 to the next morning session. We are continuing with a talk by Dr. Mariana Tanasova. She is a researcher in the group of Professor Irene Ducinova at the Medical University of Sofia. She applies molecular doctoring in molecular simulations to study ligand protein interactions. And her talk is entitled, Beyond in Cervical Timulations, a case study on attitude culinary stars inhibitors. I'm not sure that I pronounced this correctly. I apologize. You have 30 minutes. Thank you. Can you hear me? Yes. Hi, everyone. So I'll present our latest on the attitude culinary stars inhibitors. So thank you for your introduction. So a few words for Alzheimer's disease, which is one of widespread type of dementia worldwide. It's a neurodegenerative disorder characterized by progressive and irreversible loss of hippocampal and cortical neurons. The pathological hallmarks of the disease are extracellular, hard and insoluble, amyloid beta-plugs, hindering the communication between neurons. And intracellular neurofibrous tangles of abnormally hyperphosphorylated tau proteins that inhibit the transmission of nutrients and other substances within the neuron. These pathological deformations lead to synaptic degeneration in neuron that are resulting in short-term memory loss, impartment in cognitive function, as thinking, speaking, learning, orientation, judgment, communication, et cetera. So one of the biochemical factors related with cognitive dysfunction is the continuous loss of neurotransmitter at stuho-line. And therefore, increasing the stuho-line levels will improve the cognition. This can be done by inhibiting the enzyme at stuho-line estuaries. The enzyme is located in the synaptic clefts and responsible for breaking down the stuho-line into Hawaiian and stuho. This concept lies in the whole energetic hypothesis for symptomatic treatment of the disease. The binding site of stuho-line estuaries is well known. It consists of several domains, catalytic, anionic, silpo, ketene, ho, and periferial anionic site. The enzyme is also involved in non-human energy functions. One of them is acting as a nucleus in amyloidosis, forming toxic complexes with amyloid beta peptide. The current drugs acting as stuho-line estuaries inhibitors are gauntamine, rivastigmin, and donepizil. Hyperazine A is approved as a drug for treatment of Alzheimer's disease in China. And it is used as a diary supplement for memory functions improvement in USA. The drugs do not lack site effects. Therefore, the search of new stuho-line estuaries inhibitors is keep going. The aim of the study is to discover new compounds with dual-site binding to the stuho-line estuaries with potential multi-target action. We selected to design hybrids between gauntamine and cocomine because cocomine is known as a strong inhibitor of the amyloid aggregation. We designed a fragment-based combinatorial library of seven to two hybrid molecules by combining nine linkers and eight aromatic fragments. Linkers and fragments mimic parts of the cocomine structure. So initially, the hybrids were subject to 11 admi filters, nine for blood-brain permeability, and one for gastrointestinal absorption, and one for nonspecific interactions with numerous biological targets denoted with PAANs. 44 compounds passed all the initial tests. They were docketed to the stuho-line estuaries binding site using a protocol that was previously optimized in terms of scoring function, rigid or flexible binding site, and ligand radius of the binding site, presence or absence of water molecule within the binding site. Here are given the both scores for some of the hybrid. And in order to examine the relationship between the docking scores and the ad stuho-line estuaries activity, compounds with high, moderate, and low gold scores were selected for synthesis. In total, 14 compounds were synthesized. Before tested for the ad stuho-line estuaries activity, they were tested for neurotoxicity on urine neuroblastoma, neuro2A cells. As guavantamin and curcumin were used as reference compounds. Five compounds were less toxic. They are shown here in bold than curcumin, and only they were tested for ad anti-ad stuho-line estuaries activity. As can be seen, all five hybrids showed activities higher than the activity of guavantamin and are between 4 and 186 times more active than it. Next, various physical, chemical properties and pharmacokinetic parameters were calculated for these five compounds. Molecular weights of the compounds are around 500. The pKa values are close to the pKa value of guavantamin. The hybrids are more lipophilic than guavantamin according to the walk-walk-d values. Although the hybrids were more lipophilic than guavantamin, they had power surface areas wider than guavantamin, but lower if normalized unit mass. There was only one hydrogen-borne acceptors donors in the molecules. This is the hydrogen atom from the ammonium