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  • Open access
  • 13 Reads
Photo-crosslinking of human protein kinase regulatory subunit CK2β for the identification of CK2 binding partners
Published: 30 October 2019 by MDPI AG in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

Human protein kinase CK2 is a heterotetrameric Ser/Thr kinase, consisting of two catalytic (CK2α/α’) and two regulatory (CK2β) subunits. CK2 plays a key role in several physiological and pathological processes. Moreover in cancer cells it was shown that CK2 is upregulated [1]. Although the number of more than 300 substrates is still increasing, the regulation of CK2 remains unclear [2]. It is assumed that several protein-protein interactions are involved in the regulation of CK2. Thereby CK2β modulates the substrate specificity of CK2 and also functions as a docking platform for regulators and substrates. This study aims for the identification of binding partners by photo-crosslinking coupled with mass spectrometry. Therefore the unnatural amino acid p‑azidophenylalanine (pAzF) is incorporated into CK2β [3].


Here we report the establishment of the photo-crosslinking procedure with purified CK2β‑pAzF with its strongest binding partner CK2α as a proof of principle. The photo-crosslinking product of CK2β-pAzF and CK2α was detected by SDS-PAGE analysis and immunostaining. Furthermore it was shown, that the photo-crosslink reaction is specific for interaction partners and is not affected by other proteins. The site directed photo-crosslinking reaction was compared to the common used homo-bifunctional NHS-ester disuccinimidyl suberate (DSS) that crosslinks primary amino groups.

References:

[1] Tawfic, S. et al.: Histol Histopathol. 2001, 16:573-582.

[2] Meggio, F.and Pinna, L.A.: FASEB J. 2003, 17:349-368.

[3] Chin, J.W. et al.: J. Am. Chem. Soc. 2002, 124, 9026-9027.

  • Open access
  • 15 Reads
Investigation of pharmacokinetic properties of CK2 Inhibitors with an Indeno[1,2-b]indole scaffold
Published: 30 October 2019 by MDPI AG in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

The highly pleiotropic and constitutively active protein kinase CK2 plays an important role in several cellular mechanisms. Due to its overexpression and elevated activity in tumor cells, CK2 became an important target in tumor therapy nowadays. It was shown, that the kinase causes antiapoptotic and proliferation enhancing effects in neoplastic tissues [1,2]. Moreover, the reduction of CK2 activity in tumor cells leads to apoptosis while normal cells stay unaffected [3].

Indeno[1,2-b]indoles are ATP competitive CK2 inhibitors with IC50 values in the nanomolar range of concentration. NA16 was described as one of the most potent indeno[1,2-b]indoles with an IC50 value of 25 nM [4]. Therefore, the pharmacokinetic properties of NA16 were further analyzed during this study. It could be shown that NA16 reduced the growth of different tumor cell lines and induced cancer cell apoptosis. Furthermore, its effect on HUVEC cell growth was comparable with the effect of CX-4945, a CK2 inhibitor in clinical trials [5], which was used as control. Intracellular concentrations of NA16 were higher than concentrations of CX-4945 at different time points. Metabolism studies showed that NA16 is moderate metabolic stable and is not glucuronidated. These results underline the potential of NA16 as an antitumor drug.

References:

[1] Ahmed, K et al.: Trends Cell Biol 2002, 12, 226-230.
[2] Meggio, F. and Pinna, L.A.: FASEB J. 2003, 17, 349-368.
[3] Slaton, J. W. et al.: Mol Cancer Res 2004, 2, 172.
[4] Gozzi, G. J. et al.: J Med Chem 2015, 58, 265-277.
[5] Siddiqui-Jain, A. et al.: Cancer Res 2010, 70, 24.

  • Open access
  • 15 Reads
Expression profile of innate immune genes in peripheral blood mononuclear cells as early detection of breast cancer
Published: 30 October 2019 by MDPI AG in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

Early detection of breast cancer plays a key role in successful treatment and patient survival. The presence of cancer cells causes immune cells to undergo various phenotypic and functional changes and the affected immune cells kill cancer cells or promote proliferation and metastasis of cancer cells. Using peripheral blood cells (PBCs) for gene expression analysis is valuable to evaluate disease-associated and drug-response related genes. In this study, we aimed to explore the innate immune gene expression profile of PBCs in patients with breast cancer.

Material & methods: Whole blood samples were collected from 30 breast cancer patients and 42 healthy volunteers. The gene expression of PBCs was determined by RT-qPCR.

