Kitosan nano-parçacıkların pseudomonas aerugınosa üzerine antibakteriyel etkisinin ın sılıco ve ın vıtro ortamlarda incelenmesi

ÖZETKİTOSAN NANO-PARÇACIKLARIN PSEUDOMONAS AERUGINOSA ÜZERİNE ANTİBAKTERİYEL ETKİSİNİN IN SILICO VE IN VITRO ORTAMLARDA İNCELENMESİBakteriler, biyofilm formuna geçerek birçok tıbbi vakanın oluşmasına sebep olmaktadırlar. Fırsatçı patojenlerden Pseudomonas aeruginosa da biyofilm oluşturarak ciddi enfeksiyonlara sebep olabilmektedir. P. aeruginosa’nın biyofilm oluşturmasında rol oynayan Quorum Sensing (QS) mekanizmasının aydınlatılması yeni ilaçların geliştirilmesine öncülük edebilecektir. İlaç geliştirmede uygun hedefin belirlenmesi önem taşımaktadır. Son yıllarda uygun hedef bölgenin belirlenmesinde in silico çalışmalar ön plana çıkmaktadır. In silico yöntemler ile ilaç moleküllerinin in vivo ya da in vitro olarak test edilmesi için harcanan zamandan ve maliyetten tasarruf sağlanması beklenmektedir. Bu bilgiler ışığında P. aeruginosa PAO1 suşunda antimikrobiyal etkinliği bilinen kitosan ve kitosan nanoparçacıklarının antibakteriyel ve anti-QS etkisinin in silico ve in vitro ortamlarda incelenmesi amaçlanmıştır. Bu amaçla P. aeruginosa PAO1 suşunun QS ve ikili bileşen sistemine ait proteinlerinin üç boyutlu yapıları ile kitosan ve kitosan nanoparçacık moleküllerinin üç boyutlu yapıları çeşitli veri bankalarından elde edilerek moleküler kenetlenme işlemine tabi tutulmuştur. Bununla birlikte kitosanın ve kitosan nanoparçacıkların in vitro olarak P. aeruginosa PAO1 suşu üzerinde antibakteriyel ve anti-QS etkileri incelenmiştir. QS mekanizmasına ait proteinlerle gerçekleştirilen in silico işlemler sonucunda, kitosan molekülleri kitosan nanoparçacık molekülüne göre daha iyi skor vermiştir. Antibakteriyel aktivitede etkili proteinlerde ise kitosan nanoparçacık molekülü, kitosan moleküllerine göre daha iyi skor vermiştir. In vitro deneylerde uygulanan düşük dozda kitosan ve kitosan nanoparçacık çözeltilerinin P. aeruginosa PAO1 suşu üzerinde antibakteriyel veya anti-QS etkisine rastlanılmamışken, yüksek dozda uygulanan çözeltilerde sırasıyla HMW > MMW > Kitosan > LMW olarak antimikrobiyal aktivite gözlenmiştir. Sonuç olarak, in silico uygulamalarda kullanılan moleküllerin stabilitesi, boyutları, programların algoritmaları arasındaki farklılıklar gibi etmenlerin in vitro şartlarla uyum sağladığı durumlarda, in silico verilerle in vitro verilerin birbirini destekleyebileceği görülmüştür.ABSTRACTIN SILICO AND IN VITRO INVESTIGATION OF CHITOSAN NANOPARTICLES FOR ANTIBACTERIAL EFFECTSON PSEUDOMONAS AERUGINOSABacteria may cause many different medical circumstances through their biofilm forms. Pseudomonas aeruginosa, one of the opportunistic pathogens, causes serious infections via biofilm formation as well. Elucidation of the Quorum Sensing (QS) mechanism, which plays a role in biofilm formation of P. aeruginosa, will be able to lead the development of new drugs. Determination of the appropriate target is carry weight in the developement of new drugs. Recently, in silico methods and studies have come into prominence for determination of the appropriate targets. In silico methods are expected to provide saving the cost and the time spent on testing the drug molecule candidates in vitro and in vivo. In the light of this information, the aim of our study is to investigate the antibacterial and anti-QS effects of the chitosan and chitosan nanoparticles, which are known for their antimicrobial effects, both in silico and in vitro on Pseudomonas aeruginosa PAO1 strain. The 3D structures of chitosan, chitosan nanoparticles and the proteins of P. aeruginosa PAO1 Quorum Sensing and Two Component System were downloaded from several databases, and several molecular docking studies were performed to this end. In addition, the antibacterial and anti-QS activities of chitosan and chitosan nanoparticles on P. aeruginosa PAO1 strain were investigated in vitro. The in silico results showed that chitosan molecules had better scores than chitosan nanoparticles on the proteins that play a role in QS mechanism. On the other hand, the chitosan nanoparticles had a better score than chitosan molecules on the proteins that are relevant to the antibacterial activity. As a result of in vitro experiments, neither antibacterial nor anti-QS effects on P.aeruginosa PAO1 strain were observed on the lower doses of chitosan and chitosan nanoparticles solutions, but we observed antimicrobial activities on the higher doses of HMW, MMW, Chitosan, LMW solutions, respectively. In conclusion, it seems possible that in silico and in vitro data could support each other under the appropriate conditions of adapted factors such as stability, size and dimension of the molecules in addition to the differences between the algorithms of programs used for in silico methods

