activity, nonetheless the compound could be useful as an inductor of human BChE crystallization. 8-Hydroxyquinoline derivatives 8 and 11 are also good antioxidants and chelators of copper(II) ions. The complexes of compounds 8 and 11 with copper(II) are not redox active. No effect on amyloid beta self-induced aggregation has been observed, however compound 11 protects neuroblastoma SH-SY5Y cell from toxic amyloid beta fibrils. Both analogs reduce the intracellular levels of reactive oxygen species, penetrate the membrane in the Caco2 cell model via passive diffusion, and are not substrates of any efflux systems (e.g. P-glycoprotein) in the mentioned cell line. These derivatives with additional activities in comparison to the nitroxoline analogs, and good results in the cell based assays represent the next generation of multifunctional ligands for the treatment of Alzheimer\u27s disease. In addition to all of the described factors of Alzheimer\u27s disease, increased activity of monoamine oxidase is detected in particular areas of the brain. Higher level of oxidative deamination diminishes the concentrations of neurotransmitters and this further leads to disturbances in the behavior. In the metabolic reactions of monoamine oxidase additional toxic products are generated, which augment the observed oxidative stress and neurodegeneration. This enzyme thus represents another target through which the effect on the cause of the Alzheimer\u27s disease can be achieved. Often the inhibitory activity on monoamine oxidase is combined with cholinesterases inhibition. Three main classes of dual inhibitors are described in the literature: propargylamine derivatives, coumarins and derivatives of natural compounds. The first group is the most studied one, where compound ASS234 and ladostigil display promising results in preclinical studiesthe latter is also in clinical trials. The attachment of the propargyl group onto the piperidine nitrogen of the hit compound 1 resulted in compound 6, a dual micromolar BChE (IC50 = 2.600 ± 0.348 μM) and monoamine oxidase B (IC50 = 53.904 ± 4.781 μM) inhibitor. The mechanism of monoamine oxidase B inhibition is time-dependent and irreversible, which is in accordance with the literature data on propargylamine based inhibitors. Compound 6 crosses the blood brain barrier as determined in vitro in parallel artificial membrane permeability assay, is not toxic to neuroblastoma cells, and protects them against amyloid beta induced toxicity. N-propargylpiperidine based dual BChE/monoamine oxidase B inhibitors represent a solid ground for additional optimization and development of multifunctional anti-Alzheimer\u27s disease agents

JURISDICTION OF POLITICAL, MILITARY AND OTHER NATO BODIES IN DECISION MAKING AND CONDUCTING MILITARY OPERATIONS, EXAMPLES OF YUGOSLAVIA, IRAQ, AFGHANISTAN AND LIBYA