Results: A healthy sample was created using the total RNA of healthy volunteers. We investigated the expression of receptors and its ligands, transcription factors, cytokines, chemokines, interferon-stimulated and pro-oxidation genes related to the innate immune system in breast cancer and healthy samples. Deregulated expression of a few genes was found out in the blood of breast cancer patients compared to the healthy sample.

Conclusions: We have identified the genes in whole blood that classifies breast cancer patients and healthy women with good accuracy. These results suggested that the expression of innate immune genes in PBCs can be used for early detection of breast cancer and evaluation of efficiency treatment of this disease.

  • Open access
  • 151 Reads
A novel approach for ER+ breast cancer treatment: A new compound that modulates aromatase and ER

Introduction: Estrogen receptor-positive (ER+) breast cancer is the most common subtype of breast cancer worldwide. Estrogens, after being synthetized by aromatase, bind to ERα promoting breast cancer proliferation (1). Therefore, the therapies already approved act either by inhibiting aromatase or by modulating ERα. However, besides their therapeutic success, they induce several side effects (2), reason why it is crucial to discover novel therapeutic approaches.

Aim: Considering that aromatase activity and ERα activation display important roles in this type of cancer, our goal is to discover multi-target compounds able to simultaneously inhibit aromatase and modulate ERα.

Methods: We used the ChEMBL Database to retrieve all the known aromatase inhibitors (AIs) and ERα antagonists. Using the ChemAxon software we computed two types of chemical descriptors: extended connectivity fingerprints and the pharmacophore fingerprints of each compound. After this, clusters were constructed and the selected compounds were analyzed by molecular docking. Anti-aromatase activity was evaluated in human placental microsomes and ERα expression was assessed by Western-Blot in ER+ an aromatase-overexpressing breast cancer cell line (MCF-7aro).

Results: Based on clusters and after docking studies one compound (MT1) was selected to be studied in microsomes and in MCF-7aro cells. Although MT1 binds to key residues in aromatase important for its inhibition, unexpectedly, MT1 was not able to inhibit this enzyme in microsomes. However, as Exemestane, the steroidal AI used in clinic (1), MT1 induced a decrease in aromatase protein levels in MCF-7aro cells. Furthermore, MT1 impaired ERα activation, acting as an ERα antagonist.

Discussion: To the best of our knowledge, this is the first attempt to discover multi-target compounds for ER+ breast cancer, using this type of approach. In fact, the compound MT1 was able to modulate aromatase and ERα, two key targets of this type of cancer, which represents a great advantage over other molecules used in breast cancer treatment.

Acknowledgments: The authors are grateful to Fundação para a Ciência e Tecnologia (FCT) for CA Post-doc grant (SFRH/BPD/98304/2013) and for the financial support (UID/MULTI/04378/2019).

References:

  1. Amaral C, Varela CL, Mauricio J, Sobral AF, Costa SC, Roleira FMF, et al. Anti-tumor efficacy of new 7alpha-substituted androstanes as aromatase inhibitors in hormone-sensitive and resistant breast cancer cells. J Steroid Biochem Mol Biol. 2017;171:218-28.

2. Augusto TV, Correia-da-Silva G, Rodrigues CMP, Teixeira N, Amaral C. Acquired resistance to aromatase inhibitors: where we stand! Endocr Relat Cancer. 2018;25(5):R283-R301.

  • Open access
  • 18 Reads
The anti-herpetic activity of fluorine-containing compounds based on β-D-glucopyranose
Published: 30 October 2019 by MDPI AG in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

Introduction: The diseases caused by Herpes Simplex Virus 1 (HSV-1) are widely spread. The shortage of antiviral compounds due to their high toxicity and emergence of resistant viruses is a major problem in the treatment of patients. This work is related to the determination of the antiviral activity of new fluorine containing derivatives against HSV-1.

Methodology: Cytotoxicity and anti-herpetic activity of compounds 10S-25 ((1-S-thio-(1- methylsulfonyl-2-difluoromethyl-vinyl)-2,3,4,6,-tetra-O-acetyl-β-D-glucopyranose) and 10S-27 1-(β-D-glucopyranosyl)-4-(hexafluoropropyl)-5-tosyl-1H-1,2,3-triazole) were studied using MTT assay. To sort the compounds, four experimental procedures were used: co-incubation of compounds and HSV-1, addition of compounds during virus adsorption and penetration, addition of compounds post-infection. The antiviral activity was assessed using real-time PCR and virus yield reduction assay.

