Synthesis of SARS-CoV-2 Mpro inhibitors bearing a cinnamic ester warhead with in vitro activity against human coronaviruses.

COVID-19 now ranks among the most devastating global pandemics in history. The causative virus, SARS-CoV-2, is a new human coronavirus (hCoV) that spreads among humans and animals. Great efforts have been made to develop therapeutic agents to treat COVID-19, and among the available viral molecular targets, the cysteine protease SARS-CoV-2 Mpro is considered the most appealing one due to its essential role in viral replication. However, the inhibition of Mpro activity is an interesting challenge and several small molecules and peptidomimetics have been synthesized for this purpose. In this work, the Michael acceptor cinnamic ester was employed as an electrophilic warhead for the covalent inhibition of Mpro by endowing some peptidomimetic derivatives with such a functionality. Among the synthesized compounds, the indole-based inhibitors 17 and 18 efficiently impaired the in vitro replication of beta hCoV-OC-43 in the low micromolar range (EC50 = 9.14 µM and 10.1 µM, respectively). Moreover, the carbamate derivative 12 showed an antiviral activity of note (EC50 = 5.27 µM) against another hCoV, namely hCoV-229E, thus suggesting the potential applicability of such cinnamic pseudopeptides also against human alpha CoVs. Taken together, these results support the feasibility of considering the cinnamic framework for the development of new Mpro inhibitors endowed with antiviral activity against human coronaviruses.

Stimuli Responsive Features of Organic RTP Materials: An Intriguing Carbazole-Cyclic Triimidazole Derivative.

Stimuli responsive luminescent materials possessing room temperature phosphorescence (RTP) are extremely desirable for various applications. The here investigated derivative of cyclic triimidazole (TT) functionalized with carbazole (Cz), namely TT-Ph-Cz, belongs to this class. TT-Ph-Cz possesses high conformational freedom resulting in rigidochromic and multi-stimuli responsive emissive behavior. It has been isolated as MeOH-solvated and de-solvated forms characterized by distinctive emissive features. In particular, the solvated form, in which hydrogen bonds with MeOH inhibit competitive non-radiative deactivation channels, possesses a higher quantum yield associated with a strong phosphorescence contribution which is preserved in DMSO/water solutions.

Discovery of a Novel Trifluoromethyl Diazirine Inhibitor of SARS-CoV-2 Mpro.

SARS-CoV-2 Mpro is a chymotrypsin-like cysteine protease playing a relevant role during the replication and infectivity of SARS-CoV-2, the coronavirus responsible for COVID-19. The binding site of Mpro is characterized by the presence of a catalytic Cys145 which carries out the hydrolytic activity of the enzyme. As a consequence, several Mpro inhibitors have been proposed to date in order to fight the COVID-19 pandemic. In our work, we designed, synthesized and biologically evaluated MPD112, a novel inhibitor of SARS-CoV-2 Mpro bearing a trifluoromethyl diazirine moiety. MPD112 displayed in vitro inhibition activity against SARS-CoV-2 Mpro at a low micromolar level (IC50 = 4.1 µM) in a FRET-based assay. Moreover, an inhibition assay against PLpro revealed lack of inhibition, assuring the selectivity of the compound for the Mpro. Furthermore, the target compound MPD112 was docked within the binding site of the enzyme to predict the established intermolecular interactions in silico. MPD112 was subsequently tested on the HCT-8 cell line to evaluate its effect on human cells' viability, displaying good tolerability, demonstrating the promising biological compatibility and activity of a trifluoromethyl diazirine moiety in the design and development of SARS-CoV-2 Mpro binders.

Novel Small Multilamellar Liposomes Containing Large Quantities of Peptide Nucleic Acid Selectively Kill Breast Cancer Cells.

Peptide nucleic acid (PNA) may be used in various biomedical applications; however, these are currently limited, due to its low solubility in aqueous solutions. In this study, a methodology to overcome this limitation is demonstrated, as well as the effect of PNA on cell viability. We show that extruding a mixture of natural phospholipids and short (6-22 bases), cytosine-rich PNA through a 100 nm pore size membrane under mild acidic conditions resulted in the formation of small (60-90 nm in diameter) multilamellar vesicles (SMVs) comprising several (3-5) concentric lipid membranes. The PNA molecules, being positively charged under acidic conditions (due to protonation of cytosine bases in the sequence), bind electrostatically to negatively charged phospholipid membranes. The large membrane surface area allowed the encapsulation of thousands of PNA molecules in the vesicle. SMVs were conjugated with the designed ankyrin repeat protein (DARPin_9-29), which interacts with human epidermal growth factor receptor 2 (HER2), overexpressed in human breast cancer. The conjugate was shown to enter HER2-overexpressing cells by receptor-mediated endocytosis. PNA molecules, released from lysosomes, aggregate in the cytoplasm into micron-sized particles, which interfere with normal cell functioning, causing cell death. The ability of DARPin-functionalized SMVs to specifically deliver large quantities of PNA to cancer cells opens a new promising avenue for cancer therapy.

