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Implementation of Suzuki cross coupling reaction for preparation of indole and coumarin derivatives as potential inhibitors of 17ß-hydroksysteroid dehydrogenase type 1
No full textThe design and synthesis of new derivatives of 8-hydroxy-5-nitroquinoline and 4-nitrophenol as inhibitors of cathepsin B
No full textEvaluation of substituted psoralenes as inhibitors of the LMP7 subunit of immunoproteasome
Get PDFProteasom je multikatalitični kompleks, ki homeostazo celice vzdržuje preko
odstranjevanja nepravilno zvitih in poškodovanih proteinov ter drugih signalnih molekul,
ki nastanejo tekom različnih celičnih procesov. Preoteasom obstaja v več oblikah, a sta
med pomembnejšimi standardni in imunoproteasom. Slednji ima pomembno vlogo
predvsem v oblikovanju imunskega odziva. Povezujejo ga s številnimi patološkimi stanji
(avtoimunskimi, rakavimi in nevrodegenerativnimi obolenji), zato s svojimi katalitičnimi
podenotami (MECL-1, LMP2 in LMP7) predstavlja vedno bolj zanimivo farmakološko
tarčo. Tekom magistrske naloge smo izvedli 5-stopenjsko sintezo potencialnega zaviralca
LMP7 podenote i-proteasoma ter ga skupaj s še 47 drugimi, predhodno sintetiziranimi
analogi psoralenskega skeleta biokemijsko ovrednotili.
Najprej smo s Pechmannovo kondenzacijo med rezorcinolom in dietil-2-acetilsukcinatom
sintetizirali osnovni kumarinski skelet. Nato smo na mestu 7-kumarinskega skeleta
hidroksilni skupini z reakcijo nukleofilne substitucije pripeli 2-bromoacetofenon in dobili
predhodnico psoralenskega skeleta, katero smo z reakcijo kondenzacije in posledično
ciklizacijo pretvorili do slednjega. V zadnjih dveh stopnjah smo aktivirano karboksilno
skupino pretvorili v amidno skupino, kamor smo vezali klorokarbonilsulfenil klorid in
dobili končno spojino z 1,3,4-oksatiazol-2-onom kot elektrofilom.
V sklopu biokemijskega vrednotenja smo sprva pri 10 μM koncentracijah spojin določili
rezidualno aktivnost LMP7 katalitične podenote i-proteasoma. Na podlagi rezidualne
aktivnosti smo izbrali 8 spojin z najmočnejšimi zaviralnimi lastnostmi in jih podrobneje
ovrednotili s parametroma encimske kinetike Ki in IC50. Za vse spojine so bile vrednosti v
nizko mikormolarnem območju. Ugotovili smo, da je za zaviralno aktivnost i-proteasoma v
terminalnem delu psoralenskega skeleta pomemben sukcimidni ester ali oksatiazolon za
ireverzibilne zaviralce in karboksilna skupina za reverzibilne, čeprav slednja ni izkazala
močnejših zaviralnih sposobnosti. Med funkcionalnih skupinami na furanskem obroču
psoralenskega skeleta so najmočneje zavirale aromatske spojine in sicer spojine, ki imajo
2,5-dimetiltiofenski, furanski in nesubstituiran benzenov obroč. Dobljeni podatki
RANČNIK, Pia: Vrednotenje substituiranih psoralenov kot zaviralcev LMP7 podenote imunoproteasoma.
predstavljajo dobro osnovo za nadaljnje načrtovanje in sintezo zaviralcev LMP7 podenote
i-proteasoma, ki bodo imeli izboljšane zaviralne lastnosti.The most significant function of proteasome is to maintain proteins homeostasis with
removal of misfolded and damaged proteins, which occurs in different cellular processes.
His subtype, immunoproteasome, is preferentially formed under impact of
immunomodulatory cytokines. Antigene presentation to cytotoxic T lymphocytes, their
proliferation and differation as well as regulation of cytokines production and other
immune functions of immunoproteasome with his catalytic subunits represent more and
more interesting pharmacological target. Its involvment in disease progression is linked
with specific autoimmune, malignant and neurodegenerative diseases. Within this masters
degree we successfully synthesized a potential inhibitor of the LMP7 subunit of the
immunoproteasome in 5 reaction steps. The biochemical evaluation was completed with
this and also other 47 psoralen analogues that have previously been synthesized.
