Barbara Kappes

New efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids

In our ongoing search for highly active hybrid molecules exceeding their parent compounds in anticancer, antimalaria as well as antiviral activity and being an alternative to the standard drugs, we present the synthesis and biological investigations of 2nd generation 1,2,4-trioxane-ferrocene hybrids. Inxa0vitro tests against the CCRF-CEM leukemia cell line revealed di-1,2,4-trioxane-ferrocene hybrid 7 as the most active compound (IC50 of 0.01xa0μM). Regarding the activity against the multidrug resistant subline CEM/ADR5000, 1,2,4-trioxane-ferrocene hybrid 5 showed a remarkable activity (IC50 of 0.53xa0μM). Contrary to the antimalaria activity of hybrids 4-8 against Plasmodium falciparum 3D7 strain with slightly higher IC50 values (between 7.2 and 30.2xa0nM) than that of their parent compound DHA, hybrids 5-7 possessed very promising activity (IC50 values lower than 0.5xa0μM) against human cytomegalovirus (HCMV). The application of 1,2,4-trioxane-ferrocene hybrids against HCMV is unprecedented and demonstrated here for the first time.

Highly potent artemisinin-derived dimers and trimers: Synthesis and evaluation of their antimalarial, antileukemia and antiviral activities.

New pharmaceutically active compounds can be obtained by modification of existing drugs to access more effective agents in the wake of drug resistance amongst others. To achieve this goal the concept of hybridization was established during the last decade. We employed this concept by coupling two artemisinin-derived precursors to obtain dimers or trimers with increased in vitro activity against Plasmodiumfalciparum 3D7 strain, leukemia cells (CCRF-CEM and multidrug-resistant subline CEM/ADR5000) and human cytomegalovirus (HCMV). Dimer 4 (IC50 of 2.6 nM) possess superior antimalarial activity compared with its parent compound artesunic acid(3) (IC50 of 9.0 nM). Dimer5 and trimers6 and 7 display superior potency against both leukemia cell lines (IC50 up to 0.002 μM for CCRF-CEM and IC50 up to 0.20 μM for CEM/ADR5000) and are even more active than clinically used doxorubicin (IC50 1.61 μM for CEM/ADR5000). With respect to anti-HCMV activity, trimer6 is the most efficient hybrid (IC50 0.04 μM) outperforming ganciclovir (IC50 2.6 μM), dihydroartemisinin(IC50 >10 μM) and artesunic acid (IC50 3.8 μM).

Vinculin phosphorylation at residues Y100 and Y1065 is required for cellular force transmission

ABSTRACT The focal adhesion protein vinculin connects the actin cytoskeleton, through talin and integrins, with the extracellular matrix. Vinculin consists of a globular head and tail domain, which undergo conformational changes from a closed auto-inhibited conformation in the cytoplasm to an open conformation in focal adhesions. Src-mediated phosphorylation has been suggested to regulate this conformational switch. To explore the role of phosphorylation in vinculin activation, we used knock-out mouse embryonic fibroblasts re-expressing different vinculin mutants in traction microscopy, magnetic tweezer microrheology, FRAP and actin-binding assays. Compared to cells expressing wild-type or constitutively active vinculin, we found reduced tractions, cytoskeletal stiffness, adhesion strength, and increased vinculin dynamics in cells expressing constitutively inactive vinculin or vinculin where Src-mediated phosphorylation was blocked by replacing tyrosine at position 100 and/or 1065 with a non-phosphorylatable phenylalanine residue. Replacing tyrosine residues with phospho-mimicking glutamic acid residues restored cellular tractions, stiffness and adhesion strength, as well as vinculin dynamics, and facilitated vinculin–actin binding. These data demonstrate that Src-mediated phosphorylation is necessary for vinculin activation, and that phosphorylation controls cytoskeletal mechanics by regulating force transmission between the actin cytoskeleton and focal adhesion proteins. Summary: Src-mediated phosphorylation on Y100 and Y1065 is a prerequisite for vinculin activation, and controls cytoskeletal mechanics by regulating force transmission between the actin cytoskeleton and focal adhesion proteins.

