In silico Methods for Design of Kinase Inhibitors as Anticancer Drugs

Rational drug design implies usage of molecular modeling techniques such as pharmacophore modeling, molecular dynamics, virtual screening, and molecular docking to explain the activity of biomolecules, define molecular determinants for interaction with the drug target, and design more efficient drug candidates. Kinases play an essential role in cell function and therefore are extensively studied targets in drug design and discovery. Kinase inhibitors are clinically very important and widely used antineoplastic drugs. In this review, computational methods used in rational drug design of kinase inhibitors are discussed and compared, considering some representative case studies

Combined ligand and structure‐based approach in search of 5‐HT2A receptor agonists and antagonists

Serotoninski 5‐HT2A receptori su uključeni u mnogobrojne fiziološke i patofiziološke procese. Strukturno različiti ligandi (agonisti, antagonisti i inverzni agonisti) dovode do različitih konformacionih promena ovih receptora, izazivajući brojne biološke odgovore. Da bi se molekul ponašao kao agonista/antagonista potrebno je da poseduje različite funkcionalne grupe i specifične interakcije sa određenim aminokiselinama u vezujućem mestu receptora. Razumevanje i objašnjavanje različitosti u strukturi i vezivanju za receptor, kod agonista i antagonista, može biti od značaja za racionalni dizajn novih lekova. Za razumevanje strukturnih razlika u farmakoforama, kao i kinetici vezivanja i zmeđu agonista i antagonista korišćeni su ligand‐based i structure‐based pristupi. 3DQSAR (3D‐quantitative structure activity relationship) studije su izvođene na grupama od 79 agonista i 90 antagonista. Uporedo su odrađene četiri simulacije molekularne dinamike: serotoninski 5‐HT2A receptor u kompleksu sa agonistima (serotonin, lorkaserin) i antagonistima (klozapin, ziprazidon). Dobijeni statistički i validacioni parametri za modele agonista i antagonista ukazuju na pouzdanost i dobru predviđajuću moć 3D‐QSAR modela. Najznačajnije varijable formiranih modela daju nam uvid u najvažnije strukturne razlike između njih. Rezultati MD simulacije otkrivaju najvažnije razlike u konformacionim promenama uzrokovane vezivanjem agoniste/antagoniste, kao i interakcije liganada sa ključnim aminokiselinama, odgovornim za vezivanje. Pomoću trajektorije iz MD simulacije izvučeni su modeli, 3D strukture 5‐HT2A receptora u njegovom aktivnom (agonistvezujućem) i inaktivnom (antagonist‐vezujućem) stanju. Na osnovu ovih in silico rezultata moguće je zaključiti da li je jedinjenje agonista ili antagonista. Formirani modeli će dalje biti korišćeni za ligand‐based i structure‐based virtualni skrining i racionalni dizajn novih 5‐HT2A liganada.The serotonin 5‐HT2A receptors have shown a wide range of clinical implications since they are involved in various physiological and pathophysiological processes. Structurally diverse ligands (agonists, antagonists, and inverse agonists) can lead to different biological responses, by provoking different conformational changes of these receptors. To behave like an agonist/antagonist the molecule should have a set of functional groups and specific interactions with certain amino acids in the binding site. Understanding and explaining dissimilarities in agonist/antagonist structure and receptor binding would be beneficial for future rational drug design. To understand structural differences in pharmacophores as well as the binding kinetics of agonists and antagonists, we have combined ligand‐based and structurebased approaches. 3D‐quantitative structure‐activity relationship (3D‐QSAR) studies were performed on a series of 79 agonists and 90 antagonists. Simultaneously, we run four molecular dynamics (MD) simulations: 5‐HT2A in complex with agonists (serotonin, lorcaserin), and antagonists (clozapine and ziprasidone). Obtained statistical and validation parameters for agonists and antagonists model indicated the reliability and good predictive potential of the 3D‐QSAR models. The most influential variables of selected models gave us the insight into major structural dissimilarities between them. Results of MD simulation revealed major differences in conformational changes caused by agonist/antagonist binding, as well as ligand interactions with the key amino acids, responsible for them. Additionally, from MD simulation trajectory, we have extracted 3D structure models of 5‐HT2A in its active (agonist‐bound) and inactive (antagonist‐bound) state. Using these finding we will be able to discriminate whether a compound is agonist or antagonist, in silico. Furthermore, models that we have generated will be further used for ligand‐based and structure‐based virtual screening and rational drug design of novel 5‐HT2A ligands.VII Kongres farmaceuta Srbije sa međunarodnim učešćem Zajedno stvaramo budućnost farmacije Beograd, 10-14. oktobar 201