Kitosan nano-parçacıkların pseudomonas aerugınosa üzerine antibakteriyel etkisinin ın sılıco ve ın vıtro ortamlarda incelenmesi

KİTOSAN NANO-PARÇACIKLARIN PSEUDOMONAS AERUGINOSA ÜZERİNE ANTİBAKTERİYEL ETKİSİNİN IN SILICO VE IN VITRO ORTAMLARDA İNCELENMESİ Bakteriler, biyofilm formuna geçerek birçok tıbbi vakanın oluşmasına sebep olmaktadırlar. Fırsatçı patojenlerden Pseudomonas aeruginosa da biyofilm oluşturarak ciddi enfeksiyonlara sebep olabilmektedir. P. aeruginosa’nın biyofilm oluşturmasında rol oynayan Quorum Sensing (QS) mekanizmasının aydınlatılması yeni ilaçların geliştirilmesine öncülük edebilecektir. İlaç geliştirmede uygun hedefin belirlenmesi önem taşımaktadır. Son yıllarda uygun hedef bölgenin belirlenmesinde in silico çalışmalar ön plana çıkmaktadır. In silico yöntemler ile ilaç moleküllerinin in vivo ya da in vitro olarak test edilmesi için harcanan zamandan ve maliyetten tasarruf sağlanması beklenmektedir. Bu bilgiler ışığında P. aeruginosa PAO1 suşunda antimikrobiyal etkinliği bilinen kitosan ve kitosan nanoparçacıklarının antibakteriyel ve anti-QS etkisinin in silico ve in vitro ortamlarda incelenmesi amaçlanmıştır. Bu amaçla P. aeruginosa PAO1 suşunun QS ve ikili bileşen sistemine ait proteinlerinin üç boyutlu yapıları ile kitosan ve kitosan nanoparçacık moleküllerinin üç boyutlu yapıları çeşitli veri bankalarından elde edilerek moleküler kenetlenme işlemine tabi tutulmuştur. Bununla birlikte kitosanın ve kitosan nanoparçacıkların in vitro olarak P. aeruginosa PAO1 suşu üzerinde antibakteriyel ve anti-QS etkileri incelenmiştir. QS mekanizmasına ait proteinlerle gerçekleştirilen in silico işlemler sonucunda, kitosan molekülleri kitosan nanoparçacık molekülüne göre daha iyi skor vermiştir. Antibakteriyel aktivitede etkili proteinlerde ise kitosan nanoparçacık molekülü, kitosan moleküllerine göre daha iyi skor vermiştir. In vitro deneylerde uygulanan düşük dozda kitosan ve kitosan nanoparçacık çözeltilerinin P. aeruginosa PAO1 suşu üzerinde antibakteriyel veya anti-QS etkisine rastlanılmamışken, yüksek dozda uygulanan çözeltilerde sırasıyla HMW > MMW > Kitosan > LMW olarak antimikrobiyal aktivite gözlenmiştir. Sonuç olarak, in silico uygulamalarda kullanılan moleküllerin stabilitesi, boyutları, programların algoritmaları arasındaki farklılıklar gibi etmenlerin in vitro şartlarla uyum sağladığı durumlarda, in silico verilerle in vitro verilerin birbirini destekleyebileceği görülmüştür. ABSTRACT IN SILICO AND IN VITRO INVESTIGATION OF CHITOSAN NANOPARTICLES FOR ANTIBACTERIAL EFFECTS ON PSEUDOMONAS AERUGINOSA Bacteria may cause many different medical circumstances through their biofilm forms. Pseudomonas aeruginosa, one of the opportunistic pathogens, causes serious infections via biofilm formation as well. Elucidation of the Quorum Sensing (QS) mechanism, which plays a role in biofilm formation of P. aeruginosa, will be able to lead the development of new drugs. Determination of the appropriate target is carry weight in the developement of new drugs. Recently, in silico methods and studies have come into prominence for determination of the appropriate targets. In silico methods are expected to provide saving the cost and the time spent on testing the drug molecule candidates in vitro and in vivo. In the light of this information, the aim