In Vitro and In Silico Antimalarial Evaluation of FM-AZ, a New Artemisinin Derivative.

Artemisinin-based Combination Therapies (ACTs) are currently the frontline treatment against Plasmodium falciparum malaria, but parasite resistance to artemisinin (ART) and its derivatives, core components of ACTs, is spreading in the Mekong countries. In this study, we report the synthesis of several novel artemisinin derivatives and evaluate their in vitro and in silico capacity to counteract Plasmodium falciparum artemisinin resistance. Furthermore, recognizing that the malaria parasite devotes considerable resources to minimizing the oxidative stress that it creates during its rapid consumption of hemoglobin and the release of heme, we sought to explore whether further augmentation of this oxidative toxicity might constitute an important addition to artemisinins. The present report demonstrates, in vitro, that FM-AZ, a newly synthesized artemisinin derivative, has a lower IC50 than artemisinin in P. falciparum and a rapid action in killing the parasites. The docking studies for important parasite protein targets, PfATP6 and PfHDP, complemented the in vitro results, explaining the superior IC50 values of FM-AZ in comparison with ART obtained for the ART-resistant strain. However, cross-resistance between FM-AZ and artemisinins was evidenced in vitro.

Backbone and side chain NMR assignments of the H-NOX domain from Nostoc sp. in complex with BAY58-2667 (cinaciguat).

Soluble guanylate cyclase (sGC) enzyme is activated by the gaseous signaling agent nitric oxide (NO) and triggers the conversion of GTP (guanosine 5'-triphosphate) to cGMP (cyclic guanylyl monophosphate). It contains the heme binding H-NOX (heme-nitric oxide/oxygen binding) domain which serves as the sensor of NO and it is highly conserved across eukaryotes and bacteria as well. Many research studies focus on the synthesis of chemical compounds bearing possible therapeutic action, which mimic the heme moiety and activate the sGC enzyme. In this study, we report a preliminary solution NMR (Nuclear Magnetic Resonance) study of the H-NOX domain from Nostoc sp. cyanobacterium in complex with the chemical sGC activator cinaciguat (BAY58-2667). An almost complete sequence-specific assignment of its 1H, 15N and 13C resonances was obtained and its secondary structure predicted by TALOS+.

De-Novo Design of Cereblon (CRBN) Effectors Guided by Natural Hydrolysis Products of Thalidomide Derivatives.

Targeted protein degradation via cereblon (CRBN), a substrate receptor of an E3 ubiquitin ligase complex, is an increasingly important strategy in various clinical settings, in which the substrate specificity of CRBN is altered via the binding of small-molecule effectors. To date, such effectors are derived from thalidomide and confer a broad substrate spectrum that is far from being fully characterized. Here, we employed a rational and modular approach to design novel and minimalistic CRBN effectors. In this approach, we took advantage of the binding modes of hydrolyzed metabolites of several thalidomide-derived effectors, which we elucidated via crystallography. These yielded key insights for the optimization of the minimal core binding moiety and its linkage to a chemical moiety that imparts substrate specificity. Based on this scaffold, we present a first active de-novo CRBN effector that is able to degrade the neo-substrate IKZF3 in the cell culture.

Immunological Alterations due to Hemodialysis Might Interfere with Early Complications in Renal Transplantation.

BACKGROUND: Chronic or intercurrent alterations of the immune system in patients with end-stage renal disease (CKD) and intermittent hemodialysis (CKD5D, HD) have been attributed to an acute rejection of renal allograft. METHODS: Leukocyte subsets in flow cytometry, complement activation, and concentrations of TGFß, sCD30 (ELISA), and interleukins (CBA) of fifteen patients eligible for renal transplantation were analyzed before, during, and after a regular HD. RESULTS: Before HD, the median proportion of CD8+ effector cells, CD8+ CCR5+ effector cells, and HLA-DR+ regulatory T cells as well as the median concentration of soluble CD30 increased and naive CD8+ T cells decreased. During HD, there was a significant decrease in CD4- CD8- T cells (p < 0.001) and an increase in CD25+ T cells (p = 0.026), sCD30 (p < 0.001), HLA-DR+ regulatory T cells (p = 0.005), and regulatory T cells (p = 0.003). TGFß and sCD30 increased significantly over time. The activity of the classical complement pathway started to slightly increase after the first hour of HD and lasted until fifteen minutes after finishing dialysis. The decrease in the functional activity of the alternative pathway was only transient and was followed by a significant increase within 15 minutes after finishing the treatment. CONCLUSION: HD might interact with the allograft outcome by influencing T cell subsets and activation of the complement system in a biphasic course.