Starting with Pechamann condensation, reaction of diethyl acetosuccinate with resorcinol
was carried out, yielding 7-hydroxycoumarin ring. The precursor of psoralen skeleton was
synthesized by nucleophile substitution where hidroxyl group was replaced by 2-
bromoacetophenone. Afterwards, cyclisation to form psoralen skeleton has been achieved
by condensation. In the last two steps carboxyl group was modified to amide group and
chlorocarbonylsulfenyl chloride was added to obtain the final compound with 1,3,4-
oxathiazolon as an electrophile.
Determination of residual activity was carried out. 8 compounds with good inhibitory
properties were identified. For these compounds parameters of enzyme kinetics (Ki in
IC50) were examined in detail. The key structural requirements significant for the
immunoproteasome inhibition of the psoralen analogues are succinimide ester or
oxathiazolone which are essential for irreversibile inhibitors and carboxylic acid for
reversibile inhibitors, however the last one does not show strong inhibitory properties.
Among the functional groups on the furane on the psoralen skeleton, the aromatic
compounds such as 2,5-dimethyl tiofen, furan and unsubstituted phenyl group showed the
strongest inhibitory properties. These and other compounds based on psoralen skeleton
represent a topic of further structural investigations and improvement of inhibition of the
immunoproteasome subunit LMP7
Design and synthesis of psoralene-based inhibitors of the immunoproteasome
Get PDFV zadnjih 30 letih so številne študije natančno okarakterizirale strukturo in fiziološko
vlogo ubikvitin proteasomskega sistema. Gre za sofisticiran 2,5 MDa velik proteinski
sistem imenovan 26S proteasom, ki je sestavljen iz proteolitičnega osrednjega 20S
jedrnega dela in dveh terminalnih regulatornih 19S delov.
Proteasom je multikatalitični encimski kompleks, ki regulira številne ključne fiziološke in
patološke celične procese preko selektivne razgradnje proteinov. Identifikacija in
odstranitev napačno zvitih, poškodovanih in toksičnih proteinov je ključna za regulacijo in
vzdrževanje celične homeostaze.
Substrati za proteasom so tudi številni proteini, ki so del karcinogeneze in so ključnega
pomena za preživetje rakave celice. Inhibicija proteasoma v rakavih celicah tako vodi v
akumulacijo proapoptotičnih tarčnih proteinov ter indukcijo celične smrti.
Področje onkologije daje velik poudarek na identifikaciji novih in za raka specifičnih
molekularnih tarč. Eden izmed takšnih tarčnih sistemov je tudi imunoproteasom, natančno
reguliran, visoko specifičen sistem, odgovoren za procesiranje številnih proteinov znotraj
celice. Odkritje, da so tumorske celice veliko bolj občutljive na delovanje zaviralcev
proteasoma kot ostale celice, je usmerilo razvoj v načrtovanje in sintezo številnih
terapevtsko uspešnih zaviralcev. S poznavanjem strukture in delovanja predstavlja danes
ubikvitin proteasomski sistem obetavno tarčo v terapiji številnih rakavih obolenj.
V okviru magistrske naloge smo uspešno sintetizirali več potencialnih zaviralcev
imunoproteasoma. Vse pripravljene spojine imajo kot osnovo psoralenski skelet, na
katerega je na mestu 4 vezana metilna skupina. Z namenom raziskati kemijski prostor smo
na izbrana mesta na skeletu uvajali različne substituente.
Sintezo načrtovanih spojin smo izvajali v več zaporednih reakcijah. Najprej smo iz
resorcinola ter dietil 2-acetilsukcinata ali dietil 2-acetilpentandionata sintetizirali osnovni
7-hidroksikumarinski obroč. Sledila je uvedba derivatov 2-bromoacetofenona na hidroksi
skupino. Nato smo izvedli reakcijo kondenzacije, pri čemer je prišlo do ciklizacije in
nastanka psoralenskega strukturnega fragmenta. Na C-terminalni del smo nazadnje uvajali
še elektrofilne fragmente, in sicer 1-hidroksipirolidin-2,5-dion, 2-(metilamino)acetonitril ali 2-aminoacetonitril, s čimer smo preko kovalentnih interakcij s katalitično aminokislino
v aktivnem mestu želeli doseči močnejšo vezavo sintetiziranih spojin na imunoproteasom.