Synthesis of potent and selective HDAC6 inhibitors led to unexpected opening of a quinazoline ring.

Histone deacetylase (HDAC) inhibitors are highly involved in the regulation of many pharmacological responses, which results in anti-inflammatory and anti-cancer effects. In the present work, chemoinformatic analyses were performed to obtain two potent and selective aminotriazoloquinazoline-based HDAC6 inhibitors. We unexpectedly obtained an aminotriazole from a water-driven ring opening of the triazoloquinazoline scaffold. Both compounds were evaluated as HDAC6 inhibitors, resulting in subnanomolar inhibitory activity and high selectivity with respect to class I HDAC1 and HDAC8. Importantly, the compounds were about 3- and 15-fold more potent compared to the reference compound trichostatin A. Additionally, the predicted binding modes were investigated with docking. Considering that the aminotriazole scaffold has never been embedded into the chemical structure of HDAC6 inhibitors, the present study suggests that both the aminotriazoloquinazoline and aminotriazole classes of compounds could be excellent starting points for further optimization of potential anticancer compounds, introducing such novel groups into a relevant and new area of investigation.

Prompt and Long-Lived Anti-Kasha Emission from Organic Dyes.

Anti-Kasha behavior has been the subject of intense debate in the last few years, as demonstrated by the high number of papers appearing in the literature on this topic, dealing with both mechanistic and applicative aspects of this phenomenon. Examples of anomalous emitters reported in the last 10 years are collected in the present review, which is focused on strictly anti-Kasha organic molecules displaying radiative deactivation from Sn and/or Tn, with n greater than 1.

Unravelling the intricate photophysical behavior of 3-(pyridin-2-yl)triimidazotriazine AIE and RTP polymorphs.

The development of purely organic materials showing multicolor fluorescent and phosphorescent behaviour represents a formidable challenge in view of practical applications. Herein the rich photophysical behaviour of 3-(pyridin-2-yl)triimidazotriazine (TT-Py) organic molecule, comprising excitation-dependent fluorescence and phosphorescence under ambient conditions in both blended films and the crystalline phase, is investigated by means of steady state, time resolved and ultrafast spectroscopies and interpreted on the basis of X-ray diffraction studies and DFT/TDDFT calculations. In particular, by proper excitation wavelength, dual fluorescence and dual phosphorescence of molecular origin can be observed together with low energy phosphorescences resulting from aggregate species. It is demonstrated that the multiple emission properties originate from the copresence, in the investigated system, of an extended polycyclic nitrogen-rich moiety (TT), strongly rigidified by π-π stacking interactions and short C-H···N hydrogen bonds, and a fragment (Py) having partial conformational freedom.

Solid State Room Temperature Dual Phosphorescence from 3-(2-Fluoropyridin-4-yl)triimidazo[1,2-a:1',2'-c:1″,2″-e][1,3,5]triazine.

Organic room temperature persistent luminescence is a fascinating but still largely unexplored phenomenon. Cyclic-triimidazole and its halogenated (Br, I) derivatives have recently revealed as intriguing phosphors characterized by multifaceted emissive behavior including room temperature ultralong phosphorescence (RTUP) associated with the presence of H-aggregates in their crystal structure. Here, we move towards a multicomponent system by incorporating a fluoropyridinic fragment on the cyclic-triimidazole scaffold. Such chromophore enhances the molecular properties resulting in a high photoluminescence quantum yield (PL QY) in solution but preserves the solid-state RTUP. By means of X-ray diffraction (XRD) analysis, theoretical calculations, steady-state and time-resolved spectroscopy on solutions, polymethylmethacrylate (PMMA) blends and crystals, the nature of the different radiative deactivation channels of the compound has been disclosed. In particular, the molecular fluorescence and phosphorescence, this latter observed in frozen solution and in PMMA blends, are associated to deactivation from S1 and T1 respectively, while the low energy RTUP, observed only for crystals, is interpreted as originated from H aggregates.

Intrinsic and Extrinsic Heavy-Atom Effects on the Multifaceted Emissive Behavior of Cyclic Triimidazole.