Synthesis and biological investigation of (+)-3-hydroxymethylartemisinin.

Herein, we describe a biomimetic entry to (+)-3-hydroxymethylartemisinin (2) as well as to the artemisinin derivatives (+)-3-hydroxymethyl-9-desmethylartemisinin (16) and (+)-3-hydroxymethyl-9-epi-artemisinin (18), starting from the known and readily available chiral aldehyde 3 and alkyne 4. Subsequently, the synthesized compounds have been evaluated for their antimalarial activity against the drug-sensitive P. falciparum NF54 strain. All of them were inactive. In addition, they did not show any toxicity against L6 cells (a primary cell line derived from rat skeletal myoblasts). These results contribute to a better understanding of artemisinins mechanism of action.

Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 µM) than d-TC (IC50 = 0.39 µM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 µM and 7.77 µM, respectively), but BBIQA2 was similar (IC50 = 5.52 µM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1ß1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 µM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1ß1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3ß2, α4ß2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3ß2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 µM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1ß3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

Total Synthesis and Biological Investigation of (-)-Artemisinin: The Antimalarial Activity of Artemisinin Is not Stereospecific.

Here, we describe an efficient and diversity-oriented entry to both (-)-artemisinin (1) and its natural antipode (+)-artemisinin, starting from commercially and readily available S-(+)- and R-(-)-citronellene, respectively. Subsequently, we answered the still open question regarding the specificity of artemisinins action. By using a drug-sensitive Plasmodium falciparum NF54 strain, we showed that the antimalarial activity of artemisinin is not stereospecific. Our straightforward and biomimetic approach to this natural endoperoxide enables the synthesis of artemisinin derivatives that are not accessible through applying current methods and may help to address the problem of emerging resistance of Plasmodium falciparum towards artemisinin.

Use of costic acid, a natural extract from Dittrichia viscosa, for the control of Varroa destructor, a parasite of the European honey bee.

Costic acid has been isolated from the plant Dittrichia viscosa and its efficacy against Varroa destructor, a parasite of Apis mellifera, the European honey bee, has been studied. Costic acid exhibited potent in vivo acaricidal activity against the parasite. Initial experiments showed that the compound is not toxic for human umbilical vein endothelial cells (HUVEC) at concentrations of up to 230 micromolar (µM), indicating that costic acid could be used as a safe, low-cost and efficient agent for controlling varroosis in honey bee colonies.

Synthesis and cytotoxic activity of new artemisinin hybrid molecules against human leukemia cells.

A series of new artemisinin-derived hybrids which incorporate cholic acid moieties have been synthesized and evaluated for their antileukemic activity against sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 cells. The new hybrids 20-28 showed IC50 values in the range of 0.019µM-0.192µM against CCRF-CEM cells and between 0.345µM and 7.159µM against CEM/ADR5000 cells. Amide hybrid 25 proved the most active compound against both CCRF-CEM and CEM/ADR5000 cells with IC50 value of 0.019±0.001µM and 0.345±0.031µM, respectively. A relatively low cross resistance to hybrids 20-28 in the range of 5.7-fold to 46.1-fold was measured. CEM/ADR5000 cells showed higher resistance than CCRF-CEM to all the tested compounds. Interestingly, the lowest cross resistance to 23 was observed (5.7-fold), whereas hybrid 25 showed 18.2-fold cross-resistant to CEM/ADR5000 cells. Hybrid 25 which proved even more potent than clinically used doxorubicin against CEM/ADR5000 cells may serve as a promising antileukemic agent against both sensitive and multidrug-resistant cells.

Lewis Acid Mediated Nazarov Cyclization as a Convergent and Enantioselective Entry to C-nor-D-homo-Steroids.