cation. While the number of the hydrogen-borne acceptors depend on the number of the oxygen atom within the molecules, but for all active compounds, they were up to 10. According to the ionized fraction, compounds for A, for B, E and F are moderate bases as 70% ionized at physiological pH like guavantamin. While compound 8B is a very weak base with only 30% ionized. The predicted steady-state volume of distribution values of the hybrids for A, B, E and F are almost three times bigger than the volume of distribution of 8B. The tested compounds bind extensively to the plasma protein according to the predicted free fraction of drug in the plasma. And their predicted values for clearance show that the hybrids are low clearance compounds with clearances between below 37 of hepatic blood flow. Their half-lives are moderate, lowering to the convenient, allowing convenient multiple dose regimens. All these pharmacokinetic parameters were predicted using previously derived quantitative structure pharmacokinetics relationships in our lab. Here, the intermolecular interactions of the docking poses for the five non-toxic compounds are presented. The guavantamin part forms dense network of hydrogen bonds, which is shown here. Between guavantamin ammonium group and tyrosine 337, between the metoxy group and serine 203, and between the hydroxyl group gaseous 202. The carbon atom of the unsaturated part from the linker is also involved in P-pycon with tryptophan 286, or tyrosine 72, in the case of 8b and 4e. Oxygen atom from the carbonium group of the linker formed the additional hydrogen bond with tyrosine 124, in the case of 4e and b. The terminal part, aromatic parts of the molecule is well fitted in the periphery on the anionic side, different orientations. But all of them make P-py stacking with tryptophan 286, or tyrosine 72. A special attention deserves compound 4b as the most active and least toxic compound in the test. It is very close to 4a and 4b. I'll just show you their final structures where the structures are differentiated. Actually, they differentiate only the terminal part where 4b have an additional group according to A and F compound. But they are in vitro, just to show you, the two and a half times difference in their activity between 4b and 4a. Cannot, the docking protocol actually, the docking results cannot explain this difference because from the dockings, we find one and the same amount of binding. Therefore, we decided to be the complex between 4b and enzyme by molecular dynamic simulation. So then the simulation showed that this metoxy group and the phenyl group at the terminal part forms an additional hydrogen bond with tyrosine 72. At some of the initial frames, high-student 287 is also coming quite close to this group, but not enough to form a long-lasting high-student bond. So this additional hydrogen bond, which is absent in the complex between 4a and acetylcholinesterase explains the better binding of this compound to the enzyme. Next, acute toxicity assay was performed on female mice after oral administration of 4b. The median lethal 4b was 49 milligrams per kilogram and no changes in size, color and consistency of the lungs, liver, heart, kidney, stomach, spleen and intestine were observed. No abnormalities in the morphology of the gonads and brain were detected. The short term toxicity of 4b was assessed on the male mice by daily administration of 2.5 milligrams, which is one-twent of the lethal dose and five milligrams per kilogram, which is one-tenth of the lethal dose for 14 days. As positive controls, galantamine in dose of three milligrams per kilogram and curcumin in dose of 25 milligrams per kilogram, which is one-tenth of their lethal doses were used. Control or placebo group was also included in the test and no behavioral changes and signs of toxicity were observed. After that, blood was taken for complete blood count and biochemistry measurements. Atstual cholinerase inhibition, malodealdehyde and gut attion levels in the brain were measured. So the results are shown here and atstual cholinerase activity was measured for 10 minutes by Elman's methods and the results are shown here in left. Curcumin caused a mild decrease in the atstual cholinerase activity followed by galantamine and 4B. The dose of five milligrams per kilogram 4B causes 25% inhibition of the enzyme activity compared to the control group. Tested in vivo, 4B showed 186 times higher inhibitory effect on the atstual cholinerase than galantamine. The mild difference here in the ex vivo experiment of atstual cholinerase inhibition between 4B and galantamine might be indicative for a recovery of the atstual cholinerase levels due to high spontaneous reactivation of the enzyme associated with rapid synthesis and release of new enzyme from the liver. The atstual cholinerase inhibition in vivo is associated with increased oxidative