LEE- Kimya-Doktora
Bu koleksiyon için kalıcı URI
Gözat
Konu "Bilgisayar destekli hesaplama" ile LEE- Kimya-Doktora'a göz atma
Sayfa başına sonuç
Sıralama Seçenekleri
-
ÖgeMultiscale computational investigation of the kynurenine 3-monooxygenase catalyzed hydroxylation reaction(Lisansüstü Eğitim Enstitüsü, 2021) Özkılıç, Yılmaz ; Tüzün, Nurcan ; 723736 ; KimyaKynurenine pathway is the biological pathway responsible for the catabolism of tryptophan to the final product nicotinamide adenine dinucleotide (NAD). Flavin adenine dinucleotide (FAD) dependent kynurenine 3-monooxygenase (KMO) is a class A type monooxygenase and its main function is to convert the substrate L-kynurenine (L-Kyn) into 3-hydroxykynurenine (3-HK) by a hydroxylation reaction in the kynurenine pathway. The product 3-HK and its derivatives were shown to be neurotoxic agents. On the other hand, L- Kyn is also a substrate to kynurenine aminotransferase which converts L-Kyn into the neuroprotective agent kynurenic acid (KynA). However, high levels of this substance correlates with the bipolar disorder and schizophrenia. As a result, KMO regulates the levels of important bioactive substances in this pathway. Extensive experimental research has been carried out to understand the mechanism of KMO catalyzed L-Kyn hydroxylation. Although these experiments contributed a lot to unravel many questions, some others related to the short-lived intermediate states involving L-Kyn derivatives and the dynamical responses of the enzyme, when an effector is within the active site, have remained unanswered. Another branch of experimental research has been maintained on the discovery of novel KMO inhibitors. Although early inhibitors bearing on the scaffold of L-Kyn were very successful in preventing the hydroxylation of L-Kyn, they also caused the generation of free hydrogen peroxide as a by-product. Therefore, structurally different inhibitors were sought and indeed, some were shown to prevent the hydrogen peroxide production. However, most of the diseases related to KMO activity being of neurodegenerative kind imposes another restraint on the KMO inhibitors: The inhibitors are required to be blood brain barrier (BBB) permeable which altogether proved to be a difficult task. The purpose of this thesis is to enlighten the second phase of the oxidative half reaction mechanism of KMO catalyzed L-Kyn hydroxylation and the discovery of new KMO inhibitors by means of computational methods. In the first chapter of this thesis, the reaction between L-Kyn and model FAD-hydroperoxide was studied via density functional theory (DFT). Initially, the reaction mechanism was studied by performing calculations on L-Kyn and the FAD-hydroperoxide model. The calculations were then carried out in the presence of the model KMO. The model KMO was built from an X-ray structure of the protein complex (pdb code: 5NAK) with quantum cluster approach, which proved to be a very efficient method in modelling the enzyme reactions. In the cluster-model based calculations, the substrate, the FAD-hydroperoxide model and the residues within the active site were represented with 348 atoms. These calculations allowed us to deduce a mechanistic pathway for the second phase of the oxidative half reaction. According to these results, KMO-catalyzed hydroxylation reaction involves four transformations in which Asn54, Pro318, and a water molecule, originating from the X-ray structure, also take part. In the first step, the hydroxyl of the hydroperoxy moiety is delivered to L-Kyn ring, where an sp3-hybridized carbon center is formed while the hydrogen of the transferred unit is immediately captured by the proximal oxygen that situated on FAD. These consequent transformations are in line with the somersault rearrangement previously described for similar enzymatic systems. In the second step, a hydride shift results in the formation of the keto form of 3-HK via the transfer of the hydrogen that was bound to the sp3-carbon center of the substrate ring. In the next step, a water assisted tautomerization transforms the keto form into the enol, yielding 3-HK. Reoxidation of FAD is achieved through a water and 3-HK assisted dehydration, marking the final step of the oxidative half reaction. The optimizations of the cluster models were carried out at the B3LYP/6-31G(d,p) level of theory. The single point energies were obtained from the B3LYP/6-311+G(2d,2p) level of theory calculations with solvation (polarizable continuum model) and dispersion (DFT-D3(BJ)) corrections. In the second chapter of this thesis, BBB permeable KMO inhibitors were sought via in silico methods. According to the experimental findings, Cl- anion stabilizes the enzyme and significantly decreases the limiting rate of the reduction. The rate of