Correlating Basal Gene Expression across Chemical Sensitivity Data to Screen for Novel Synergistic Interactors of HDAC Inhibitors in Pancreatic Carcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies. Development of the chemoresistance in the PDAC is one of the key contributors to the poor survival outcomes and the major reason for urgent development of novel pharmacological approaches in a treatment of PDAC. Systematically tailored combination therapy holds the promise for advancing the treatment of PDAC. However, the number of possible combinations of pharmacological agents is too large to be explored experimentally. In respect to the many epigenetic alterations in PDAC, epigenetic drugs including histone deacetylase inhibitors (HDACi) could be seen as the game changers especially in combined therapy settings. In this work, we explored a possibility of using drug-sensitivity data together with the basal gene expression of pancreatic cell lines to predict combinatorial options available for HDACi. Developed bioinformatics screening protocol for predictions of synergistic drug combinations in PDAC identified the sphingolipid signaling pathway with associated downstream effectors as a promising novel targets for future development of multi-target therapeutics or combined therapy with HDACi. Through the experimental validation, we have characterized novel synergism between HDACi and a Rho-associated protein kinase (ROCK) inhibitor RKI-1447, and between HDACi and a sphingosine 1-phosphate (S1P) receptor agonist fingolimod

Biological activity of Aronia melanocarpa antioxidants pre-screening in an intervention study design

The beneficial effects of black chokeberry fruits and juices in health promotion and prevention of chronic diseases shown in both epidemiological and dietary intervention studies are often connected with their antioxidant activity. The aim of this study was to investigate the total phenolics and anthocyanins content, chemical antioxidant activity (DPPH-assay), antioxidant protection in erythrocytes and anti-platelet activity in vitro of three different chokeberry products: commercial and fresh pure chokeberry juice and a crude lyophilized water-ethanol extract of chokeberry fruits, as part of their pre-clinical evaluation. The obtained results indicated differences in chemical composition and antioxidant activity of the investigated products. Cellular effects, including both in vitro anti-platelet and antioxidant effects, were not directly correlated with the chemical antioxidant activity and the results obtained in vitro for antiplatelet effects were only partially consistent with the results obtained in vivo, in a pilot intervention trial. In conclusion, chemical analyses and in vitro experiments on foods and their bioactive substances are a valuable pre-screening tool for the evaluation of their biological activity. However, extrapolation of the obtained results to the in vivo settings is often limited and influenced by the bioavailability and metabolism of native dietary compounds or interactions with differrent molecules within the human body

Cytotoxic activity of amino acid esters of vitamin E against breast and lung cancer cell lines

U velikom broju studija pokazana je antitumorska aktivnost prirodnih izomera vitamina E, a naročito njihovih polusintetskih derivata. Cilj ove studije je bio ispitivanje citotoksične aktivnosti estara α‐tokoferola sa aminokiselinama lizinom, prolinom, glutaminom, asparaginom i estara γ‐tokotrienola sa lizinom, prolinom i glutaminom na MCF7 i MDA‐MB 231 ćelijskim linijama tumora dojke i A549 ćelijskoj liniji tumora pluća. Sve ćelijske linije tretirane su koncentracijama ispitivanih jedinjenja u opsegu 0,62‐50 μM u toku 48 sati. Preživljavanje ćelija nakon tretmana ispitivanim jedinjenjima je određeno MTT‐testom. Najveći uticaj na preživljavanje malignih ćelija su imali α‐tokoferil lizin, α‐tokoferil asparagin u formi nitrila i γ‐tokotrienil lizin. α‐ Tokoferil lizin je ispoljio snažnu antitumorsku aktivnost na A549 (IC50=10,6 μM) i MCF7 (IC50=8,6 μM) ćelijama, dok je γ‐tokotrienil lizin je jedini od ispitivanih jedinjenja koji je ispoljio aktivnost na sve tri maligne ćelijske linije, sa IC50 vrednostima 20,6 μM (MCF7), 28,6 μM (MDA‐MB‐231) i 19 μM (A549). Asparaginski estar α‐tokoferola u formi nitrila je je doveo do snažne inhibicije preživljavanja MDA‐MB‐231 ćelija (IC50=9,2 μM) koje se odlikuju višestrukom rezistencijom na lekove koji se koriste u terapiji tumora dojke. Ispitivana jedinjenja nisu ispoljila toksičnost ka MRC‐5 zdravoj ćelijskoj liniji fetalnih fibroblasta pluća. Zahvaljujući pokazanoj in vitro citotoksičnoj aktivnosti i selektivnosti za maligne ćelije, aminokiselinski estri α‐tokoferola i γ‐tokotrienola predstavljaju dobre kandidate za buduća in vivo ispitivanja.In recent studies, the antitumor activity of vitamin E derivatives has been demonstrated. The aim of this study was to investigate the cytotoxic activity of α‐ tocopherol esters with amino acids lysine, proline, glutamine, asparagine and γ‐ tocotrienol esters with lysine, proline and glutamine on MCF7 and MDA‐MB 231 breast cancer cell lines and A549 lung cancer cell line. All cell lines were treated with concentrations of the test compounds in the range of 0.62‐50 μM for 48 hours. Cell survival after treatment with the investigated compounds was determined by MTT test. The greatest influence on the survival of malignant cells was observed with α‐ tocopheryl lysine, α‐tocopheryl asparagine in the form of nitrile and γ‐tocotrienyl lysine. α‐Tocopheryl lysine exhibited strong cytotoxic activity on A549 (IC50 = 10.6 μM) and MCF7 (IC50 = 8.6 μM) cells, while γ‐tocotrienyl lysine is the only compound that exhibited activity on all three cancer cell lines, with IC50 values of 20.6 μM (MCF7), 28.6μM (MDA‐MB‐231) and 19 μM (A549). The α‐tocopheryl asparagine nitrile led to a strong inhibition of the survival of MDA‐MB‐231 cells (IC50 =9.2 μM) that are characterized by multiple resistance to drugs used for treatment of breast cancer. All investigated compounds did not exhibit toxicity to normal MRC‐5 cell line of the fetal fibroblasts of the lungs. Based on the shown in vitro cytotoxic activity and selectivity for tumor cells, α‐ tocopherol and γ‐tocotrienol amino acid esters represent promising candidates for future in vivo studies.VII Kongres farmaceuta Srbije sa međunarodnim učešćem Zajedno stvaramo budućnost farmacije Beograd, 10-14. oktobar 201