Na Katedri za klinično biokemijo na Fakulteti za farmacijo so nato sintetizirane spojine še
biokemijsko ovrednotili. Določevali so rezidualno aktivnost kimotripsinske podenote
imunoproteasoma po dodatku spojine. Po pridobljenih podatkih ima največje zaviralno
delovanje spojina z oznako 5c. Na podlagi rezultatov lahko sklepamo, da je ključnega
pomena tvorba aktiviranega estra, ki lahko tvori kovalentno interakcijo s treoninom v
aktivnem mestu imunoproteasoma. Pretvorba C-terminalne karboksilne kisline v amid ter
uvedba N-cianometilamidov kot elektrofilov ne vodita v boljše zaviralno delovanje. Prav
tako vezava broma na para mesto fenilnega obroča ne pripomore k povečanju afinitete za
vezavo na imunoproteasom.In the past 30 years there were several studies that have thoroughly characterized the
structure and the physiological function of the ubiquitin proteasome system. This is a
sophisticated 2.5 MDa protein complex named 26S proteasome, which consists of 20S core
particle and two terminal 19S regulatory particles.
The proteasome is a complex multicatalytic enzymatic system that regulates vital
physiological and pathological cellular processes through selective breakdown of the
proteins. Identification and removal of misfolded, damaged and toxic proteins is crucial for
the regulation and maintenance of cell homeostasis.
Proteins that are involved in the processes of carcinogenesis and cancer cell survival are
also substrates for the proteasome. Its inhibition in cancer cells therefore leads to
accumulation of pro-apoptotic proteins and induction of cell death.
The cancer drug discovery puts a significant emphasis on the identification of new and
cancer-specific molecular targets. One of the most recently discovered and very promising
targets is also the immunoproteasome, tightly regulated, highly-specific system that is
responsible for cellular protein turnover. The discovery that cancer cells are more
susceptive to proteasome inhibition than other cells had a strong impact on the discovery
and synthesis of novel therapeutically useful inhibitors. The knowledge and recent findings
associated with the structure and the functions of the ubiquitin proteasome system make
this enzyme machinery a promising target in cancer treatment.
Within this masters degree, we successfully synthesized several potential
imunoproteasome inhibitors. The prepared compounds had a psoralen core scaffold with
methyl group bound at the position 4. With the aim to explore the chemical space, we
introduced several carefully planned substituents on the main psoralen structure.
The synthesis of compounds was performed in several consecutive reaction steps. Firstly,
we synthesized the 7-hydroxycoumarin ring by reacting diethyl 2-acethylsuccinate or
diethyl 2-acetylpentanedionate with resorcinol. Then, different 2-bromoacetophenones
were introduced to properly modify the hydroxyl group. Afterwards, the condensation
reaction was carried out, leading to the formation of the core psoralen ring. Finally, we introduced three different electrophilic moieties at the C-terminus, such as 1-
hydroxypyrrolidine-2,5-dione, 2-aminoacetonitrile or 2-(methylamino)acetonitrile. The
purpose of this last step was to achieve covalent bonding with the catalytic amino acid in
the immunoproteasome active site.
All compounds were biochemically evaluated at the Department of Clinical Biochemistry.
The residual activities of the chymotrypsin-like imunoproteasome subunit after the
addition of compounds were determined. Based on the data, compund 5c showed the most
potent inhibitory activity. On the basis of results from inhibition assays, we can also
conclude that the transformation of the C-terminal carboxylic acid into an activated ester is
crucial for improving inhibitionmost probably because of the formation of covalent bonds
within the active site of the immunoproteasome. Introduction of an amide bond and Ncianomethylamides
as electrophiles did not improve inhibitory effect. In addition, bromine
at the para position of the phenyl fragment led to diminished affinity for binding into the
active site of the immunoproteasome
Retrosynthesis-driven in silico optimization of antiviral agents targeting 3C and 3C-like proteases
Get PDFObstaja nujna potreba po razvoju novih rešitev za zdravljenje virusnih bolezni brez komercialno dostopnih zdravil. Raziskave protivirusnih učinkovin ostajajo ključnega pomena in ponujajo osnovo za spopadanje z izbruhi virusnih bolezni v prihodnosti, tveganje katerih se ne sme ponovno zanemariti. 3C proteaza (3Cpro) ter funkcionalno in strukturno podobna 3C podobna proteaza (3CLpro) igrata ključno vlogo pri virusni replikaciji in imata izrazito ohranjeno aktivno mesto med različnimi vrstami virusov, zaradi česar sta odlični tarči za zdravljenje pikornavirusnih, kalicivirusnih in/ali koronavirusnih okužb.