Synthesis of Novel Hybrids of Quinazoline and Artemisinin with High Activities against Plasmodium falciparum, Human Cytomegalovirus, and Leukemia Cells

Many quinazoline derivatives have been synthesized over the last few decades with great pharmacological potential, such as antimalarial, anti-inflammatory, antimicrobial, anticancer, and antiviral. But so far, no quinazoline–artemisinin hybrids have been reported in the literature. In the present study, five novel quinazoline–artemisinin hybrids were synthesized and evaluated for their in vitro biological activity against malarial parasites (Plasmodium falciparum 3D7), leukemia cells (CCRF-CEM and CEM/ADR5000), and human cytomegalovirus. Remarkably, hybrid 9 (EC50 = 1.4 nM), the most active antimalarial compound of this study, was not only more potent than artesunic acid (EC50 = 9.7 nM) but at the same time more active than the clinically used drugs dihydroartemisinin (EC50 = 2.4 nM) and chloroquine (EC50 = 9.8 nM). Furthermore, hybrids 9 and 10 were the most potent compounds with regard to anticytomegaloviral activity (EC50 = 0.15–0.21 μM). They were able to outperform ganciclovir (EC50 = 2.6 μM), which is the relevant standard drug of antiviral therapy, by a factor of 12–17. Moreover, we identified a new highly active quinazoline derivative, compound 14, that is most effective in suppressing cytomegalovirus replication with an EC50 value in the nanomolar range (EC50 = 50 nM). In addition, hybrid 9 exhibited an antileukemia effect similar to that of artesunic acid, with EC50 values in the low micromolar range, and was 45 times more active toward the multidrug-resistant CEM/ADR5000 cells (EC50 = 0.5 μM) than the standard drug doxorubicin.

Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

Synthesis of Thymoquinone–Artemisinin Hybrids: New Potent Antileukemia, Antiviral, and Antimalarial Agents

A series of hybrid compounds based on the natural products artemisinin and thymoquinone was synthesized and investigated for their biological activity against the malaria parasite Plasmodium falciparum 3D7 strain, human cytomegalovirus (HCMV), and two leukemia cell lines (drug-sensitive CCRF-CEM and multidrug-resistant subline CEM/ADR5000). An unprecedented one-pot method of selective formation of C-10α-acetate 14 starting from a 1:1 mixture of C-10α- to C-10β-dihydroartemisinin was developed. The key step of this facile method is a mild decarboxylative activation of malonic acid mediated by DCC/DMAP. Ether-linked thymoquinone-artemisinin hybrids 6a/b stood out as the most active compounds in all categories, while showing no toxic side effects toward healthy human foreskin fibroblasts and thus being selective. They exhibited EC50 values of 0.2 μM against the doxorubicin-sensitive as well as the multidrug-resistant leukemia cells and therefore can be regarded as superior to doxorubicin. Moreover, they showed to be five times more active than the standard drug ganciclovir and nearly eight times more active than artesunic acid against HCMV. In addition, hybrids 6a/b possessed excellent antimalarial activity (EC50 = 5.9/3.7 nM), which was better than that of artesunic acid (EC50 = 8.2 nM) and chloroquine (EC50 = 9.8 nM). Overall, most of the presented thymoquinone-artemisinin-based hybrids exhibit an excellent and broad variety of biological activities (anticancer, antimalarial, and antiviral) combined with a low toxicity/high selectivity profile.

Deeper Insight into the Six‐Step Domino Reaction of Aldehydes with Malononitrile and Evaluation of Antiviral and Antimalarial Activities of the Obtained Bicyclic Products

