Combined molecular dynamics and virtual screening studies to identify novel sirtuin 2 inhibitors

Sirtuins are highly conserved class of NAD+-dependent lysine deacetylases. Altered function of sirtuin 2 (Sirt2) is related to pathogenesis of cancer, inflammation and neurodegeneration, which makes Sirt2 very attractive drug target in novel epigenetic research [1]. A number of Sirt2 inhibitors have been recently developed, but for most of them are missing structural information of their interaction with the enzyme [2, 3]. Our molecular dynamic (MD) study was performed on recently resolved crystal structures of selective ligand-Sirt2 complexes [1]. In the MD study were defined significant interactions with novel inhibitors, one of key residues responsible for conformational stability of cofactor-binding pocket, and residue acting as gate-keeper for cofactor-binding loop. Some residues completely changed orientation after the MD simulation, compared to the starting crystal structures. This result indicates on the errors in the X-ray structures that may have influence on structure-based design of novel inhibitors. After clustering of MD trajectory, 20 conformations (centroids) from 20 clusters of Sirt2 have been selected as representative conformations for retrospective structure based virtual screening. The virtual screening performances were significantly improved by use of the ensemble of conformations, selected with this MD methodology, compared to screening against available X-ray structures.XXV EFMC International Symposium on Medicinal Chemistry, Ljubljana, Slovenia, September 2-6, 201

Molecular dynamics‐based virtual screening of sirtuin 2 inhibitors

Modulacija aktivnosti epigenetičkog brisača, NAD‐zavisne protein deacetilaze sirtuina 2 (SIRT2), poslednjih godina se pokazala kao obećavajuća strategija u lečenju Parkinsonove bolesti, depresije, određenih tipova kancera, ishemijsko‐reperfuzionih povreda itd. Nasuprot terapijskom potencijalu, još uvek nijedan predstavnik ove grupe farmakološki aktivnih supstanci nije našao svoje mesto na tržištu. Najčešći problemi sa dosada opisanim SIRT2 inhibitorima predstavljaju niska potentnost, loša selektivnost, kao i loše farmakokinetičke osobine što dalje opravdava razvoj novih predstavnika. Cilj ovog rada je bio ispitivanje konformacionih promena SIRT2 u prisustvu inhibitora i dalje poboljšanje dostupnih kristalografskih modela u cilju razvoja efikasnijeg protokola virtual screening‐a. Polazeći od 5 različitih kristalografskih struktura SIRT2‐inhibitor kompleksa, ukupno 1,5 μs simulacija molekulske dinamike u eksplicitnom solventu je izvedeno. 3D deskriptori zasnovani na GRID‐u i linearna diskriminantna analiza su korišćeni za virtual screening (VS) studiju. Konformaciona fleksibilnost SIRT2‐inhibitor kompleksa zabeležena tokom simulacija ukazuje na značajnu fleksibilnost aktivnog mesta i posledično na multiple vezivne modove inhibitora. Nakon procedure klasterovanja trajektorije, nekoliko relevantnih modela kompleksa je izdvojeno i uključeno u dalju VS studiju. VS modeli generisani pomoću tri relevantna kompleksa dobijena studijom molekulske dinamike su pokazali značajno bolje performanse u poređenju sa modelima dobijenim pomoću do danas opisanih kristalografskih struktura. Performanse generisanog VS protokola su značajno poboljšane i u odnosu na do danas publikovane protokole. Rezultati ove studije jasno ukazuju na značaj uračunavanja fleksibilnosti aktivnog mesta u racionalni dizajn SIRT2 inhibitora. Novi hemotipovi potenijalnih inhibitora SIRT2 su izdvojeni iz baza komercijalno dostupnih jedinjenja primenom generisanih VS modela. U ovoj studiji formirani su realističniji modeli aktivnog mesta sirtuina 2 kojima su značajno poboljšane performanse virtual screening‐a u odnosu na do danas publikovane studije. Rezultati ove studije, uključujući i opisane konformacione promene doprinose sveobuhvatnom razumevanju odnosa strukture i aktivnosti SIRT2 inhibitora i dodatno racionalizuju dizajn selektivnijih i potentnijih inhibitora.Modulation of activity of epigenetic eraser, NAD‐dependent protein deacetylase sirtuin 2 (SIRT2), recently emerged as promising therapeutic strategy for the treatment of many diseases, including Parkinson’s disease, depression, some types of cancers, necrotic injuries (ischemic stroke, myocardial infarction) etc. Contrary to therapeutic potential, none of SIRT2 inhibitors reported to date has been approved for the market. Some of the most common problems with current SIRT2 inhibitors include poor potency, selectivity and pharmacokinetic properties which justify further development of novel inhibitors. The Aim of this study was to explore conformational space of sirtuin2‐inhibitor complexes and further refinement of available crystallographic structures in order to develop more efficient virtual screening (VS) protocol. Starting from five different crystallographic structures of SIRT2 co‐crystalized with inhibitors, total of 1.5 μs of molecular dynamics (MD) simulations in explicit solvent has been performed. GRID‐based 3D descriptors and linear discriminant analysis were used for virtual screening. Significant conformational flexibility of SIRT2‐inhibitor complexes was observed during simulations indicating overall binding site flexibility and multiple binding modes of inhibitors. Several atomistic models of SIRT2‐inhibitor complexes were extracted and used for structure‐based VS study. VS models generated from three extracted SIRT2‐inhibitor complexes were significantly better compared to VS models generated from available crystallographic structures. Generated VS protocol was also better in performance compared to published virtual screening studies. These results clearly indicate importance of considering flexibility of binding site in rational design of SIRT2 inhibitors. Obtained models were used for screening of commercial databases of compounds. Several chemotypes of potential novel SIRT2 inhibitors have been identified. Refined atomistic models of SIRT2‐inhibitor complexes have been generated and significant improvement of virtual screening performance has been achieved. These results further rationalize design of SIRT2 inhibitors with improved selectivity and potency.VII Kongres farmaceuta Srbije sa međunarodnim učešćem Zajedno stvaramo budućnost farmacije, Beograd, 10-14. oktobar 201