Combined 3D-QSAR modeling, molecular dynamics and molecular docking studies in rational drug design of novel 5-HT2A antagonists

Serotonin 5-HT2A receptors are widely distributed in the human brain where they play a key role in many physiological functions. Numerous neurological disorders caused by 5-HT2A malfunction have made it a very attractive target. Therefore, analysis of 3D-structure of the pharmacophore as well as binding kinetics of 5-HT2A antagonists would be beneficial for future rational drug design. Three dimensional quantitative structure-activity relationship (3D-QSAR) study was combined with molecular docking and molecular dynamic (MD) simulation in order to find crucial structural features responsible for high binding affinity and selectivity of 5-HT2A antagonists. This study was performed on wide range of structurally diverse antagonists that were divided into three different clusters: clozapine, ziprasidone, and CHEMBL240876 derivates. We have used 50ns MD simulations to obtain inactive, antagonist bound, conformations of each cluster representative. Subsequently, these conformations were used as templates for docking studies in order to find virtually bioactive conformations of examined compounds. Selected virtually bioactive conformations were used for generation of specific molecular descriptors (Grid Independent Descriptors- GRIND) and 3D-QSAR model building. The 3D-QSAR approach was used to identify the most important structural determinants responsible for the antagonistic activity and to propose structural modifications for novel antagonists of serotonin 5-HT2A receptors. Furthermore, diverse internal and external validation methods were applied. Obtained statistical parameters indicated the reliability and good predictive potential of the created model. Following these findings we have identified differences and similarities in the binding mode and pharmacophores of structurally diverse 5-HT2A antagonists as well as conformational changes they provoke

Combined ligand-based and structure-based approaches in rational drug design of novel 5-HT2A receptor antagonists

Serotonin 5-HT2A receptors (5-HT2AR) are highly expressed in human prefrontal cortex, essential for learning and cognition [1]. Consequently, antagonists of these receptors are effective in treatment of various neuropsychiatric disorders, such as depression, insomnia, schizophrenia, anxiety, and cent progress in molecular modelling studies has led to significant success in drug discovery using ligand and target-based methods. To design novel potent 5-HT2AR antagonists, we report a strategy of combining three-dimensional quantitative structure-activity relationship (3DQSAR) modelling with molecular docking and molecular dynamic (MD) simulation. Based on the common structural features, data set of 75 compounds was divided into three clusters. Firstly, MD simulations were carried out for each cluster representative in complex with 5-HT2AR, providing important molecular level insight into their structure and dynamics. Afterward, to provide more accurate information about binding modes in the active site of the receptor, obtained conformations were used for docking studies and generation of the virtually bioactive conformations of all studied ligands. In addition, 3D-QSAR study, utilizing selected conformers, was carried out to gain further insights into the structural requirements that affect their antagonistic activity. Besides, some commercially available 5-HT2AR antagonists were examined through in vitro PAMPA essay, as well as in silico computational methods not only to improve BBB permeability of new designed compounds, but also to establish promising tool to study their membrane permeability in detail. Overall, these and future results will provide new methodologies that could be used as guidelines for rational drug design of novel 5-HT2AR antagonists

Structure and ligand based drug design strategies in the development of novel serotonin 5-HTt2a receptor antagonists

The serotonin 5-HT2A receptors are widely distributed throughout the central and the peripheral nervous system where they play a key role in many physiological functions. Abnormal activity of 5-HT2A receptors is associated with various neurological disorders, such as depression, schizophrenia, anxiety, and Parkinson disease. In order to analyze 3D-structure of the pharmacophore as well as binding kinetics of 5-HT2A-R antagonists, we have combined ligand and structure based approaches. Three-dimensional quantitative structure-activity relationship (3D-QSAR) study in combination with molecular docking and molecular dynamic (MD) simulation was used to identify key substituents responsible for high binding affinity and selectivity of 5-HT2A antagonists. The study was performed on wide range of structurally diverse antagonists that were divided into three different clusters: clozapine, ziprasidone, and CHEMBL240876 derivates. We have obtained three different inactive, antagonistbound, conformations of this receptor by using the 50ns long MD simulations with each cluster representative. Subsequently, these conformations were used as templates for docking studies in order to find virtually bioactive conformations of ligands. Selected virtually bioactive conformations were used for calculation of specific molecular descriptors (Grid Independent Descriptors- GRIND) and 3D-QSAR model building. The 3D-QSAR approach was used to select the most influential variables which were used for clarifying the structural features required for 5-HT2A antagonists. The reliability and predictive power of the model was assessed using an external test set compounds and showed reasonable external predictability. The study provides valuable information about the key structural features that are required in the rational drug design of novel 5-HT2A antagonists