stress. In response, the increased oxidative stress mediates an increase in the atstual cholinerase activity. The antioxidant activity of the tested compounds after 40-day treatment was assessed by measuring the levels of myodealdehyde and glutathione in mice brain homogenates. Both compounds are specific markers for oxidative stress. It is evident that in both tests, 4B has equal or even better antioxidant activity than galantamine. Additionally, the antioxidant activity of galantamine, cocomine and 4B was assessed by two methods for radical scavenging, DPPH and ABTS. And one method for reducing power, FRAP, as butyohydroxidol was used as a positive control. In the DPPH activity, the DPPH activity range from 2.6 to 3.4 galantamine to 10.034 cocomine. 4B showed an intermediate activity with 7.32. The results were expressed as micro-molar TROX equivalent. In the ABTSSA, 4B did the highest capacity for 101.22, which is stronger even than the positive control of butyohydroxidol. In the FRAP assay, cocomine showed the highest activity regarding to its reducing power, which is followed by 4B and galantamine. The inhibition of lipid peroxidation was monitored for five. The increase in the absorbance indicates peroxide formation. Such an increase was observed initially for galantamine and cocomine on the day two, which galantamine here is in red and cocomine is in yellow, which is... And after that, the absorbance remained constant. And the second increase was observed for cocomine on day five. On the contrary, 4B, which is shown in green, has lower absorbance that showed actually immediately increase in the absorbance on day two and then a gradual increase of the level of cocomine on day four and on day five. Actually, 4B has lower absorbance than both galantamine and cocomine. Thus, 4B protects better against peroxide formation than its late galantamine and cocomine. In general, the in vitro antioxidant axes, DPPH, ABTS, FRAP and inhibition of lipid peroxidation confirmed the results found in the ex vivo experiments. And finally, the effects of 4B and galantamine were tested on human neuroblastoma cells by amyloid beta. Four scenarios were designed. Compounds were administered as single treatment, administered simultaneously with amyloid beta peptides and administered before or after amyloid beta peptides. The single administration indicates that amyloid beta is toxic for the cells as the virus is by 35%. Both galantamine and 4B themselves are non-toxic for the cells. Industries simultaneously with amyloid beta, galantamine and 4B show an immediate protection on the cells. Protection by 4B is higher than the protection of galantamine. In the third scenario, the cells were incubated in the tested compounds for 24 hours, followed by the addition of amyloid beta peptides. And the aim of this test is to examine the ability of the compounds to prevent amyloid beta cytotoxicity. It is evident that both compounds prevent amyloid beta toxicity, as 4B performs slightly better than galantamine. And in the fourth scenario, the cells were incubated with amyloid beta peptides for 24 hours and then the tested compounds were added. The results here are indicative for the ability of the compounds to repair the damage caused by the amyloid beta and to restore the cell viability. Here again, the two compounds revive the cells as galantamine slightly for B. As a possible amount of this protection, we suggest an aosteric modulation of alpha-7 nicotinic steel haline receptors, as there are such an experimental data for galantamine. In summary, 72 compounds were designed and tested for admitt filters. 44 of them passed the filters and were docked into the binding site of the acetylcholinesterase. 14 compounds were synthesized for neurotoxicity, five for non-toxic and were tested for anti-acetylcholinesterase activity. The compounds showed better between 41 and 186 times more active than galantamine. The non-toxic and most active compound for B was further investigated in vivo, ex vivo and in vitro, and the results indicate that compound for B is comparable to galantamine. It is low toxic with anti-acetylcholinesterase anti-oxidant and anti-amiwhite beta activity. Therefore, for B has emerged as a promising multi-target agent for a complex treatment of neurodegenerative disorder. So this study, the publications related with the study is presented here. Acknowledgement for the support for this research and curatation, attention. So I'm here for questions. Thank you. I saw one question in the chat. How was the clearance and plasma protein binding were calculated? Q-cell? So yes, this is by models that are previously derived in our lab, so just me to find this slide. Here are the papers that these pharmacokinetic parameters, the models that were derived, published already. Q-cell? Do you put the references in the chat so that the- Yes, copy done. Thank you.