of our study is to investigate the antibacterial and anti-QS effects of the chitosan and chitosan nanoparticles, which are known for their antimicrobial effects, both in silico and in vitro on Pseudomonas aeruginosa PAO1 strain. The 3D structures of chitosan, chitosan nanoparticles and the proteins of P. aeruginosa PAO1 Quorum Sensing and Two Component System were downloaded from several databases, and several molecular docking studies were performed to this end. In addition, the antibacterial and anti-QS activities of chitosan and chitosan nanoparticles on P. aeruginosa PAO1 strain were investigated in vitro. The in silico results showed that chitosan molecules had better scores than chitosan nanoparticles on the proteins that play a role in QS mechanism. On the other hand, the chitosan nanoparticles had a better score than chitosan molecules on the proteins that are relevant to the antibacterial activity. As a result of in vitro experiments, neither antibacterial nor anti-QS effects on P.aeruginosa PAO1 strain were observed on the lower doses of chitosan and chitosan nanoparticles solutions, but we observed antimicrobial activities on the higher doses of HMW, MMW, Chitosan, LMW solutions, respectively. In conclusion, it seems possible that in silico and in vitro data could support each other under the appropriate conditions of adapted factors such as stability, size and dimension of the molecules in addition to the differences between the algorithms of programs used for in silico methods

Screening of Clinically Approved and Investigation Drugs as Potential Inhibitors of SARS-CoV-2 Main Protease and Spike Receptor-Binding Domain Bound with ACE2 COVID19 Target Proteins: A Virtual Drug Repurposing Study

In this virtual drug repurposing study, we used 7922 FDA approved drugs and compounds in clinical investigation from NPC database. Both apo and holo forms of SARS-CoV-2 Main Protease as well as Spike Protein/ACE2 were used for virtual screening. Initially, docking was performed for these compounds at target binding sites. The compounds were then sorted according to their docking scores which represent binding energies. The first 100 compounds from each docking simulations were initially subjected to short (10 ns) MD simulations (in total 300 ligand-bound complexes), and average binding energies during MD simulations were calculated using the MM/GBSA method. Then, the selected promising hit compounds based on average MM/GBSA scores were used in long (100-ns and 500-ns) MD simulations. In total around 15 µs MD simulations were performed in this study. Both docking and MD simulations binding free energy calculations showed that holo form of the target protein is more appropriate choice for virtual drug screening studies. These numerical calculations have shown that the following 8 compounds can be considered as SARS-CoV-2 Main Protease inhibitors: Pimelautide, Rotigaptide, Telinavir, Ritonavir, Pinokalant, Terlakiren, Cefotiam and Cefpiramide. In addition, following 5 compounds were identified as potential SARS-CoV-2 ACE-2/Spike protein domain inhibitors: Denopamine, Bometolol, Naminterol, Rotigaptide and Benzquercin. These compounds can be clinically tested and if the simulation results validated, they may be considered to be used as treatment for COVID-19