Regulation of S100As Expression by Inflammatory Cytokines in Chronic Lymphocytic Leukemia

The calcium-binding proteins S100A4, S100A8, and S100A9 are upregulated in chronic lymphocytic leukemia (CLL), while the S100A9 promotes NF-κB activity during disease progression. The S100-protein family has been involved in several malignancies as mediators of inflammation and proliferation. The hypothesis of our study is that S100A proteins are mediators in signaling pathways associated with inflammation-induced proliferation, such as NF-κB, PI3K/AKT, and JAK/STAT. The mononuclear cells (MNCs) of CLL were treated with proinflammatory IL-6, anti-inflammatory IL-10 cytokines, inhibitors of JAK1/2, NF-κB, and PI3K signaling pathways, to evaluate S100A4, S100A8, S100A9, and S100A12 expression as well as NF-κB activation by qRT-PCR, immunocytochemistry, and immunoblotting. The quantity of S100A4, S100A8, and S100A9 positive cells (p < 0.05) and their protein expression (p < 0.01) were significantly decreased in MNCs of CLL patients compared to healthy controls. The S100A levels were generally increased in CD19+ cells compared to MNCs of CLL. The S100A4 gene expression was significantly stimulated (p < 0.05) by the inhibition of the PI3K/AKT signaling pathway in MNCs. IL-6 stimulated S100A4 and S100A8 protein expression, prevented by the NF-κB and JAK1/2 inhibitors. In contrast, IL-10 reduced S100A8, S100A9, and S100A12 protein expressions in MNCs of CLL. Moreover, IL-10 inhibited activation of NF-κB signaling (4-fold, p < 0.05). In conclusion, inflammation stimulated the S100A protein expression mediated via the proliferation-related signaling and balanced by the cytokines in CLL

In silico reconstruction of human dopamine transporter and design of novel neuroprotective drugs for Parkinson’s disease

Abnormally folded alpha-synuclein protein, dysfunctional mitochondria, increased oxidative stress and reduced dopamine neurotransmitter synthesis are a ll extremely well characterized phenomena in Parkinson’s disease (PD) and are thought to be interconnected. While direct targeting of these areas has demonstrated neuroprotection in vitro and in vivo, there has been a major lack of success in clinical trials. A critical component in the failure of these clinical trials is the inability to specifically target drugs to dopamine producing neurons in the brain. New drugs targeting the dopaminergic neurons by specific uptake through the human dopamine transporter (hDAT) could represent a viable strategy for establishing selective neuroprotection. Molecules able to increase the bioactive amount of extracellular dopamine, thereby enhancing and compensating a loss of dopaminergic neurotransmission, and to exert neuroprotective response because of their accumulation in the cytoplasm, are required. By means of homology modeling, molecular docking and molecular dynamics simulations, we have generated 3D structure models of hDAT in complex with substrate and inhibitors. Our results clearly reveal differences in binding kinetics of these compounds to the hDAT in the open and closed conformations, critical for future drug design. The established in silico approach allowed the identification of three promising substrate compounds that were subsequently analyzed for their efficiency in inhibiting hDAT-dependent fluorescent substrate uptake, through in vitro live cell imaging experiments. Taken together, our work presents the first implementation of a combined in silico/in vitro-approach enabling the selection of promising dopaminergic neuron specific substrates.Rad je osvojio prvu nagradu na 2. Simpozijumu iz biomedicine, 2019

Inflammation Promotes Oxidative and Nitrosative Stress in Chronic Myelogenous Leukemia