V tej nalogi opisujemo retrosintezno voden pristop k optimizaciji spojine MLC3 - novega tipa inhibitorja 3Cpro Enterovirusa 68, ki z encimom tvori Meisenheimerjev kompleks. MLC3 domnevno zaseda samo S1 žepek aktivnega mesta. Ker je struktura S1 žepka ena glavnih skupnih značilnosti 3Cpro in 3CLpro, smo po enterovirusno usmerjeni optimizaciji in silico ovrednotili potencial širokospektralnega protivirusnega delovanja izbranih derivatov MLC3.
Naš pristop k optimizaciji je bil sestavljen iz dveh metod. Prva metoda je »rast fragmenta« - dodajanje substituentov, ki tvorijo dodatne interakcije z aktivnim mestom, hkrati pa ohranjajo že obstoječe interakcije MLC3 ogrodja. Z retrosintezo smo prepoznali komercialno dostopne primarne reaktante za sintezo derivatov in z uporabo analitične platforme ustvarili virtualno knjižnico z 153.675 derivati spojine MLC3. Predstavljamo dve hipotezi o vezavi MLC3 in na njuni podlagi dva farmakoforja, tvorjena z namenom virtualnega rešetanja MLC3 derivatov. Rezultati virtualnega rešetanja so bili sidrani v aktivno mesto 3Cpro. Vsako spojino smo nato ocenili glede na prileganje značilnostim izvirne farmakoforne poizvedbe. Izrazito ugodne interakcije z encimom smo prepoznali pri devetnajstih derivatih, ki so predstavljeni v nalogi.
Druga metoda optimizacije je analiza razmerja med strukturno in aktivnostjo »ogrodja« - MLC3. V katalogih komercialno dostopnih spojin smo odkrili trinajst strukturnih analogov MLC3, ki so navedeni za nadaljnje in vitro preskuse.There is an urgent unmet need for the development of therapies to treat a wide range of devastating viral diseases for which there are no accessible curative medications. Antiviral research remains essential and provides critical resources for tackling viral disease outbreaks in the future, the risk of which should not be underestimated again. The 3C protease (3Cpro) and the functionally and structurally similar 3C like protease (3CLpro) play an essential role in viral replication and have a remarkably conserved active site among different virus species, making them an excellent target for the treatment of picornavirus , calicivirus-, and/or coronavirus induced diseases.
In this thesis, we describe a retrosynthesis-based approach to the optimization of MLC3 – a novel Meisenheimer complex-forming inhibitor of Enterovirus 68 3Cpro which is presumed to occupy only the S1 pocket of the active site. Since the S1 pocket structure is one of the main characteristics linking the 3CLpro to the 3Cpro, the broad-spectrum inhibitor potential of the selected derivatives was examined in silico following the enterovirus-focused optimization.
The optimization is planned by a combination of two approaches. The first one is fragment growing – the addition of substituents that form additional interactions with the active site, while retaining the preexisting ones of the MLC3 scaffold. Retrosynthesis and an analytics platform were used to identify available starting materials allowing the synthesis of 153,675 derivatives. Two binding hypotheses of MLC3 are presented, and two pharmacophores optimized for the screening of the derivatives were created based on them. The hits of the pharmacophore-based virtual screenings were docked and energy minimized. Each compound was scored by how well it fulfilled the features of the original query pharmacophore. Favorable enzyme-substituent interactions were recognized by visual inspection in nineteen derivatives.