Epigenetic drug discovery: fragment-based drug design of novel 1-benzhydryl-piperazine derivatives as selective histone deacetylase 6 inhibitors

Selective histone deacetylase 6 (HDAC6) inhibition with small molecules is regarded as a rational strategy to develop safer anti-cancer drugs compared to non-selective HDAC inhibitors1. To date, structural motifs that are important for HDAC inhibitory activity and selectivity are defined as: surface recognition group (CAP group), aliphatic or aromatic linker and zinc-binding group (ZBG). Herein, we describe a comprehensive protocol for the computational fragment search of novel surface-recognition (CAP) groups aimed to design selective Histone Deacetylase 6 (HDAC6) inhibitors (Figure 1)2. Identified heterocyclic CAP group, 1-benzhydryl piperazine was employed to synthesize novel HDAC inhibitors with small structural perturbations in the hydrocarbon linker. Enzymatic in vitro HDAC screening identified two selective HDAC6 inhibitors (6b, IC50 = 186 nM and 9b, IC50 = 31 nM), as well as two non-selective nanomolar HDAC inhibitors (7b and 8b). The influence of linker chemistry of synthesized inhibitors on HDAC6 potency was studied using structure-based molecular modelling. References 1. J. Amengual, J. Lue, H. Ma, R. Lichtenstein, B. Shah, S. Cremers, S. Jones, A. Sawas, The Oncologist, 2021, 26(3), 184 e366. 2. D. Ruzic, M. Petkovic, D. Agbaba, A. Ganesan, K. Nikolic, Mol. Inform., 2019, 38(5), 1800083. Acknowledgments The authors acknowledge a Ministry of Education, Science and Technological Development of the Republic of Serbia Faculty of Pharmacy project (451-03-68/2022- 14/200161).8th Conference of Young Chemists of Serbia 29th October 2022 University of Belgrade, Faculty of Chemistry Book of Abstracts M32 DRuzic Abstracts_8 CYCS BGD 202

Telmisartan induces melanoma cell apoptosis and synergizes with vemurafenib in vitro by altering cell bioenergetics