Chronic inflammation is characterized by the production of reactive oxygen species (ROS), reactive nitrogen species, and inflammatory cytokines in myeloproliferative neoplasms (MPNs). In addition to these parameters, the aim of this study was to analyze the influence of ROS on the pro-liferation-related AKT/mTOR signaling pathway and the relationship with inflammatory factors in chronic myelogenous leukemia (CML). The activity of the antioxidant enzymes superoxide dis-mutase, glutathione peroxidase, and catalase is reduced in erythrocytes while levels of the oxidative stress markers malondialdehyde and protein carbonyl are elevated in the plasma of patients with CML. In addition, nitrogen species (nitrotyrosine, iNOS, eNOS) and inflammation markers (IL-6, NFkB, and S100 protein) were increased in granulocytes of CML while anti-inflammatory levels of IL-10 were decreased in plasma. CML granulocytes exhibited greater resistance to cytotoxic H2O2 activity compared to healthy subjects. Moreover, phosphorylation of the apoptotic p53 protein was reduced while the activity of the AKT/mTOR signaling pathway was increased, which was further enhanced by oxidative stress (H2O2) in granulocytes and erythroleukemic K562 cells. IL-6 caused oxidative stress and DNA damage that was mitigated using antioxidant or inhibition of inflammatory NFkB transcription factor in K562 cells. We demonstrated the presence of oxidative and ni-trosative stress in CML, with the former mediated by AKT/mTOR signaling and stimulated by in-flammation

VEGF Regulation of Angiogenic Factors via Inflammatory Signaling in Myeloproliferative Neoplasms

Background: Chronic inflammation has been recognized in neoplastic disorders, including myeloproliferative neoplasm (MPN), as an important regulator of angiogenesis. Aims: We investigated the influence of vascular endothelial growth factor (VEGF) and pro-inflammatory interleukin-6 (IL-6) on the expression of angiogenic factors, as well as inflammation-related signaling in mononuclear cells (MNC) of patients with MPN and JAK2V617F positive human erythroleukemic (HEL) cells. Results: We found that IL-6 did not change the expression of angiogenic factors in the MNC of patients with MPN and HEL cells. However, IL-6 and the JAK1/2 inhibitor Ruxolitinib significantly increased angiogenic factors—endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor-1 alpha (HIF-1α)—in patients with polycythemia vera (PV). Furthermore, VEGF significantly increased the expression of HIF-1α and eNOS genes, the latter inversely regulated by PI3K and mTOR signaling in the MNC of primary myelofibrosis (PMF). VEGF and inhibitors of inflammatory JAK1/2, PI3K, and mTOR signaling reduced the eNOS protein expression in HEL cells. VEGF also decreased the expression of eNOS and HIF-1α proteins in the MNC of PMF. In contrast, VEGF increased eNOS and HIF-1α protein expression in the MNC of patients with PV, which was mediated by the inflammatory signaling. VEGF increased the level of IL-6 immunopositive MNC of MPN. In summary, VEGF conversely regulated gene and protein expression of angiogenic factors in the MNC of PMF, while VEGF increased angiogenic factor expression in PV mediated by the inflammation-related signaling. Conclusion: The angiogenic VEGF induction of IL-6 supports chronic inflammation that, through positive feedback, further promotes angiogenesis with concomitant JAK1/2 inhibition

Inhibition of proinflammatory signaling impairs fibrosis of bone marrow mesenchymal stromal cells in myeloproliferative neoplasms

Although bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been identified as a major cellular source of fibrosis, the exact molecular mechanism and signaling pathways involved have not been identified thus far. Here, we show that BM-MSCs contribute to fibrosis in myeloproliferative neoplasms (MPNs) by differentiating into αSMA-positive myofibroblasts. These cells display a dysregulated extracellular matrix with increased FN1 production and secretion of profibrotic MMP9 compared to healthy donor cells. Fibrogenic TGFβ and inflammatory JAK2/STAT3 and NFκB signaling pathway activity is increased in BM-MSCs of MPN patients. Moreover, coculture with mononuclear cells from MPN patients was sufficient to induce fibrosis in healthy BM-MSCs. Inhibition of JAK1/2, SMAD3 or NFκB significantly reduced the fibrotic phenotype of MPN BM-MSCs and was able to prevent the development of fibrosis induced by coculture of healthy BM-MSCs and MPN mononuclear cells with overly active JAK/STAT signaling, underlining their involvement in fibrosis. Combined treatment with JAK1/2 and SMAD3 inhibitors showed synergistic and the most favorable effects on αSMA and FN1 expression in BM-MSCs. These results support the combined inhibition of TGFβ and inflammatory signaling to extenuate fibrosis in MPN