A straightforward synthesis of C-nor-D-homo steroids starting from (+)-Wieland-Miescher ketone is reported. This convergent synthetic strategy utilizes a scalable diastereoselective Nazarov cyclization of functionalized chiral aryl vinyl ketones, allowing for further functionalization. The ability to conduct this key transformation on a multi-gram scale paves the way for the synthesis of a variety of completely new C-nor-D-homo steroids, without the need of a classic steran steroid rearrangement or achiral linear reaction sequences.

Synthesis of novel C-9 carbon substituted derivatives of artemisinin.

Several artemisinin derivatives carrying several groups (alkyl, hydroxyalkyl, allyl or azide) at position 9 have been synthesized starting from artemisinin via enolate formation and subsequent reaction with appropriate electrophiles.

Curare Alkaloids: Constituents of a Matis Dart Poison.

A phytochemical study of dart and arrow poison from the Matis tribe led to the identification of D-(-)-quinic acid, L-malic acid, ethyldimethylamine, magnoflorine, and five new bisbenzyltetrahydroisoquinoline alkaloids (BBIQAs), 1-5. D-Tubocurarine could not be identified among these products. BBIQA (3) contains a unique linkage at C-8 and C-11'. All structures were characterized by a combination of NMR and HRESIMS data. The effects of Matis poison and individual BBIQAs (1-3) on rat muscle nAChR expressed in Xenopus oocytes have been investigated using the two-electrode voltage clamp technique.

Synthesis of a hexasaccharide partial sequence of hyaluronan for click chemistry and more.

In the present work, the synthesis of a hexasaccharide partial sequence of hyaluronan equipped with a terminal azido moiety is reported. This hexasaccharide can be used for the attachment on surfaces by means of click chemistry and after suitable deprotection for biophysical studies.

The Veratrum and Solanum alkaloids.

This survey on steroidal alkaloids of the Veratrum and Solanum family isolated between 1974 and 2014 includes 187 compounds and 197 references. New developments in the chemistry and biology of this family of natural products with a special focus on the medicinal relevance of the jervanine alkaloid cyclopamine are discussed.

Sensitive and site-specific identification of carboxymethylated and carboxyethylated peptides in tryptic digests of proteins and human plasma.

Glycation refers to a nonenzymatic post-translational modification formed by the reaction of amino groups and reducing sugars. Consecutive oxidation and degradation can produce advanced glycation end products (AGEs), such as N(ε)-(carboxyethyl)lysine (CEL) and N(ε)-(carboxymethyl)lysine (CML). Although CEL and CML are considered to be markers of arteriosclerosis, diabetes mellitus, and aging, the modified proteins and the exact modification sites are mostly unknown due to their low frequency and a lack of enrichment strategies. Here, we report characteristic fragmentation patterns of CML- and CEL-containing peptides and two modification-specific reporter ions for each modification (CML, m/z 142.1 and 187.1; CEL, m/z 156.1 and 201.1). The protocol allowed sensitive and selective precursor ion scans to detect the modified peptides in complex sample mixtures. The corresponding m/z values identified eight CEL/CML-modification sites in glycated human serum albumin (HSA) by targeted nano-RPC-MS/MS. The same strategy revealed 21 CML sites in 17 different proteins, including modified lysine residues 88 and 396 of human serum albumin, in a pooled plasma sample that was obtained from patients with type 2 diabetes mellitus.

Guanylyl cyclase activation reverses resistive breathing-induced lung injury and inflammation.

Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure-volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC-cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.

Synthesis and evaluation of anticancer activity in cells of novel stoichiometric pegylated fullerene-doxorubicin conjugates.