Objective: Despite recent advancements in targeted therapy and immunotherapies, prognosis for metastatic melanoma patients remains extremely poor. Development of resistance to previously effective treatments presents a serious challenge and new approaches for melanoma treatment are urgently needed. The objective of this study was to examine the effects of telmisartan, an AGTR1 inhibitor and a partial agonist of PPAR gamma, on melanoma cells as a potential agent for repurposing in melanoma treatment. Methods: Expression of AGTR1 and PPAR gamma mRNA in melanoma patient tumor samples was examined in publicly available datasets and confirmed in melanoma cell lines by qRT-PCR. A panel of melanoma cell lines was tested in viability, apoptosis and metabolic assays in presence of telmisartan by flow cytometry and immunocytochemistry. A cytotoxic effect of combinations of telmisartan and targeted therapy vemurafenib was examined using the Chou-Talalay combination index method. Results: Both AGTR1 and PPAR gamma mRNA were expressed in melanoma patient tumor samples and decreased compared to the expression in the healthy skin. In vitro, we found that telmisartan decreased melanoma cell viability by inducing cell apoptosis. Increased glucose uptake, but not utilization, in the presence of telmisartan caused the fission of mitochondria and release of reactive oxygen species. Telmisartan altered the cell bioenergetics, thereby synergizing with vemurafenib in vitro, and even sensitized vemurafenib-resistant cells to the treatment. Conclusions: Given that the effective doses of telmisartan examined in our study can be administered to patients and that telmisartan is a widely used and safe antihypertensive drug, our findings provide the scientific rationale for testing its efficacy in treatment of melanoma progression

Targeting Histone Deacetylases: Opportunities for Cancer Treatment and Chemoprevention

The dysregulation of gene expression is a critical event involved in all steps of tumorigenesis. Aberrant histone and non-histone acetylation modifications of gene expression due to the abnormal activation of histone deacetylases (HDAC) have been reported in hematologic and solid types of cancer. In this sense, the cancer-associated epigenetic alterations are promising targets for anticancer therapy and chemoprevention. HDAC inhibitors (HDACi) induce histone hyperacetylation within target proteins, altering cell cycle and proliferation, cell differentiation, and the regulation of cell death programs. Over the last three decades, an increasing number of synthetic and naturally derived compounds, such as dietary-derived products, have been demonstrated to act as HDACi and have provided biological and molecular insights with regard to the role of HDAC in cancer. The first part of this review is focused on the biological roles of the Zinc-dependent HDAC family in malignant diseases. Accordingly, the small-molecules and natural products such as HDACi are described in terms of cancer therapy and chemoprevention. Furthermore, structural considerations are included to improve the HDACi selectivity and combinatory potential with other specific targeting agents in bifunctional inhibitors and proteolysis targeting chimeras. Additionally, clinical trials that combine HDACi with current therapies are discussed, which may open new avenues in terms of the feasibility of HDACi’s future clinical applications in precision cancer therapies

Rilmenidine binds to and inhibits the activity of MDR pumps in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the sixth leading cause of death worldwide and the fourth in Europe with a 5-year survival rate. The common cause of treatment failure in PDAC patients is multidrug resistance (MDR) due to the increased expression of plasma membrane efflux pumps that limit the intracellular uptake and retention of numerous xeno- and endobiotics. As the 93.3% of pancreatic carcinomas expressed P-glycoprotein (P-gp-MDR1/ABCB1) and 31% co-expressed multidrug resistance protein 1 (MRP1/ABCC1) with MDR1 P-gp, the inhibition of these pumps may be the target for novel anticancer drugs. We used the FRED 3.2.0.2 software to predict the affinity of I1-imidazoline receptor ligand rilmenidine within the binding site of P-gp-MDR1/ABCB1 and MRP1/ABCC1, and flow cytometry to evaluate the effect of rilmenidine phosphate and rilmenidine fumarate on the efflux pumps in PDAC cells in vitro. The results of the molecular docking studies indicate that rilmenidine has the binding affinity for both P-gp-MDR1/ABCB1 and MRP1/ABCC1 efflux pumps. While, in vitro studies show that rilmenidine fumarate has better potential to inhibit Calcein AM efflux than rilmenidine phosphate, and it did so in a dose-dependent manner. Our results indicate that rilmenidine has the affinity to bind to MDR efflux pumps and to inhibit their activity. This potential of rilmenidine to overcome multidrug resistance in PDAC should be further investigated in order to develop more effective PDAC therapy

Antibody Epitope Specificity for dsDNA Phosphate Backbone Is an Intrinsic Property of the Heavy Chain Variable Germline Gene Segment Used