Results: Compounds 10S-25 and 10S-27 demonstrated high toxicity for cells and their IC50 values were 13 and 250 µg/ml, respectively. It was determined, that only 10S-27 inhibited the formation CPE of the HSV-1 (EC50 value is 48 µg/ml). The absence of virucidal activity and prevention of the adsorption and penetration of HSV-1 into cells were shown for this compound. But in the presence of 10S-27 in higher concentration, HSV-1 DNA replication was inhibited and the viral DNA copy number was reduced to 38 %. Moreover, it was found that 10s-27 in concentrations 4 - 150 μg/ml reduced the titer of the virus obtained de novo by 39 - 98%.

Conclusion: Taken together, our results showed that 10S-27 possesses an anti-HSV-1 activity at non-toxic concentrations with multiple mechanisms, but further investigation is needed to explore this action in detail.

  • Open access
  • 47 Reads
Microencapsulation of bioactive leaf extracts of Eucalyptus camaldulensis by freeze drying technology using sodium alginate and sodium carboxymethyl cellulose as coating materials
Published: 30 October 2019 by MDPI AG in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

Bioactive crude ethanolic extracts of Eucalyptus camaldulensis was encapsulated with alginate–CMC using freeze drying technique. The microcapsules were characterized for particle size, morphology, physicochemical parameters such as solubility, swelling index, and micromeritics properties. FTIR was used to evaluate the interactions of the polymer and the extract. Antioxidant and antimicrobial activities of the microcapsules were also demonstrated. Results revealed and irregular shaped microparticles with mean diameter ranging from 6.7–26.6 µm. Zeta potential and polydispersity index ranged from -17.01–2.23 mV and 0.344–0.489 respectively. Percentage yield and encapsulation efficiency ranged between 70.4–81.5 % and 74.2±0.011 – 82.43±0.772 %. In addition, the microcapsules exhibited high swelling index with poor solubility. Antioxidant activity of the microcapsules evaluated using DPPH and ABTS assays increased with increase in the concentration of the extract. Minimum inhibitory and minimum bactericidal concentrations of the microcapsules against gram-positive foodborne pathogens ranged from 0.19–3.12 mg/mL and 0.19–12.25 mg/mL respectively. Moreover, the microcapsules at concentration of 1 mg/mL did not show cytotoxic effects on human colon cell CaCo-2. Alginate–CMC showed good encapsulation properties that preserved the bioactivity of the extract, thus might be employed for application of natural products in processing systems.

  • Open access
  • 52 Reads
Lactose-based Fatty Acid Monoesters: Synthesis, antimicrobial activity and permeability enhancement studies

We present the synthesis and the in vitro assaying of a series of lactose-based non-ionic surfactants, highlighting the relationship between their structure and biological effect. Using tensiometric measurements the critical micelle concentrations (CMCs) of the surfactants were determined and demonstrate that increasing hydrophobic chain length reduces surfactant CMC. In vitro testing on Caco-2 intestinal and Calu-3 airway epithelia revealed that cytotoxicity is present, for most of the surfactants, at concentrations greater than their CMCs. Importantly, through the culture of epithelial monolayers on Transwell® supports, the surfactants demonstrate the ability to reversibly modulate transepithelial electrical resistance (TEER), and thus open tight junctions, at non-toxic concentrations. The surfactants were then tested for their ability to improve the in vitro permeability of Ovalbumin and Dextran, confirming their potential application as safe permeability enhancers in vivo. Moreover, the synthesized compounds exhibit antimicrobial activity versus eight pathogenic species belonging to Gram-positive, Gram-negative microorganisms and fungi.

  • Open access
  • 22 Reads
Coupling deoxy sugars to polyphenols: Neuroprotection and bioavailability
Published: 30 October 2019 by MDPI AG in 5th International Electronic Conference on Medicinal Chemistry session Posters