The second approach is the structure-activity relationship analysis of the MLC3 scaffold. Thirteen MLC3 structural analogs have been found in the catalogs of commercially available compounds and are listed for subsequent in vitro assays
Discovery of novel ß-ketoacyl-ACP reductase (FABG) inhibitors from Escherichia coli using virtual high-throughput screening
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Synthesis of coumarin derivatives as potencial inhibitors of 17ß-hydroxysteroid dehydrogenase type 1
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Structure-based design and evaluation of potential immunomodulatory and neuroprotective agents
No full textPomembnost računalniških metod v farmacevtski kemiji narašča, medtem ko je virtualno rešetanje postalo sestavni del procesa odkrivanja novih spojin zadetkov na različnih terapevtskih tarčah. Knjižnica spojin predstavlja izhodišče za izvedbo virtualnega rešetanja, njena kvaliteta pa je ključnega pomena za uspeh le-tega. V okviru doktorskega dela smo razvili postopen protokol, kjer smo s pomočjo različne programske opreme pripravili kvalitetne, filtrirane in obogatene knjižnice spojin, ki so nato predstavljale čvrsto osnovo za izvedbo sledečih virtualnih rešetanj na osnovi strukture tarče in na osnovi znanega liganda. Nove spojine zadetke smo s pomočjo metod virtualnega rešetanja iskali na terapevtsko pomembnih tarčah, kot so receptor TLR4 in encimi proteasom, imunoproteasom in butirilholin-esteraza. Receptor TLR4 v kompleksu s pomožnim proteinom MD-2 specifično veže komponento bakterijske celične stene po gramu negativnih bakterij (LPS), kar vodi v sproščanje vnetnih citokinov in aktivacijo imunskih celic, ki je ključnega pomena za obrambo pred infekcijami. Vendar pa prekomerna stimulacija receptorja TLR4 lahko vodi v pretiran odgovor imunskega sistema, kar povzroči poslabšanje stanja pri določenih patoloških procesih, zato receptor TLR4 predstavlja atraktivno tarčo za zdravljenje hude sepse in nevropatske bolečine. V procesu iskanja novih antagonistov receptorja TLR4 smo izvedli virtualno rešetanje na osnovi znanega derivata beta-amino alkoholov. Sočasno smo izvedli tudi strukturno-podprto virtualno rešetanje obogatene knjižnice spojin s ciljem motenja interakcije med proteinoma TLR4 in MD-2. Vzporedno virtualno rešetanje je vodilo v odkritje treh spojin z obetavnimi antagonističnimi aktivnostmi na receptorju TLR4 z vrednostmi IC50 v mikromolarnem območju. Te spojine imajo tudi imunomodulatorne lastnosti, saj zavirajo sproščanje citokinov iz človeških mononuklearnih celic periferne krvi. Specifično afiniteto najbolj aktivnega virtualnega zadetka (IC50 = 16,6 μM) do receptorja TLR4 smo potrdili tudi s poskusi direktne vezave s pomočjo površinske plazmonske resonance, zato odkritje te spojine predstavlja zelo obetavno izhodišče za razvoj novih antagonistov receptorja TLR4.
Proteasom je multi-encimski kompleks, ki igra pomembno vlogo v nelizosomski razgradnji proteinov. Upoštevajoč strukturo aktivnega žepka in mesto izražanja razlikujemo konstitutivni, imuno- in timoproteasom. Zaviralci proteasomov izkazujejo pomembne protivnetne in protitumorne lastnosti, predvsem preko zaviranja aktivacije jedrnega dejavnika Nf-κB in spodbujanja apoptoze hitro delečih se celic. Na osnovi strukture znanega aminopiperidinskega liganda, ki preko zaviranja konstitutivnega proteasoma deluje selektivno toksično na celice Burkittovega limfoma, smo izvedli tridimenzionalno virtualno rešetanje z namenom odkritja ligandov z izboljšano aktivnostjo in drugačnim skeletom. Identificirali smo spojino s podobno razporeditvijo strukturnih elementov v prostoru, vendar drugačnim osnovnim skeletom, ki ima povečano selektivno toksičnost do celic Burkittovega