Analysis of protein sequences by the informational spectrum method (ISM) enables characterization of their specificity according to encoded information represented with defined frequency (F). Our previous data showed that F(0.367) is characteristic for variable heavy chain (VH) domains (a combination of variable (V), diversity (D) and joining (J) gene segments) of the anti-phosphocholine (PC) T15 antibodies and mostly dependent on the CDR2 region, a site for PC phosphate group binding. Because the T15 dsDNA-reactive U4 mutant also encodes F(0.367), we hypothesized that the same frequency may also be characteristic for anti-DNA antibodies. Data obtained from an analysis of 60 spontaneously produced anti-DNA antibody VH domain sequences supported our hypothesis only for antibodies, which use V gene segment in germline configuration, such as S57(VH31), MRL-DNA22, and VH11, members of the VH1 (J558) and VH7 (S107) gene families. The important finding is that out of seven V gene segments used by spontaneous anti-DNA antibodies, F(0.367) is only expressed by the germline configuration of these three V gene segments. The data suggest that antibody specificity for the phosphate group moiety delineated as F(0.367) is the intrinsic property of the V germline gene segments used, whereas paratope/epitope interaction with antigens bearing this epitope, such as PC or dsDNA, requires corresponding antibody VH conformation that is susceptible to somatic mutation(s). © 2018 Srdic-Rajic, Kohler, Jurisic and Metlas

New benzothiazolamine derivatives as inducers of an early apoptosis in MCF-7 human breast cancer cell line

Background Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012.1 Current chemotherapy targets the proliferative advantage of tumor cells over healthy cells, but the lack of selectivity of chemotherapeutic agents usually leads to serious side effects. A major challenge in the development of effective and safe cancer treatment is to identify the agents that could affect cellular processes essential for, or greatly enhanced in, malignant cells only. Aims Benzothiazole derivatives represent a series of compounds of an undoubted interest because of the broad spectrum of biological effects associated with this scaffold.2 In addition, benzothiazoles have attracted considerable attention in anticancer research and a lot of structural modifications on their core nuclei have been made to improve the antitumor activity. Therefore, we have synthesized novel benzothiazolamine derivatives and investigated their anticancer potential against MCF-7 human breast cancer cell line. Methods We have synthesized a series of novel benzothiazolamine carbamates and amides starting from 1-chloro4-nitrobenzene and an appropriate alkylthiol, 3 followed by cyclization to benzothiazolamine and further derivatization of amino-group. The selected compounds were subjected to a panel of NCI-60 cell line for in vitro determination of antitumor activity. For better insight into possible mechanism of antiproliferative activity, we have examined the cell cycle phase distribution and apoptosis in MCF-7 human breast cancer cell line using flow cytometry methods, after treatment with synthesized compounds. Our research continued towards examination of our compounds’ influence on the reactive oxygen species level, mitochondrial membrane potential, as well as cell cycle regulators. Results The cell cycle phase distribution and apoptosis in MCF-7 human breast cancer cell line were investigated after exposure to IC50 concentrations, obtained in vitro, of four selected compounds (Figure 1.b) for 24 and 48 hours, respectively. Using flow cytometry after PI staining we showed that our compounds affect cell cycle distribution in a time dependent manner. After 24 h treatment, the portion of cells in G2/M phase increased, suggesting cell cycle arrest in mitosis. After 48 hours, the number of sub G1 phase cells increased, which indicates apoptosis. Following incubation with selected compounds for 48 hours, the proapoptotic effect was reflected by the increase of portion of early apoptotic cells up to 63 % measured by bivariate Annexin V/PI flow cytometry (Figure 1.a). Moreover, we observed the loss of mitochondrial membrane potential, which could indicate that our compounds promote apoptosis via the mitochondrial pathway in MCF-7 cells. In addition, reactive oxygen species level in MCF-7 cells significantly decreased after treatment with benzothiazolamine derivatives. Conclusion Benzothiazolamine carbamates and amides showed great potency for promoting highly specific programmed cell death apoptosis in MCF-7 cancer cell line. Further examination will eventually provide identification of molecular targets of benzothiazolamines. Our data offer a significant contribution to the search for medicinally active compounds and may lead to discovery of a new potent antitumor agent

The absence of immunoreactivity to donkey’s milk in patients with recurrent aphthous ulcers and immunoreactivity to cow’s milk

Despite the numerous benefits of milk constituents for human health a considerable number of the general population follow a milk-restricted diet due to clinically confirmed or self-assessed adverse reactions to cow’s milk consumption. Recurrent aphthous ulcers (RAU) are currently one of the most common oral disorders, with a worldwide distribution and insufficiently defined etiology, which, among other factors, implies the immunological reaction to food proteins. The aim of this study was to determine the immune-reactivity to donkey’s milk proteins in patients with RAU and compare it to the reactivity towards the proteins from cow’s and goat’s milks, in a set of simultaneous experiments. Levels of serum IgA, IgG and IgE antibodies to the same quantity of the examined antigens were determined by enzyme-linked immunosorbent assay. The results indicate that patients with RAU with increased immunity to cow’s milk proteins could consider the use of donkey’s milk as the best protein source