Exploring conformational changes of sirtuin 2- molecular dynamic approach

Sirtuin 2 (Sirt2) se poslednjih godina sve više povezuje sa patogenezom kancera, neurodegeneracijom i inflamacijom zbog čega predstavlja atraktivan target za razvoj novih lekova. Većina do danas okarakterisanih inhibitora Sirt2 ne poseduje odgovarajuću aktivnost i/ili selektivnost. Kristalografske strukture objavljene u poslednjih par godina obezbedile su određeni pomak u razvoju novih inhibitora, međutim i dalje postoje nedoumice u vezi sa mehanizmom inhibicije koje su posledica visoke konformacone fleksibilnosti Sirt2 enzima. U cilju sticanja detaljnijeg uvida u konformacionu fleksibilnost Sirt2, izvedeno je sedamnaest simulacija molekulske dinamike u trajanju od po 100ns. Rezultati ove studije ukazali su na neke od ključnih rezidua neophodnih za stabilizaciju kompleksa Sirt2/inhibitor. Takođe, opisane konformacije daju jasniji uvid u katalitički ciklus deacilovanja. Opisan je i značaj konformacije kofaktorske petlje na konformaciju vezivnog mesta inhibitora. Rezultati ove studije će omogućiti racionalniji pristup dizajnu selektivnijih i potentnijih inhibitora sirtuina2.Sirtuin 2 (Sirt2) has been implicated in pathogenesis of cancer, inflammation and neurodegeneration, which makes Sirt2 attractive target for development of novel therapeutics. A number of small molecule Sirt2 inhibitors have been reported, but most of them lack selectivity and/or high potency. Very recently published X-ray structures of selective ligand/Sirt2 complexes have revealed more details about mechanism of action of inhibitors, but some information still missing. Here, we will try to answer on questions regarding the conformational changes during enzyme catalysis, based on our recent molecular dynamic study of Sirt2. Total of seventeen 100 ns simulations have been performed on five different crystal structures. Results of this study have suggested one of key residues responsible for conformational stability of cofactor-binding loop and significant interaction with novel inhibitors. Further, “closing” of Sirt2 induced by presence of substrate has been described as major factor responsible for conformational changes in substrate-binding site. In conclusion, this study provides dynamic information on Sirt2 and may facilitate the rational design on novel, more potent and selective inhibitors.Fifth Conference of Young Chemists of Serbia. September 29-30. 2017. Belgrade, Serbi

Sirtuin functions and modulation: from chemistry to the clinic

Sirtuins are NAD+ -dependent histone deacetylases regulating important metabolic pathways in prokaryotes and eukaryotes and are involved in many biological processes such as cell survival, senescence, proliferation, apoptosis, DNA repair, cell metabolism, and caloric restriction. The seven members of this family of enzymes are considered potential targets for the treatment of human pathologies including neurodegenerative diseases, cardiovascular diseases, and cancer. Furthermore, recent interest focusing on sirtuin modulators as epigenetic players in the regulation of fundamental biological pathways has prompted increased efforts to discover new small molecules able to modify sirtuin activity. Here, we review the role, mechanism of action, and biological function of the seven sirtuins, as well as their inhibitors and activators

