Optimized isolation of 7,7′-biphyscion starting from Cortinarius rubrophyllus, a chemically unexplored fungal species rich in photosensitizers

Mushrooms such as the dermocyboid Cortinarius rubrophyllus are characterized by strikingly colorful fruiting bodies. The molecular dyes responsible for such colors recently experienced a comeback as photoactive compounds with remarkable photophysical and photobiological properties. One of them—7,7′-biphyscion—is a dimeric anthraquinone that showed promising anticancer effects in the low nanomolar range under blue-light irradiation. Compared to acidic anthraquinones, 7,7′-biphyscion was more efficiently taken up by cells and induced apoptosis after photoactivation. However, seasonal collection of mushrooms producing this compound, low extraction yields, and tricky fungal identification hamper further developments to the clinics. To bypass these limitations, we demonstrate here an alternative approach utilizing a precursor of 7,7′-biphyscion, i.e., the pre-anthraquinone flavomannin-6,6′-dimethyl ether, which is abundant in many species of the subgenus Dermocybe. Controlled oxidation of the crude extract significantly increased the yield of 7,7′-biphyscion by 100%, which eased the isolation process. We also present the mycochemical and photobiological characterization of the yet chemically undescribed species, i.e. C. rubrophyllus. In total, eight pigments (1–8) were isolated, including two new glycosylated anthraquinones (1 and 2). Light-dependent generation of singlet oxygen was detected for the first time for emodin-1-O-β-d-glucopyranoside (3) [photophysical measurement: Φ∆ = 0.11 (CD3OD)]. Furthermore, emodin (7) was characterized as promising compound in the photocytotoxicity assay with EC50-values in the low micromolar range under irradiation against cells of the cancer cell lines AGS, A549, and T24. Graphical abstract

Selection of Promising Novel Fragment Sized S. aureus SrtA Noncovalent Inhibitors Based on QSAR and Docking Modeling Studies

Sortase A (SrtA) of Staphylococcus aureus has been identified as a promising target to a new type of antivirulent drugs, and therefore, the design of lead molecules with a low nanomolar range of activity and suitable drug-like properties is important. In this work, we aimed at identifying new fragment-sized starting points to design new noncovalent S. aureus SrtA inhibitors by making use of the dedicated molecular motif, 5-arylpyrrolidine-2-carboxylate, which has been previously shown to be significant for covalent binding SrtA inhibitors. To this end, an in silico approach combining QSAR and molecular docking studies was used. The known SrtA inhibitors from the ChEMBL database with diverse scaffolds were first employed to derive descriptors and interpret their significance and correlation to activity. Then, the classification and regression QSAR models were built, which were used for rough ranking of the virtual library of the synthetically feasible compounds containing the dedicated motif. Additionally, the virtual library compounds were docked into the “activated” model of SrtA (PDB:2KID). The consensus ranking of the virtual library resulted in the most promising structures, which will be subject to further synthesis and experimental testing in order to establish new fragment-like molecules for further development into antivirulent drugs.

A Handy and Accessible Tool for Identification of Sn(II) in Toothpaste: Inclination to an Eco-Friendly Environment

An easily accessible colorimetric probe, a carbazole-naphthaldehyde conjugate (CNP), was successfully prepared for the selective and sensitive recognition of Sn(II) in different commercially-available toothpaste and mouth wash samples. The binding mechanism of CNP for Sn2+ was confirmed by UV-Vis, 1H and 13C NMR titrations. The proposed sensing mechanism was supported by quantum chemical calculations. Selective detection of Sn(II) in the nanomolar range (85 nM), among other interfering metal ions, makes it exclusive. Moreover, Sn2+ can be detected with a simple paper strip from toothpaste, which makes this method handy and easy accessible. The potential application of this system for monitoring Sn2+ can be used as an expedient tool in environmental and industrial purpose.

Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection

C-reactive protein (CRP) is an inflammation biomarker that should be quantified accurately during infections and healing processes. Nanobodies are good candidates to replace conventional antibodies in immunodiagnostics due to their inexpensive production, simple engineering, and the possibility to obtain higher binder density on capture surfaces. Starting from the same pre-immune library, we compared the selection output resulting from two independent panning strategies, one exclusively exploiting the phage display and another in which a first round of phage display was followed by a second round of yeast display. There was a partial output convergence between the two methods, since two clones were identified using both panning protocols but the first provided several further different sequences, whereas the second favored the recovery of many copies of few clones. The isolated anti-CRP nanobodies had affinity in the low nanomolar range and were suitable for ELISA and immunoprecipitation. One of them was fused to SpyTag and exploited in combination with SpyCatcher as the immunocapture element to quantify CRP using electrochemical impedance spectroscopy. The sensitivity of the biosensor was calculated as low as 0.21 μg/mL.

Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca2+i]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.

Amine- and Amino Acid-Based Compounds as Carbonic Anhydrase Activators

After being rather neglected as a research field in the past, carbonic anhydrase activators (CAAs) were undoubtedly demonstrated to be useful in diverse pharmaceutical and industrial applications. They also improved the knowledge of the requirements to selectively interact with a CA isoform over the others and confirmed the catalytic mechanism of this class of compounds. Amino acid and amine derivatives were the most explored in in vitro, in vivo and crystallographic studies as CAAs. Most of them were able to activate human or non-human CA isoforms in the nanomolar range, being proposed as therapeutic and industrial tools. Some isoforms are better activated by amino acids than amines derivatives and the stereochemistry may exert a role. Finally, non-human CAs have been very recently tested for activation studies, paving the way to innovative industrial and environmental applications.

Synthesis and multifaceted pharmacological activity of novel quinazoline NHE-1 inhibitors

The Na+/H+ exchanger isoform 1 (NHE-1) attracts ongoing attention as a validated drug target for the management of cardiovascular and ocular diseases owing to cytoprotective, anti-ischemic and anti-inflammatory properties of NHE-1 inhibitors. Herein we report novel NHE-1 inhibitors realized via functionalization of N1-alkyl quinazoline-2,4(1H,3H)-dione and quinazoline-4(3H)-one with N-acylguanidine or 3-acyl(5-amino-1,2,4-triazole) side chain. Lead compounds show activity in a nanomolar range. Their pharmacophoric features were elucidated with neural network modeling. Several compounds combine NHE-1 inhibition with antiplatelet activity. Compound 6b reduces intraocular pressure in rats and effectively inhibits the formation of glycated proteins. Compounds 3e and 3i inhibit pro-inflammatory activation of murine macrophages, LPS-induced interleukin-6 secretion and also exhibit antidepressant activity similar to amiloride. Hence, novel compounds represent an interesting starting point for the development of agents against cardiovascular diseases, thrombotic events, excessive inflammation, long-term diabetic complications and glaucoma.