PURPOSE: To synthesize pegylated stoichiometrically and structurally well-defined conjugates of fullerene (C60) with doxorubicin (DOX) and investigate their antiproliferative effect against cancer cell lines. METHODS: Stoichiometric (1:1 and 1:2) pegylated conjugates of C60 with DOX were synthesized using the Prato reaction to create fulleropyrrolidines equipped with a carboxyl function for anchoring a polyethylene glycol (PEG) moiety and either a hydroxyl group for attaching one molecule of DOX or a terminal alkyne group for attaching two molecules of DOX through a click reaction. In both conjugates, the DOX moieties are held through a urethane-type bond. Drug release was studied in phosphate buffer (PBS, pH 7.4) and MCF-7 cancer cells lysate. The uptake of the conjugates by MCF-7 cancer cells and their intracellular localization were studied with fluorescence microscopy. The antiproliferative activity of the conjugates was investigated using the WST-1 test. RESULTS: One or two DOX molecules were anchored on pegylated C60 particles to form DOX-C60-PEG conjugates. Drug liberation from the conjugates was significantly accelerated in the presence of tumor cell lysate compared to PBS. The conjugates could be internalized by MCF-7 cells. DOX from the conjugates exhibited much delayed, compared to free DOX, localization in the nucleus and antiproliferative activity. CONCLUSION: Pegylated DOX-C60 conjugates (1:1) and (2:1) with well-defined structure were successfully synthesized and found to exhibit comparable, but with a delayed onset, antiproliferative activity with free DOX against MCF-7 cancer cells. The results obtained justify further investigation of the potential of these conjugates as anticancer nanomedicines.

Novel Nor-Homo- and Spiro-Oxetan- Steroids Target the Human Androgen Receptor and Act as Antiandrogens.

The prostate adenocarcinoma is the cancer with the highest incidence for men in Western countries. Targeting the androgen receptor (AR) by antagonists is used as hormone therapy for prostate cancer (PCa), however, eventually therapy resistance occurs in most patients. In most of these cancer the AR signaling is active and thus AR remains an important drug target. Since many years we are characterizing novel chemical structural platforms to provide a broader possibility for compounds that bind to and act as AR antagonists. Here, we describe the chemical synthesis of a battery of novel steroidal derivatives as nor-homo-, spiro-oxolan- and spiro-oxetan- steroids. They modulate the transcriptional activity of the human AR. As AR antagonists, the spiro-oxetan- steroid derivatives seem to be the most potent steroid derivatives. They inhibit the transcriptional activity of both wild-type AR as well as the AR mutant T877A. In line with this, these compounds bind to the human AR and inhibit the proliferation of the human androgen-dependent growing PCa cell line LNCaP. Interestingly, the castration-resistant AR expressing human PC3-AR cells are also growth inhibited. On mechanistic level, fluorescence resonance energy transfer (FRET) assays with living cells indicate that the androgen-induced N/C terminal interaction of the AR is inhibited by the investigated compounds. Using fluorescence recovery after photobleaching (FRAP) assays in living cells suggest a higher mobility of the AR in the cell nuclei in the presence of spiro-oxetan- steroidal antagonists. Together, these findings suggest that spiro-oxetan- steroids are very useful as a chemical platform for novel AR antagonists.

A novel approach to decrease sialic acid expression in cells by a C-3-modified N-acetylmannosamine.

Due to its position at the outermost of glycans, sialic acid is involved in a myriad of physiological and pathophysiological cell functions such as host-pathogen interactions, immune regulation, and tumor evasion. Inhibitors of cell surface sialylation could be a useful tool in cancer, immune, antibiotic, or antiviral therapy. In this work, four different C-3 modified N-acetylmannosamine analogs were tested as potential inhibitors of cell surface sialylation. Peracetylated 2-acetylamino-2-deoxy-3-O-methyl-D-mannose decreases cell surface sialylation in Jurkat cells in a dose-dependent manner up to 80%, quantified by flow cytometry and enzyme-linked lectin assays. High-performance liquid chromatography experiments revealed that not only the concentration of membrane bound but also of cytosolic sialic acid is reduced in treated cells. We have strong evidence that the observed reduction of sialic acid expression in cells is caused by the inhibition of the bifunctional enzyme UDP-GlcNAc-2-epimerase/ManNAc kinase. 2-Acetylamino-2-deoxy-3-O-methyl-D-mannose inhibits the human ManNAc kinase domain of the UDP-GlcNAc-2-epimerase/ManNAc kinase. Binding kinetics of the inhibitor and human N-acetylmannosamine kinase were evaluated using surface plasmon resonance. Specificity studies with human N-acetylglucosamine kinase and hexokinase IV indicated a high specificity of 2-acetylamino-2-deoxy-3-O-methyl-D-mannose for MNK. This substance represents a novel class of inhibitors of sialic acid expression in cells, targeting the key enzyme of sialic acid de novo biosynthesis.