Considering that heavy halogen atoms can be used to tune the emissive properties of organic luminogens, the understanding of their role in photophysics is fundamental for materials engineering. Here, the extrinsic and intrinsic heavy-atom effects on the photophysics of organic crystals were separately evaluated by comparing cyclic triimidazole (TT) with its monoiodo derivative (TTI) and its co-crystal with diiodotetrafluorobenzene (TTCo). Crystals of TT showed room-temperature ultralong phosphorescence (RTUP) originated from H-aggregation. TTI and TTCo displayed two additional long-lived components, the origin of which is elucidated through single-crystal X-ray and DFT/TDDFT studies. The results highlight the different effects of the I atom on the three phosphorescent emissions. Intrinsic heavy-atom effects play a major role on molecular phosphorescence, which is displayed at room temperature only for TTI. The H-aggregate RTUP and the I⋅⋅⋅N XB-induced (XB=halogen bond) phosphorescence on the other side depend only on packing features.

Chemical shift assignment of mono- and di-bromo triimidazo[1,2-a:1',2'-c:1″,2″-e][1,3,5]triazine derivatives by DFT/NMR integrated approach.

Mono- and di-bromo derivatives of triimidazo[1,2-a:1',2'-c:1″,2″-e][1,3,5]triazine have been proposed as new organic molecules presenting a very rich and complex photophysical behavior. Thus, we afforded the correct chemical shift assignment by integrating the experimental data with DFT calculation of NMR parameters. Our findings lay foundation for a structural reference in the organic synthesis and characterization of new congeners of this intriguing class of molecules.

Selective staining and eradication of cancer cells by protein-carrying DARPin-functionalized liposomes.

Since their discovery, liposomes have been widely employed in biomedical research. These nano-size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand-targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80-90 nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme-containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9-29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub-nanomolar concentrations HER2-positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor-positive cells demonstrated here makes the DARPin-functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy.

Cyclic Triimidazole Derivatives: Intriguing Examples of Multiple Emissions and Ultralong Phosphorescence at Room Temperature.

The performance of solid luminogens depends on both their inherent electronic properties and their packing status. Intermolecular interactions have been exploited to achieve persistent room-temperature phosphorescence (RTP) from organic molecules. However, the design of organic materials with bright RTP and the rationalization of the role of interchromophoric electronic coupling remain challenging tasks. Cyclic triimidazole has been shown to be a promising scaffold for such purposes owing to its crystallization-induced room-temperature ultralong phosphorescence (RTUP), which has been associated with H-aggregation. Herein, we report three triimidazole derivatives as significant examples of multifaceted emission. In particular, dual fluorescence, RTUP, and phosphorescence from the molecular and supramolecular units were observed. H-aggregation is responsible for the red RTUP, and Br substituents favor yellow molecular phosphorescence while halogen-bonded Br⋅⋅⋅Br tetrameric units are involved in the blue-green phosphorescence.

Heteronanoparticles by self-Assembly of Doxorubicin and Cyclopamine Conjugates.

The preparation of heteronanoparticles (NPs) with doxorubicin (DOXO) and cyclopamine (CYP) conjugates is presented. Biological evaluation on A431 cell lines confirms the maintenance of the activity of the parental drugs. The in vivo study shows that self-assembled NPs reduce tumor growth and toxicity of chemotherapy.

Tumor Targeting with an isoDGR-Drug Conjugate.

Herein we report the first example of an isoDGR-drug conjugate (2), designed to release paclitaxel selectively within cancer cells expressing integrin αV ß3 . Conjugate 2 was synthesized by connecting the isoDGR peptidomimetic 5 with paclitaxel via the lysosomally cleavable Val-Ala dipeptide linker. Conjugate 2 displayed a low nanomolar affinity for the purified integrin αV ß3 receptor (IC50 =11.0 nm). The tumor targeting ability of conjugate 2 was assessed in vitro in anti-proliferative assays on two isogenic cancer cell lines characterized by different integrin αV ß3 expression: human glioblastoma U87 (αV ß3 +) and U87 ß3 -KO (αV ß3 -). The isoDGR-PTX conjugate 2 displayed a remarkable targeting index (TI=9.9), especially when compared to the strictly related RGD-PTX conjugate 4 (TI=2.4).

H-Aggregates Granting Crystallization-Induced Emissive Behavior and Ultralong Phosphorescence from a Pure Organic Molecule.

Solid-state luminescent materials with long lifetimes are the subject of ever-growing interest from both a scientific and a technological point of view. However, when dealing with organic compounds, the achievement of highly efficient materials is limited by aggregation-caused quenching (ACQ) phenomena on one side and by ultrafast deactivation of the excited states on the other. Here, we report on a simple organic molecule, namely, cyclic triimidazole (C9H6N6), 1, showing crystallization-induced emissive (CIE) behavior and, in particular, ultralong phosphorescence due to strong coupling in H-aggregated molecules. Our experimental data reveal that luminescence lifetimes up to 1 s, which are several orders of magnitude longer than those of conventional organic fluorophores, can be realized under ambient conditions, thus expanding the class of organic materials for phosphorescence applications.