Resveratrol and methyl caffeate health-promoting role in chronic and degenerative diseases is well known, however, their efficiency is affected by their low bioavailability in humans due to poor water solubility. Since these compounds have a peculiar role in the inhibition of Aβ oligomeric cytotoxicity and thus reduction of neuronal cell death [1,2], we were encouraged to investigate a new approach to overcome these issues. We disclose herein the benefits discovered by coupling sugars to these polyphenols, aiming to improve compound polarity, facilitated by the presence of additional OH groups of the sugar moiety. A robust synthetic route was developed in order to couple 2-deoxyglycosides to these natural molecules- resveratrol and caffeic acid, using glycals as easily accessed glycosyl donors. The resveratrol 2-deoxyglycosides 1 and 2 are more effective at protecting neuronal cells from peroxide-induced cytotoxicity than resveratrol itself, and caffeic acid ester glycoside 3 was also highly active as neuroprotective agent. Compounds were not cytotoxic to neuroblastoma, intestinal or liver cells at all concentrations tested. Coefficient partition measurements (logD, pH=7.4) confirmed the moderate lipophilicity of these resveratrol glycosides, which log D values are typical of CNS drug and for BBB penetration. This work illustrates the benefits of coupling sugars to polyphenols, in particular deoxy sugars, to improve polyphenol bioactivity and physicochemical properties.

  • Open access
  • 40 Reads
The in silico identification of potent natural bioactive anti-dengue agents by targeting the human hexokinase 2 enzyme
Published: 30 October 2019 by MDPI AG in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

Background:Hexokinase 2 (HKII) is a rate-limiting and the first key enzyme of glycolysis, responsible for the biosynthesis of glucose-6-phospate (G6P) and is up- regulated in dengue virus (DENV) infected cells. During DENV infections, the glycolytic pathway of the host is activated by the pathogens, and inhibition of glycolysis by targeting HKII enzyme can significantly block the infectious DENV production.

Objectives: The main aim of this study was to computer-aided identification of natural bioactive anti-dengue agents that can inhibit the activity of human HKII enzyme.

Methods: A ligand-based pharmacophore model (LBPM) was developed using previously known inhibitors of HKII enzymes to ensure the optimal molecular interactions with the specific target. Virtual screening (VS), molecular docking (MD) and the absorption, distribution, metabolism, excretion, and toxicity (ADMET) approaches were used to identify potential and specific natural human HKII inhibitors.

Result: Based on MD results and binding interaction analysis, four compounds D-Glucose hydrate, (2R,3R,4S,5S)-2,3,4,5,6-Pentahydroxyhexanal, (2S)-2-Amino-3-hydroxy-N'-(2,3,4-trihydroxybenzyl) propanehydrazide hydrochloride, (2S)-2-Amino-3-hydroxy-N’, N'-bis[(2,3,4-trihydroxyphenyl)methyl]propanehydrazide were predicted to be the basis for lead optimization. They bind to the active site of human HKII and virtually behave as strong competitive inhibitors.

Interpretation and conclusion: The results demonstrated 4 hits compatible with the active site of HKII enzymes. The current results will be further evaluated in the wet lab by both in vitro and in vivo testing for the development of potential DENV inhibitor.

  • Open access
  • 34 Reads
Design, synthesis, X-ray structure and evaluation of functionalized hexacyclic carbazoles as new inhibitors of ABCG2 transporter
Published: 30 October 2019 by MDPI AG in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

Cancer is one of the diseases with the highest mortality rates worldwide and the emergence of neoplasms presenting resistance to chemotherapy, also known as multidrug resistance (MDR), makes this conjuncture even worse. The overexpression of transmembrane proteins named ABC transporters is considered the main cause of this clinical condition [1]. These transporters (e.g. ABCB1, ABCC1, ABCG2) can recognize and promote the efflux of a broad spectrum of antineoplastic agents; thus, many studies have been carried out to develop compounds and evaluate their ability to inhibit this activity. Despite its pronounced relation with MDR, there are still no promising inhibitors of ABCG2 to be forwarded to clinical steps of drug development, which endorses the urgency to identify and characterize new selective inhibitors of this protein.

Carbazole skeleton is a key structural motif of many biologically active compounds including natural and synthetic products [2]. Starting from the tricyclic-carbazole motif to fused tetra-, penta-, hexa- and heptacyclic carbazoles, this skeleton could enable the design of new inhibitors of ABCG2 transporter.

In this study, a one-pot method for the synthesis of novel hexacyclic carbazole derivatives from readily available starting materials using a sequential multicomponent reaction/Fisher indolization strategy is described. Then five carbazole derivatives were tested to inhibit ABCG2 activity.

  1. O. Briz et al. Expert Opin Drug Metab Toxicol. 2019, 15(7):577-593.

2. S. Issa et al. J Enzyme Inhib. Med. Chem. 2019, 34(1):1321-1346.

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