hydroxylation is also reduced when the experiments were carried out in NaCl solutions. The inhibitor candidates are expected to be similar to the aminobenzyl group containing substrate L-Kyn, to some extent. However, to appreciably eliminate the electron donating ability of the ring, any kind of amine must be excluded and an electron withdrawing group must be included as a substituent. Using the tranche browser of Zinc15, 7561938 in-stock molecules with drug-like properties were obtained. Using Open Babel, only the molecules containing a chlorophenyl group that is not substituted by an amine were selected. This reduced the number of the molecules in the library to 501777. Using AutoDock Vina, the binding affinity ranking of this set of molecules were determined. Starting from the highest scoring molecules, the ADMET properties of these substances were checked using the web-based SwissADME program. ZINC_71915355 passed all the evaluations considered. ZINC_19827377 and ZINC_19458579 were not BBB permeable but were found to be suitable for peripheral administration. The KMO complexes with these molecules were further tested via MD simulations to see if they retain the active site conformational state, which is an important criterion to eliminate H2O2 production. Only ZINC_71915355 and ZINC_19827377 were found to retain the conformational state of the enzyme in the active site. If experimentally confirmed, these two molecules can inhibit KMO without hydrogen peroxide production. After the publication of our results of the first mechanistic investigation, a new X-ray structure of KMO–L-Kyn complex (pdb id: 6FOX) was published. The authors of the communication suggested that the transferred oxygen is not the distal, but the proximal one. This idea stemmed from the positions of the two water molecules which together seem to hold the place of the hydroperoxy moiety. The water oxygen, that seems to hold the place of the proximal oxygen, is closer to L-Kyn and in a more appropriate position to participate in the reaction, in comparison to the one that was associated with the distal oxygen. This idea was one of the two inquiries investigated in the third chapter of this thesis. To be able to correctly represent the structural differences, which include slightly different alignment of FAD's isoalloxazine ring system and L-Kyn, the active site of the new X-ray structure was used in the cluster approach calculations. In this new model, the active site was represented with 386 atoms and the proximal (P-mechanism) versus distal (D-mechanism) oxygen transfer mechanisms were compared. In addition, these transformations were analyzed via natural bonding orbital (NBO) calculations which allowed us to deepen the difference between the two possible transformations and further explain how the working mechanism consistently enables a stable environment for its intermediate states. According to these results, the transformation via P-mechanism requires significantly higher barrier energy and additionally, results in an unexpected L-Kyn derivative. The somersault intermediate of the D-mechanism guarantees the prevention of such derivatives. To further strengthen the thesis against the P-mechanism, the stability of the supposed alignment of the hydroperoxy moiety which paves the way for the P-mechanism was questioned by molecular dynamics (MD) simulations. Starting from that alignment, three 100 ns production runs were carried out. According to the results of these simulations, the alignment was not conserved and therefore the positions of the water molecules were found not to be related to that of the hydroperoxy moiety. In another recent experimental paper, the authors have proposed that L-Kyn participates in its base form in the reaction. The investigation of this interpretation is the last part of the third chapter of this thesis. Although DFT calculations confirmed a much more facile reaction with the base form of L-Kyn, a mechanism that would allow the deprotonation of the L-Kyn before the oxygen transfer could not be determined with the X-ray based positions. A concerted mechanism with both the oxygen transfer and the deprotonation required a high barrier energy. A stepwise mechanism involving the deprotonation of L-Kyn was found, starting from an MD frame. The overall barrier of the oxygen transfer step of this model was found to be in the range of that of with neutral L-Kyn. MD simulations supported the idea of ineffectiveness of the nearby shell surrounding the utilized active site core on the deprotonation of L-Kyn. With this thesis, we hope to help the researchers, who seek novel therapeutic strategies, by elucidating the details of the enzymatic reaction and proposing a potentially BBB permeable noneffector inhibitor candidate.