limfoma, hkrati pa močneje zavira rast celic kot izhodna spojina (IC50 = 6,7 μM). Študije mehanizma delovanja nakazujejo tudi drugačen tarčni profil, ki ne vključuje zaviranja proteasoma ali signalne poti NFκB. Najnovejše študije so pokazale, da lahko specifično zaviranje imunoproteasoma predstavlja novo strategijo za zdravljenje avtoimunih bolezni, kot so revmatoidni artritis, vnetne bolezni črevesja in lupus. Nepeptidni in reverzibilni zaviralci imunoproteasoma so redki, zato smo izvedli virtualno rešetanje na osnovi razrešene kristalne strukture, kjer smo ciljali podenoto β5i. Odkrili smo derivat psoralena, ki specifično in selektivno zavira samo β5i podenoto imunoproteasoma (Ki = 15 μM), medtem ko zaviranja konstitutivnega proteasoma nismo zaznali. Preiskovanje odnosa med strukturo in delovanjem je vodilo v odkritje spojine 37, z izboljšanim delovanjem (Ki = 1,6 μM) in manjšo molekulsko maso. Študije sidranja psoralenskih derivatov so nakazovale smiselnost uvedbe elektrofilnih funkcionalnih skupin v bližino terminalne -COOH skupine, kar je omogočilo sintezo specifičnih kovalentnih zaviralcev imunoproteasoma z aktivnostjo v nanomolarnem območju. Odkriti derivati psoralena tako predstavljajo osnovo za intenzivno preučevanje vloge imunoproteasoma in za razvoj potencialnih novih imunomodulatornih spojin. Alzheimerjeva bolezen je napredujoča nevrodegenerativna motnja centralnega živčnega sistema, ki povzroči odmiranje nevronov in izgubo kognitivnih fukcij. Encim BChE je obetavna tarča za lajšanje simptomov Alzheimerjeve bolezni, saj se v poznih stadijih bolezni povečata tako ekspresija kot aktivnost samega encima. Z namenom odkritja novih reverzibilnih zaviralcev BChE smo razvili protokol za hierarhično virtualno rešetanje na osnovi strukture, s pomočjo katerega smo identificirali tri spojine, ki zaviranjo hidrolizno aktivnost BChE. Za najbolj aktivno spojino 1 (IC50 = 21,3 nM) smo razvili sintezno pot preko ortogonalno zaščitenih derivatov piperidin-3-ilmetanamina, nato pa smo s pomočjo kiralne HPLC resolucije pridobili še čista enantiomera spojine 1, od katerih evtomer (+)-1 izkazuje izrazito stereoselektivnost in 2,7 nM konstantno inhibicije. Razrešena kristalna struktura človeške BChE v kompleksu s spojino (+)-1 je razkrila način vezave in nam omogočila strukturno-podprto načrtovanje izboljšanih analogov na osnovi piperidina. Sulfonamidni derivat piperidina 4 ima izboljšano aktivnost (IC50 = 4,95 nM), manjšo stereoselektivnost obeh enantiomerov, razrešena kristalna struktura kompleksa BChE-zaviralec pa je razkrila tudi spremenjen način vezave v aktivno mesto encima. Spojina 4 izkazuje tudi pozitivne učinke na skopolaminskem modelu kognitivnega pomanjkanja in vivo. S spremembo metoksietilne verige spojine 1 smo v molekulo uvedli dodaten bazični center, ki je sposoben tvorbe kation-π interakcij v holin-vezavnem žepku. Spojina GUK942 je najbolj aktiven zaviralec v tej seriji s konstanto inhibicije v nizkem pikomolarnem območju. V okviru doktorskega dela smo optimizirali metodo za histološko barvanje po Koelleju, ki omogoča detekcijo aktivnosti holin-esteraz na možganskih rezinah. S pomočjo optimizirane metode po Koelleju smo v možganih našli specifična področja z visoko aktivnostjo BChE, na katerih smo dokazali zaviranje BChE s piperidinskimi derivati (spojini 1 in 4) ex vivo.
Spojino 1 smo uporabili tudi za validacijo programa LiSiCA, ki išče 2D in 3D podobnost ligandov na osnovi iskanja maksimalnih klik. LiSiCA je izjemno učinkovito orodje, saj je omogočila odkritje petih novih zaviralcev BChE z aktivnostjo v nanomolarnem območju. Vsi zadetki vsebujejo drugačen skelet in manjšo molekulsko maso v primerjavi s spojino 1, kar nakazuje na uporabnost programa za t.i. \u27\u27scaffold-hopping\u27\u27.