Novi protokol za poboljšano uzorkovanje dinamike vezivnih mesta i njegova integracija u virtuelni skrining inhibitora sirtuina 2

Inhibitors of sirtuin 2 (SIRT2i) represent a promising group of novel therapeutics in treatment of the age-related disorders, including carcinomas, neurodegenerative diseases, metabolic syndrome etc. (1). Despite the promising preclinical results, none of the known SIRT2i reached clinical trials. One of the main obstacles in the structure-based drug design of novel SIRT2i represents intricate conformational dynamics of sirtuin 2 (SIRT2) binding pocket (2). In order to facilitate the discovery of novel SIRT2i, we have developed the protocol for enhanced sampling of the binding pocket dynamics and validated it by integration into structure-based virtual screening (SBVS) pipeline for discovery of novel SIRT2i. Developed protocol relies on well-tempered metadynamics simulations using the set of pocket-related collective variables derived from time-lagged independent component analysis (tICA). Our protocol outperformed classical molecular dynamics in search for alternative conformational states of the binding pocket. Additionally, the protocol was able to reveal the existence of cryptic subpocket inside SIRT2 binding pocket. To validate our findings, the protocol was implemented into SBVS which resulted in significant expansion of SIRT2i chemical space. To further probe the potential of expanded chemical space in discovery of chemically novel groups of SIRT2i, a few virtual hit molecules were selected and tested in vitro. Compound NDJ18 was shown to be potent and selective SIRT2i with anticancer activity on triple-negative breast cancer cell line MDA-MB-231. Experimental validation supported future generalization of the protocol by application on wider scope of challenging protein targets.Inhibitori sirtuina 2 (SIRT2i) predstavljaju obećavajuću grupu novih terapeutika u terapiji poremećaja povezanih sa starenjem, poput malignih oboljenja, neurodegenerativnih oboljenja, metaboličkog sindroma itd. (1). Uprkos obećavajućim rezultatima prekliničkih ispitivanja, nijedan SIRT2i nije došao do kliničkih studija. Jedna od glavnih prepreka u strukturno-zavisnom dizajnu novih SIRT2i predstavlja kompleksna konformaciona dinamika vezivnog mesta sirtuina 2 (SIRT2) (2). U cilju povećanja efikasnosti racionalnog dizajna novih SIRT2i, razvijen je protokol za poboljšano uzorkovanje konformacione dinamike vezivnog mesta SIRT2 koji se zasniva na metadinamičkim simulacijama uz set kolektivnih varijabli izvedenih iz analize nezavisnih komponenti vremenskih zaostataka dinamike vezivnog mesta (tICA). Razvijeni protokol nadmašio je klasičnu molekulsku dinamiku u efikasnosti pretrage alternativnih konformacionih stanja vezivnog mesta. Dodatno, primenom razvijenog protokola otkriveno je postojanje skrivenog džepa unutar vezivnog mesta SIRT2. U cilju validacije, protokol je implementiran u strukturno-zavisni virtuelni skrining SIRT2i što je rezultovalo u značajnoj ekspanziji postojećeg hemijskog prostora SIRT2i. U daljem testiranju potencijala proširenog hemijskog prostora u otkriću potpuno novih hemijskih skeleta SIRT2i, nekoliko najbolje rangiranih molekula je selektovano i evaluirano in vitro. Jedinjenje NDJ18 se pokazalo kao potentan i selektivan novi SIRT2i. Testiranjima na trostruko-negativnoj ćelijskoj liniji kancera dojke MDA-MB-231 utvrđen je značajan antikancerski potencijal navedenog jedinjenja. Eksperimentalnom validacijom podržan je dalji razvoj i generalizacija protokola kroz primenu na širem spektru proteinskih targeta izazovnih sa aspekta tehnika racionalnog dizajna lekova.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Computer-aided drug design of selective histone deacetylase inhibitors

The concept of gene expression is continuously explained with epigenetic modification. Post-translational histone acetylation and DNA methylation are dominant epigenetic alterations of the genome. Histone deacetylases (HDAC) play essential role in this process and therefore are very intensively investigated drug targets. The alteration in the structure and function of HDAC isoforms are identified in the pathogenesis of inflammation, cancer, and neurodegeneration. Eleven human HDAC isoforms are sharing a highly conserved catalytic domain. Among them, HDAC6 and SIRT2 are important for a wide range of diseases, due to their unique physiological functions. In our research, we have applied pharmacophore modelling, virtual screening, molecular docking and molecular dynamic methodologies for design and identification of selective HDAC6 and SIRT2 inhibitors. Recently resolved the crystal structure of catalytic domain II of human HDAC6 discovered a wide binding site essential for the substrate recognition. We have successfully used these structural features of human HDAC6 catalytic domain II to rationally design selective HDAC6 inhibitors. Newly published X-ray structures of selective ligand-SIRT2 complexes have revealed high conformational flexibility of this enzyme, and gave us more details about mechanism of action of sirtuin 2 inhibitors. Based on these findings we have performed molecular dynamic study of SIRT2 and tried to explain the conformational changes during enzyme catalysis. Since small number of selective HDAC modulators have been reported so far, rational design of HDAC6 and SIRT2 inhibitors are essential for further progress in discovery of epigenetic drugs.Journal of organic & inorganic chemistr