PRISTOJNOSTI POLITIČNIH, VOJAŠKIH IN DRUGIH TELES NATO PRI ODLOČANJU IN IZVAJANJU VOJAŠKIH OPERACIJ – PRIMERI JUGOSLAVIJE, IRAKA, AFGANISTANA IN LIBIJE Organizacija Severnoatlantske pogodbe – NATO (North Atlantic Treaty Organization) je mednarodno vojaško-politična organizacija, ki predstavlja čezatlantsko vez – povezavo varnosti v Severni Ameriki z varnostjo v Evropi. Kolektivna prizadevanja članic so v podporo skupnim varnostnim interesom. Spreminjanje razmer v mednarodnem varnostnem okolju je narekovalo tudi prilagajanje zveze. Njen obstoj je povezan z njeno večnamenskostjo in prilagodljivostjo spremembam v okolju. Severnoatlantsko zavezništvo je bilo ustanovljeno 4. aprila 1949 z Washingtonsko pogodbo med 12 državami članicami, ki so k njej pristopile prostovoljno, na podlagi javne razprave in ustreznega parlamentarnega postopka. Leta 2012 je v zvezo NATO vključenih 28 držav članic. Pogodba podpira njihove individualne pravice ter mednarodne obveznosti v skladu z 51. členom Ustanovne listine Združenih narodov. Ključni členi Severnoatlantske pogodbe so: 4., 5. ter 6. člen. V času svojega obstajanja je Nato za svoje delovanje oblikoval in sprejel več strateških konceptov. Zadnji je bil sprejet novembra 2010. S spreminjanjem vloge Nata se spreminjajo in s tem širijo tudi njegove pristojnosti. V času hladne vojne in sovjetske nevarnosti je bila v ospredju vojaško-obrambna naravnanost. Po koncu hladne vojne in razširitvah pa je zavezništvo omogočilo mirnodobno sodelovanje med državami podpisnicami tako na vojaškem kot tudi na političnem, gospodarskem, znanstvenem, omejeno celo na kulturnem področju. Njegov namen je poleg zagotavljanja varnosti svojim članicam, prispevati k miru, stabilnosti, sodelovanju in demokratičnemu razvoju tudi na drugih celinah v evroatlantskem prostoru in s tem prispevati k svetovnemu miru. Dejavnosti Organizacije severnoatlantske pogodbe so podprte z zapleteno civilno in vojaško strukturo. Delovna telesa, ki se ustanavljajo in spreminjajo glede na obseg in potrebe, imajo določene pristojnosti in naloge. Slovenija je v Natu od leta 2004 ter sodeluje s predstavniki v delovnih telesih. Ima svoje obveznosti in pristojnosti. Slovenska vojska se vključuje v mednarodne operacije in misije Nata. Kot organizacija za kolektivno samoobrambo in varnostno sodelovanje deluje zveza NATO pod vodstvom Varnostnega sveta OZN ali vsaj usklajeno z njim. Pri različnih posegih v primeru kriz izven ozemlja zavezništva gre za naloge izven določil 5. člena Severnoatlantske pogodbe. Mirovne operacije obravnavajo v Natu kot del kriznega upravljanja. Ustanovili so sile za posredovanje, ki so v stalni visoki pripravljenosti. Posredovanja, ki so potekala izven območij držav članic zavezništva, so tudi posredovanja v Jugoslaviji, Iraku, Afganistanu in Libiji. Natovi posegi, ki niso povezani s kolektivno obrambo, niso nezakoniti, če so v skladu z odločitvami VS ZN. Problem legalnosti posega Nata pa se pojavi ob posredovanjih, ki nimajo jasnega mandata VS ZN za operacijo. Natovi vojaški napadi v Bosni in Hercegovini, Afganistanu in Libiji so bili izvedeni na podlagi resolucij VS ZN. Oborožena akcija zoper Zvezno republiko Jugoslavijo v zvezi s položajem na Kosovu ni imela mandata VS ZN. Pomoč Natovih sil Makedoniji pri razoroževanju Osvobodilne vojske Kosova in skupin albanskih skrajnežev prav tako ni imela podlage v mandatu ZN. Brez predhodne odobritve VS ZN je bila tudi invazija Amerike in Velike Britanije na Irak, pri kateri Nato kot organizacija ni imel nobene vloge pri odločitvi za izvedbo napada in nekatere članice zveze ga tudi niso podprle. Dodatne zaščite Turčiji pred morebitnimi zračnimi napadi Iraka ni odobril Severnoatlantski svet, ki je najvišje politično telo za odločanje in posvetovanje v zvezi Nato, ker so temu nasprotovale tri članice zavezništva. Sklep o ukrepih za zaščito Turčije je sprejel Komite za obrambno načrtovanje Nata. Slednje nakazuje problem znotraj zavezništva v zvezi z odločanjem ob posegu izven območja držav članJURISDICTION OF POLITICAL, MILITARY AND OTHER NATO BODIES IN DECISION MAKING AND CONDUCTING MILITARY OPERATIONS – EXAMPLES OF YOUGOSLAVIA, IRAQ, AFGHANISTAN AND LIBYA NATO (North Atlantic Treaty Organization) is an international military-political organization, which represents a transatlantic bond – it is a link of safety in North America on one hand and in Europe on the other. Collective efforts of its member states support their common security interests. Changes in the international security environment demand its flexibility. Its existence is connected with its versatility and adaptability to changes of the environment. North-Atlantic Alliance was established on 4th April, 1949 by The Washington Treaty among the twelve member states. They joined the alliance voluntarily on the basis of public debate and parliamentary procedure. There are twenty-eight member states of NATO in 2012. The treaty supports their individual rights and international obligations in accordance with Article 51 of the UN Charter. Articles 4, 5 and 6 are key articles of the North Atlantic Treaty. NATO has formulated and adopted several strategic concepts during its existence. The last one was adopted in November 2010. The role of NATO changes and along with it its jurisdiction changes and expands. During the cold war and the soviet threat the military-defensive stance was at the forefront. The end of the cold war and the expansion of the alliance enabled peacetime cooperation of the member states on the military as well as on other fields: policy, economy, science, even, though a limited cooperation, in the field of culture. Its purpose is to ensure safety of all the member states, as well as contribute to peace, stability, cooperation and democratic development in other continents of the Euro-Atlantic area and thus contribute to global peace. Activities of NATO are supported by a complex civil and military structure. Working bodies, which are established and change according to size and needs, have certain jurisdiction and duties. Slovenia has been in NATO since 2004 and has been cooperating with representatives of its working bodies. It has its obligations and responsibilities. Slovenian Armed Forces are included in international operations and NATO missions. As an organization for collective self-defence and security cooperation NATO functions under the direction of UN Security Council or at least in coordination with it. Different interventions in cases of crises off the territory of the alliance represent tasks which are not included in Article 5 of the North-Atlantic Treaty. Peacekeeping operations are understood by NATO as part of crisis management. Forces for interventions have been founded and they are in permanent high readiness. Interventions which have taken place off the area of the alliance are also those in Yugoslavia, Iraq, Afghanistan and Libya. NATO interventions, which are not related to collective defence, are not illegal, provided they comply with the decisions of the UN Security Council. The problem of the legality of a NATO intervention appears at those interventions that have no clear mandate of the UN Security Council. The military attacks of NATO in Bosnia and Herzegovina, Afghanistan and Libya were conducted in the framework of UN Security Council resolutions. The armed campaign against Federal Republic of Yugoslavia regarding the situation in Kosovo had no mandate of UN Security Council. And the help of NATO forces to Macedonia at the disarmament of the Liberation Army of Kosovo and groups of Albanian extremists as well had no basis in the Security Council mandate. The invasion of Iraq by the USA and the UK had no prior approval of UN Security Council. In this case NATO as an organization had no role in the decision about the carrying out of the attackit was not supported by some alliance members. Additional protection of Turkey in case of possible Iraqi air strikes was not approved by North-Atlantic Council, which is the highest political body for making decisions and consultation in NATO, because three a