Abstract The straightforward and efficient synthesis of complex aza‐ and carbobicyclic compounds, which are of importance for medicinal chemistry, is a challenge for modern chemical methodology. An unprecedented metal‐free six‐step domino reaction of aldehydes with malononitrile was presented in our previous study to provide, in a single operation, these bicyclic nitrogen‐containing molecules. Presented here is a deeper investigation of this atom‐economical domino process by extending the scope of aldehydes, performing post‐modifications of domino products, applying bifunctional organocatalysts and comprehensive NMR studies of selected domino products. The thermodynamic aspects of the overall reaction are also demonstrated using DFT methods in conjunction with a semi‐empirical treatment of van der Waals interactions. Furthermore, biological studies of seven highly functionalized and artemisinin‐containing domino products against human cytomegalovirus (HCMV) and Plasmodium falciparumu20053D7 are presented. Remarkably, inu2005vitro tests against HCMV revealed five domino products to be highly active compounds (EC50 0.071–1.8u2005μm), outperforming the clinical reference drug ganciclovir (EC50 2.6u2005μm). Against P.u2005falciparumu20053D7, three of the investigated artemisinin‐derived domino products (EC50 0.72–1.8u2005nm) were more potent than the clinical drug chloroquine (EC50 9.1u2005nm).

Quantification of human complement factor H binding to asexual malaria blood stages by an enzyme-linked immunosorbent assay

The human complement system is the most effective defense mechanism of the human innate immune system. One major negative regulator of the alternative pathway in human blood is complement factor H (FH). It binds to autologous cells and thus, prevents complement attack against body-cells or tissues. Various pathogens are known to escape complement recognition by recruiting FH to provide protection against the hosts immune system. This immune evasion mechanism was recently qualitatively reported for asexual malaria blood stages. To indirectly evaluate the stage-specific potential of FH-receptor proteins as vaccine candidates, we quantified the FH molecules bound to the surface of different malaria blood stage parasites by Western blot and a commercially available FH-ELISA, which was originally designed to measure the FH concentration in human serum. Host-cell-free merozoites and intracellular mature schizont (here called segmenter) stages bind significantly more FH molecules than earlier parasite stages.

Frontispiece: Synthesis of Artemisinin-Derived Dimers, Trimers and Dendrimers: Investigation of Their Antimalarial and Antiviral Activities Including Putative Mechanisms of Action

Generation of dimers, trimers and dendrimers of bioactive compounds is an approach that has recently been developed for the discovery of new potent drug candidates. Herein, we present the synthesis of new artemisinin-derived dimers and dendrimers and investigate their action against malaria parasite Plasmodium falciparum 3D7 strain and human cytomegalovirus (HCMV). Dimer 7 was the most active compound (EC50 1.4u2005nm) in terms of antimalarial efficacy and was even more effective than the standard drugs dihydroartemisinin (EC50 2.4u2005nm), artesunic acid (EC50 8.9u2005nm) and chloroquine (EC50 9.8u2005nm). Trimer 4 stood out as the most active agent against HCMV in vitro replication and exerted an EC50 value of 0.026u2005μm, representing an even higher activity than the two reference drugs ganciclovir (EC50 2.60u2005μm) and artesunic acid (EC50 5.41u2005μm). In addition, artemisinin-derived dimer 13 and trimer 15 were for the first time both immobilized on TOYOPEARL AF-Amino-650M beads and used for mass spectrometry-based target identification experiments using total lysates of HCMV-infected primary human fibroblasts. Two major groups of novel target candidates, namely cytoskeletal and mitochondrial proteins were obtained. Two putatively compound-binding viral proteins, namely major capsid protein (MCP) and envelope glycoprotein pUL132, which are both essential for HCMV replication, were identified.

Access to new highly potent antileukemia, antiviral and antimalarial agents via hybridization of natural products (homo)egonol, thymoquinone and artemisinin

Hybridization of natural products has high potential to further improve their activities and may produce synergistic effects between linked pharmacophores. Here we report synthesis of nine new hybrids of natural products egonol, homoegonol, thymoquinone and artemisinin and evaluation of their activities against P. falciparum 3D7 parasites, human cytomegalovirus, sensitive and multidrug-resistant human leukemia cells. Most of the new hybrids exceed their parent compounds in antimalarial, antiviral and antileukemia activities and in some cases show higher in vitro efficacy than clinically used reference drugs chloroquine, ganciclovir and doxorubicin. Combined, our findings stress the high potency of these hybrids and encourages further use of the hybridization concept in applied pharmacological research.