Chasing "Zampanalogs": Advancing the Synthesis of the Zampanolide Macrocycle

<p>(-)-Zampanolide (1), a natural product isolated from a marine sponge, is a microtubule-stabilizing agent that exhibits activity in the nanomolar range against various cancer cells, including in P-gp pump overexpressing cells. This attribute makes (-)-zampanolide an interesting target for further investigation. In this work, a new method for a modular and convergent total synthesis of optically pure zampanolide was investigated, which would also allow the generation of “zampanalogs” following the same basic strategy. Their biological activity may then be assessed to allow the elucidation of structure-activity relationships of (-)-zampanolide and its analogs in tubulin binding. The synthetic plan consisted of the modular combination of four major fragments, which would be connected in the late stages of the synthesis and could therefore be easily exchanged to allow the generation of analogs. The C15-C16 bond would be connected via an alkynylation reaction, and a subsequent reductive methylation would install the trisubstituted alkene. The connections at C1 and C3 could be achieved through a Bestmann ylid linchpin reaction, while the macrolactonization would be completed using a ring-closing metathesis to form the C8-C9 alkene. The side chain could be attached at C20 using one of the established aza-aldol methods. The fragments necessary for the formation of the macrocycle were synthesized successfully. The purification strategy throughout the synthetic route was rationalized and provides an improvement with respect to yield and time compared to work previously done in this research group. Alongside these fragments, modified fragments that were originally intended to serve as model systems were synthesized, which could also be used as building blocks in the synthesis of “zampanalogs”. Several methods for a stereoselective alkynylation at C15 were tested. These led to only meager successes, so an approach using a non-stereoselective alkynylation, followed by oxidation and a stereoselective CBS-reduction, was chosen. For the installation of the trisubstituted alkene a reductive methylation with vitride was tested, but this only led to the reduction of the alkyne without methylation. This product may be employed for the synthesis of C17-desmethyl analogs. The reductive methylation at C16-C17 was ultimately achieved using the Gilman reagent in a similar manner to the installation of the C5 methyl group in the C3-C8 fragment. A linchpin strategy with the Bestmann ylid simultaneously formed the connectivity at C1 and C3. This process was successfully performed on multiple substrates arising from the model systems used in the alkynylation and reductive methylation reactions, yielding precursors to the ring-closing metathesis and potentially enabling the synthesis of various analogs. The ring-closing metathesis proved to be difficult in analogs lacking the C17 methyl group and cis-tetrahydropyran ring, and due to this tendency further investigations are necessary. Once the macrocycle has been closed, a global deprotection and oxidation of hydroxy groups is necessary to allow for the installation of the sidechain.</p>

Chasing "Zampanalogs": Advancing the Synthesis of the Zampanolide Macrocycle

<p>(-)-Zampanolide (1), a natural product isolated from a marine sponge, is a microtubule-stabilizing agent that exhibits activity in the nanomolar range against various cancer cells, including in P-gp pump overexpressing cells. This attribute makes (-)-zampanolide an interesting target for further investigation. In this work, a new method for a modular and convergent total synthesis of optically pure zampanolide was investigated, which would also allow the generation of “zampanalogs” following the same basic strategy. Their biological activity may then be assessed to allow the elucidation of structure-activity relationships of (-)-zampanolide and its analogs in tubulin binding. The synthetic plan consisted of the modular combination of four major fragments, which would be connected in the late stages of the synthesis and could therefore be easily exchanged to allow the generation of analogs. The C15-C16 bond would be connected via an alkynylation reaction, and a subsequent reductive methylation would install the trisubstituted alkene. The connections at C1 and C3 could be achieved through a Bestmann ylid linchpin reaction, while the macrolactonization would be completed using a ring-closing metathesis to form the C8-C9 alkene. The side chain could be attached at C20 using one of the established aza-aldol methods. The fragments necessary for the formation of the macrocycle were synthesized successfully. The purification strategy throughout the synthetic route was rationalized and provides an improvement with respect to yield and time compared to work previously done in this research group. Alongside these fragments, modified fragments that were originally intended to serve as model systems were synthesized, which could also be used as building blocks in the synthesis of “zampanalogs”. Several methods for a stereoselective alkynylation at C15 were tested. These led to only meager successes, so an approach using a non-stereoselective alkynylation, followed by oxidation and a stereoselective CBS-reduction, was chosen. For the installation of the trisubstituted alkene a reductive methylation with vitride was tested, but this only led to the reduction of the alkyne without methylation. This product may be employed for the synthesis of C17-desmethyl analogs. The reductive methylation at C16-C17 was ultimately achieved using the Gilman reagent in a similar manner to the installation of the C5 methyl group in the C3-C8 fragment. A linchpin strategy with the Bestmann ylid simultaneously formed the connectivity at C1 and C3. This process was successfully performed on multiple substrates arising from the model systems used in the alkynylation and reductive methylation reactions, yielding precursors to the ring-closing metathesis and potentially enabling the synthesis of various analogs. The ring-closing metathesis proved to be difficult in analogs lacking the C17 methyl group and cis-tetrahydropyran ring, and due to this tendency further investigations are necessary. Once the macrocycle has been closed, a global deprotection and oxidation of hydroxy groups is necessary to allow for the installation of the sidechain.</p>