Screening of big pharma’s library against various in-house biological targets

Open innovation initiatives provide opportunities for collaboration and sharing of knowledge and experience between industry, academia, and government institutions. Through open innovation, Merck is offering a Mini Library of 80 carefully selected compounds from previous research and development projects to a broader scientific community for testing in academic drug discovery projects. These compounds are predominantly drug-like and cover a broad range of molecular targets. They could potentially interact with other enzymes, receptors, transporters, and ion channels of interest. The Mini Library was tested on seven in-house enzymes (bacterial MurA, MurC ligase, and DdlB enzyme, human MAO-A/B, human BChE, and murine AChE), and several hits were identified. A follow-up series of structural analogues provided by Merck gave a more detailed insight into the accessibility and the quality of the hit compounds. For example, sartan derivatives were moderate inhibitors of MurC, whereas bisarylureas were potent, selective, nanomolar inhibitors of hMAO-B. Importantly, 3-n-butyl-substituted indoles were identified as low nanomolar selective inhibitors of hBChE. All in all, the hit derivatives provide new starting points for the further exploration of the chemical space of high-quality enzyme inhibitors

Redox active or thiol reactive?

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action

Tunable heteroaromatic nitriles for selective bioorthogonal click reaction with cysteine

The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking molecules in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroaromatic nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chemical space of heteroaromatic nitriles for bioconjugation under physiologically relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2-cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles containing electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compounds were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2-cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chemical biology, and nanomaterial science

4-phenethyl-1-propargylpiperidine-derived dual inhibitors of butyrylcholinesterase and monoamine oxidase B

The multi-target-directed ligands (MTDLs) strategy is encouraged for the development of novel modulators targeting multiple pathways in the neurodegenerative cascade typical for Alzheimer’s disease (AD). Based on the structure of an in-house irreversible monoamine oxidase B (MAO-B) inhibitor, we aimed to introduce a carbamate moiety on the aromatic ring to impart cholinesterase (ChE) inhibition, and to furnish multifunctional ligands targeting two enzymes that are intricately involved in AD pathobiology. In this study, we synthesized three dual hMAO-B/hBChE inhibitors 13–15, with compound 15 exhibiting balanced, low micromolar inhibition of hMAO-B (IC$_{50}$ of 4.3 µM) and hBChE (IC$_{50}$ of 8.5 µM). The docking studies and time-dependent inhibition of hBChE confirmed the initial expectation that the introduced carbamate moiety is responsible for covalent inhibition. Therefore, dual-acting compound 15 represents an excellent starting point for further optimization of balanced MTDLs