Amiloidni plaki so glavna značilnost Alzeimerjeve bolezni, zvrsti amiloida beta pa povezujejo z nevrotoksičnostjo. Spojine, ki zavirajo spontano agregacijo amiloida beta, lahko delujejo nevroprotektivno in vplivajo na nastanek Alzheimerjeve bolezni. Odkrili smo, da novi zaviralci holin-esteraz, vplivajo tudi na spontano agregacijo amiloida beta do fibrilov. Spojina 1, ki je selektivni nanomolarni zaviralec BChE, izkazuje 61,7% zaviranje agregacije pri koncentraciji 10 μM, medtem ko spojina 13b kot selektivni nanomolarni zaviralec AChE pri tej koncentraciji izkazuje 66,0% zaviranje. Obe spojini delujeta tudi nevroprotektivno in zaščitita celice SH-SY5Y pred toksičnimi vplivi zvrsti amiloida beta. Upoštevajoč pomembno vlogo kovinskih ionov pri razvoju Alzheimerjeve bolezni, smo v piperidinske zaviralce BChE uvedli strukturne elemente, ki so sposobni kelacije dvovalentnih kovinskih ionov (Cu, Zn in Fe), pri tem pa smo ohranili zaviranje BChE in spontane agregacije amiloida beta. Spojina 8g je selektivni nanomolarni zaviralec BChE (IC50 = 215 nM), ki selektivno kompleksira Cu2+ ione in zavira spontano agregacijo amiloida beta do fibrilov pri koncentraciji 10 μM. Razrešena kristalna struktura 8g kompleksu s humano BChE je razkrila vezavo kelirajočega nitroksolinskega fragmenta v acil-vezavni žepek encima. Nitroksolinski derivati predstavljajo odlično izhodišče za nadaljnji razvoj multiplih ligandov za zdravljenje Alzheimerjeve bolezni.Computational methods grow in importance in the field of medicinal chemistry. In the last decade, virtual screening has become a routine procedure in the process of hit identification on various therapeutic targets. Generally, a compound library represents a starting point of each virtual screening, while the quality of the former is crucial for the success of the latter. Our work was focused on a design of a hierarchical protocol for the construction of high-quality, filtered and enriched compound libraries, which served as starting points for subsequent structure- and ligand-based virtual screening campaigns. Virtual screening methods were applied in the discovery of novel hit compounds on various therapeutically important targets, such as receptor TLR4 and enzymes proteasome, immunoproteasome and butyrylcholinesterase.
Receptor TLR4 in complex with its accessory protein MD-2 specifically binds a cell-wall component of gram-negative bacteria (lipopolysaccharide), resulting in a cytokine release and immune cell activation, which is of crucial importance for the defence against infections. Given the fact that the dysregulation of TLR4 signaling has been directly implicated in an array of acute and chronic human diseases, receptor TLR4 represents an attractive target for the treatment of severe sepsis and neuropathic pain. In the process of the discovery of novel TLR4 antagonists, ligand-based virtual screening was performed which considered the structural features of known beta-amino alcohol derivative. Simultaneously, structure-based virtual screening of enriched compound library was carried out aiming to disturb protein-protein interactions between TLR4 and MD-2. Parallel virtual screening approach allowed us to identify three novel compounds with promising TLR4 antagonistic activities with IC50 values in micromolar range. These compounds exhibited immunomodulatory properities with the ability to suppress the cytokine secretion by human peripheral blood mononuclear cells. The specific affinity of the most potent hit (IC50 = 16.6 μM) was confirmed by surface plasmon resonance direct-binding experiments. The results of our study represent a very promising starting point for the development of novel small-molecule antagonists of TLR4. Proteasome is a multi-protein complex, which plays an important role in nonlysosomal protein degradation. Based on the structure and site of expression, three major classes of proteasomes can be identified: constitutive, immuno- and thymoproteasome. Proteasome inhibitors display important anti-inflamatory and antitumor activities via nuclear factor Nf-κB inhibition and induction of apoptosis of rapidly dividing cells. 3D ligand-based virtual screening was accomplished using the structure of aminopiperidine ligand with previously described selective cytotoxicity for Burkitt’s lymphoma cells through proteasome inhibition. Our aim was to discover ligands with improved potency and different scaffold compared to the query ligand. Virtual screening led to the identification of the chemically different compound exhibiting similar spatial conformation and improved selectivity and potency (IC50 = 6.7 μM) towards Burkitt’s lymphoma cells. Mechanism-of-action studies revealed different target profile in comparison with the previous query ligand, which does not involve the inhibition of the proteasome or the NFκB pathway. Recent studies revealed that the specific immunoproteasome