Proposing Novel MAO‑B Hit Inhibitors Using Multidimensional Molecular Modeling Approaches and Application of Binary QSAR Models for Prediction of Their Therapeutic Activity, Pharmacokinetic and Toxicity Properties

Monoamine oxidase (MAO) enzymes MAO-A and MAO-B play a critical role in the metabolism of monoamine neurotransmitters. Hence, MAO inhibitors are very important for the treatment of several neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS). In this study, 256 750 molecules from Otava Green Chemical Collection were virtually screened for their binding activities as MAO-B inhibitors. Two hit molecules were identified after applying different filters such as high docking scores and selectivity to MAO-B, desired pharmacokinetic profile predictions with binary quantitative structure–activity relationship (QSAR) models. Therapeutic activity prediction as well as pharmacokinetic and toxicity profiles were investigated using MetaCore/MetaDrug platform which is based on a manually curated database of molecular interactions, molecular pathways, gene–disease associations, chemical metabolism, and toxicity information. Particular therapeutic activity and toxic effect predictions are based on the ChemTree ability to correlate structural descriptors to that property using recursive partitioning algorithm. Molecular dynamics (MD) simulations were also performed to make more detailed assessments beyond docking studies. All these calculations were made not only to determine if studied molecules possess the potential to be a MAO-B inhibitor but also to find out whether they carry MAO-B selectivity versus MAO-A. The evaluation of docking results and pharmacokinetic profile predictions together with the MD simulations enabled us to identify one hit molecule (ligand <b>1</b>, Otava ID: 3463218) which displayed higher selectivity toward MAO-B than a positive control selegiline which is a commercially used drug for PD therapeutic purposes

Biological Insights of the Dopaminergic Stabilizer ACR16 at the Binding Pocket of Dopamine D2 Receptor

The dopamine D2 receptor (D2R) plays an important part in the human central nervous system and it is considered to be a focal target of antipsychotic agents. It is structurally modeled in active and inactive states, in which homodimerization reaction of the D2R monomers is also applied. The ASP2314 (also known as ACR16) ligand, a D2R stabilizer, is used in tests to evaluate how dimerization and conformational changes may alter the ligand binding space and to provide information on alterations in inhibitory mechanisms upon activation. The administration of the D2R agonist ligand ACR16 [³H]­(+)-4-propyl-3,4,4<i>a</i>,5,6,10<i>b</i>-hexahydro-2<i>H</i>-naphtho­[1,2-<i>b</i>]­[1,4]­oxazin-9-ol ((+)­PHNO) revealed <i>K</i>_i values of 32 nM for the D2^highR and 52 μM for the D2^lowR. The calculated binding affinities of ACR16 with post processing molecular dynamics (MD) simulations analyses using MM/PBSA for the monomeric and homodimeric forms of the D2^highR were −9.46 and −8.39 kcal/mol, respectively. The data suggests that the dimerization of the D2R leads negative cooperativity for ACR16 binding. The dimerization reaction of the D2^highR is energetically favorable by −22.95 kcal/mol. The dimerization reaction structurally and thermodynamically stabilizes the D2^highR conformation, which may be due to the intermolecular forces formed between the TM4 of each monomer, and the result strongly demonstrates dimerization essential for activation of the D2R