Tetrazole as a Replacement of the Electrophilic Group in Characteristic Prolyl Oligopeptidase Inhibitors

4-Phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines were studied as prolyl oligopeptidase inhibitors. The compounds were more potent than expected from the assumption that the tetrazole would also here be a bioisostere of the carboxylic acid group and the corresponding carboxylic acids are at their best only weak inhibitors. The aminoacyl groups L-prolyl and L-alanyl gave potent inhibitors with IC50 values of 12 and 129 nM, respectively. This was in line with typical prolyl oligopeptidase inhibitors; however, we did observe a difference with N-methyl-L-alanyl, which gave potent inhibitors in typical prolyl oligopeptidase inhibitors but not in our novel compound series. Furthermore, all studied 4-phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines decreased alpha-synuclein dimerization at the concentration of 10 mu M, also when they were only weak inhibitors of the proteolytic activity of the enzyme with an IC50 value of 205 mu M. Molecular docking studies revealed that the compounds are likely to bind differently to the enzyme compared to typical prolyl oligopeptidase inhibitors represented in this study by 4-phenylbutanoyl-aminoacyl-2(S)-cyanopyrrolidines.Peer reviewe

Rational design of selective histone deacetylase inhibitors

The concept of gene expression is continuously explained with epigenetic modifications. One of the most studied enzymes which have an influence on histone posttranslational modifications are histone deacetylases (HDACs). The overexpression and alterations in the structure of HDACs isoforms are described in the pathogenesis of cancer, inflammation and neurodegeneration. With different cellular function and tissue distribution of HDACs, scientists introduced them as attractive targets used in novel drug discovery protocols. Rational drug design of novel small molecules is usually guided by computational approaches. In our laboratory, we use ligand-based (pharmacophore modeling and virtual screening) and structure-based (molecular docking and molecular dynamics) drug design methodologies. The main focus in our drug design project is identification of selective HDAC6 and SIRT2 inhibitors. With respect to all published data, very small number of selective HDAC modulators has been reported so far. Here, we present rational design of novel selective HDAC6 and SIRT2 inhibitors as promising drug candidates for further development and structure optimization.http://www.socphyschemserb.org/en/events/physical-chemistry-2018

The Identification of a SIRT6 Activator from Brown Algae Fucus distichus

peer-reviewedBrown seaweeds contain many bioactive compounds, including polyphenols, polysaccharides, fucosterol, and fucoxantin. These compounds have several biological activities, including anti-inflammatory, hepatoprotective, anti-tumor, anti-hypertensive, and anti-diabetic activity, although in most cases their mechanisms of action are not understood. In this study, extracts generated from five brown algae (Fucus dichitus, Fucus vesiculosus (Linnaeus), Cytoseira tamariscofolia, Cytoseira nodacaulis, Alaria esculenta) were tested for their ability to activate SIRT6 resulting in H3K9 deacetylation. Three of the five macroalgal extracts caused a significant increase of H3K9 deacetylation, and the effect was most pronounced for F. dichitus. The compound responsible for this in vitro activity was identified by mass spectrometry as fucoidan

Rational design, synthesis and in vitro testing of selective HDAC6 and SIRT2 inhibitors

Histone deacetylases (HDACs) are epigenetic enzymes involved in regulation of histone posttranslational modifications and gene expression. Since changed function of HDACs is involved in pathogenesis of cancer 1-3, the HDAC inhibitors are extensive examined as promising anticancer agents. In our in silico study we have combined structure-based, ligand-based, and fragment-based methodologies to design selective inhibitors against cytoplasmic isoforms of HDAC, such as histone deacetylase 6 inhibitors (HDAC6) and SIRT2. The drug design study has defined several promising selective HDAC6 and SIRT2 inhibitors for further synthesis and in vitro testing. Based on the in vitro activities of the novel compounds in a panel of biochemical HDAC assays as well as various cell-based assays were selected the most promising candidates for further investigation.XXVI EFMC International Symposium on Medicinal Chemistry (EFMC-ISMC 2021), Virtual Event, August 29-September 2, 202