inhibition represents an emerging strategy for the treatment of autoimmune disorders, such as rheumatoid arthritis, inflammatory bowel disease and lupus. Given the fact that non-peptide and reversible immunoproteasome inhibitors are rare, structure-based virtual screening was performed targeting β5i subunit. A psoralen derivative with marked specifity and selectivity towards β5i subunit of immunoproteasome (Ki = 15 μM) was identified, whereas no inhibition of constitutive proteasome was observed. Structure-activity relationship studies led us to identify compound 37 with improved potency (Ki = 1 μM) and lower molecular weight. Docking simulations of the psoralen derivatives proposed the incorporation of electrophilic moiety in proximity of the terminal –COOH functional group, which allowed us to recognize specific covalent inhibitors of immunoproteasome with activity in nanomolar range. Alzheimer’s disease is a progressive neurodegenerative disorder of central nervous system, which lead to neuron death and loss of cognitive functions. Enzyme BChE is regarded as a promising drug target as its levels and activity significantly increase in the late stages of Alzheimer’s disease. To discover novel BChE inhibitors, we used a hierarchical virtual screening protocol, which allowed us to identify three compounds with significant inhibitory activities against BChE. The most potent compound 1 (IC50 = 21.3 nM) was resynthesized through orthogonally protected piperidin-3-ylmethanamine and resolved into its pure enantiomers exposing significant stereoselective activity and a dissociation constant of 2.7 nM for the most potent stereoisomer (+)-1. The crystal structure of human BChE in complex with compound (+)-1 was solved, revealing the binding mode and providing clues for structure-based optimization of piperidine derivatives. Sulfonamide analogue 4 diplayed improved potency (IC50 = 4.95 nM) and lower degree of stereoselectivity, while the solved complex crystal structure BChE-inhibitor exposed distinctive mode of binding. Compound 4 exhibited positive effects in scopolamine model of cognitive deficit in mice in vivo. Through methoxyetyl chain modification of compound 1, one additional basic centre was incorporated in the structure, which enabled cation-π interactions in choline-binding pocket. The resulting compound GUK942 is the most potent inhibitor in this seris with dissociation constant in low picomolar concentration range. In this work, Koelle\u27s histochemical staining method for cholinesterase activity detection on brain slices was optimized. Optimized Koelle\u27s method allowed us to identify specific brain regions with highest BChE activity, which were further used to prove the ability of piperidine derivatives (compounds 1 and 4) to inhibit brain BChE on rat brain slices ex vivo. Compound 1 was further used to validate the usefulness of a novel software LiSiCA, which examines 2D and 3D similarity on the basis of clique algorithm. LiSiCA was demonstrated to be an effective tool in drug discovery, as it enabled the discovery of five new inhibitors of BChE with potency in nanomolar range. All hits possess different scaffolds and lower molecular weight, if compared to the compound 1, thus proving the ability of LiSiCA for scaffold hopping. Amyloid plaques are one of the hallmarks of Alzheimer\u27s disease, while amyloid beta species are linked to the neurotoxicity. Compounds capable of inhibiting amiloid beta self-induced aggregation might exhibit neuroprotective effect and influence the progress of Alzheimer\u27s disease. Studies revealed, that our novel cholinesterase inhibitors affected the amyloid beta self-induced aggregation into fibrils. Compound 1, which is a selective nanomolar inhibitor of BChE, displayed 61.7% inhibition of aggregation at 10 μM, while compound 13b as selective nanomolar inhibitor of AChE exhibited 65.96% inhibition at the same concentration. Both compounds possessed neuroprotective effect and protected SH-SY5Y cell-line against amlyoid beta species toxicity. Considering the important role of metal ions in progression of Alzheimer\u27s disease, functional groups with the ability to chelate the divalent metal ions (Cu, Zn and Fe) were incorporated in the structures of the piperidine inhibitors, whereas the BChE activity and effect on amyloid beta self-induced aggregation was retained. Compound 8g showed selective nanomolar inhibition of BChE (IC50 = 215 nM), Cu2+ selective complexation, and the ability to inhibit amyloid beta self-induced aggregation at 10 μM. The resolved crystal structure of 8g in complex with BChE revealed the binding of metal chelating nitroxoline moiety in acyl-binding pocket of the enzyme. Nitroxoline derivatives thus represent an encouraging starting point for further development of multifunctional candidates for the treatment of AD
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