Preparation, Characterization and Manipulation of Conjugates between Gold Nanoparticles and DNA.

Here we described the preparation and characterization by atomic force microscopy of dumbbell-shaped conjugates between 450 bp double-stranded DNA polymer, poly(dG)-poly(dC), and 5 nm gold nanoparticles (GNPs). We have demonstrated that the size of the nanoparticles in the conjugates can be increased in a controlled fashion. Application of the conjugates for measuring the electrical conductivity of DNA is discussed.

The N-terminus of mature human frataxin is intrinsically unfolded.

Frataxin is a highly conserved nuclear-encoded mitochondrial protein whose deficiency is the primary cause of Friedreich's ataxia, an autosomal recessive neurodegenerative disease. The frataxin structure comprises a well-characterized globular domain that is present in all species and is preceded in eukaryotes by a non-conserved N-terminal tail that contains the mitochondrial import signal. Little is known about the structure and dynamic properties of the N-terminal tail. Here, we show that this region is flexible and intrinsically unfolded in human frataxin. It does not alter the iron-binding or self-aggregation properties of the globular domain. It is therefore very unlikely that this region could be important for the conserved functions of the protein.

Synthesis, characterization, and evaluation of a novel 99mTc(CO)3 pyrazolyl conjugate of a peptide nucleic acid sequence.

The 16-mer peptide nucleic acid sequence H-A GAT CAT GCC CGG CAT-Lys-NH2 (1), which is complementary to the translation start region of the N-myc oncogene messenger RNA, was synthesized and conjugated to a pyrazolyl diamine bifunctional chelator (pz). The novel conjugate pz-A GAT CAT GCC CGG CAT-Lys-NH2 (2) was labeled with technetium tricarbonyl, yielding quantitatively the complex fac-[99mTc(CO)3(kappa3-pz-A GAT CAT GCC CGG CAT-Lys-NH2)]2+ (4). Complex 4 was obtained with high radiochemical purity and high specific activity, revealing high stability in human serum and in cell culture medium. The identity of 4 was confirmed by comparing its reversed-phase high performance liquid chromatography profile with that of the rhenium analog fac-[Re(CO)3(kappa3-pz-A GAT CAT GCC CGG CAT-Lys-NH2)]2+ (3), prepared by conjugation of fac-[Re(CO)3(3,5-Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2)]+ to 1, using solid-phase techniques. UV melting experiments of 1 and 3 with the complementary DNA sequence led to the formation of stable duplexes, indicating that the conjugation of 1 to the pyrazolyl chelator and to the metal fragment fac-[M(CO)3]+ did not affect the recognition of the complementary sequence as well as the duplex stability. For a first screening, SH-SY5Y human neuroblastoma cells, which express N-myc, were treated with 4. The results show that 4 internalizes (7% of the activity goes into the cells, after 4 h at 37 degrees C), presenting also a relatively high cellular retention (only 40% of internalized activity is released from the cells after 5 h).

Modulatory effect of bolinaquinone, a marine sesquiterpenoid, on acute and chronic inflammatory processes.

The marine metabolite bolinaquinone is a novel inhibitor of secretory phospholipase A(2) (sPLA(2)), with a potency on the human synovial enzyme (group II) higher than that of manoalide. This activity on the sPLA(2) was confirmed in vivo in the 8-h zymosan rat air pouch on the secretory enzyme accumulation in the pouch exudate. Additionally, bolinaquinone decreased potently the synthesis and release of leukotriene B(4) (LTB(4)) in calcimycin (A23187)-stimulated human neutrophils as a consequence of the inhibition of 5-lipoxygenase activity, as well as PGE(2) and NO production on zymosan-stimulated mouse peritoneal macrophages. This compound exerted anti-inflammatory effects by topical and oral routes on the mouse ear edema induced by 12-O-tetradecanoylphorbolacetate, with ID(50) values of 76.7 microg/ear and 5.6 mg/kg, respectively, with a significant decrease in PGE(2), LTB(4), and tumor necrosis factor-alpha (TNF-alpha) levels being more effective than indomethacin. This effect was confirmed in the mouse paw carrageenan edema after oral administration. Moreover, bolinaquinone was able to reduce the inflammatory response of adjuvant arthritis by inhibiting PGE(2), NO, and TNF-alpha production in paw homogenates without affecting PGE(2) levels in the stomach. Additionally, bolinaquinone inhibited inducible nitric oxide synthase expression and reduced the degree of bone resorption, soft tissue swelling, and osteophyte formation.

Crellastatin A: A Cytotoxic Bis-Steroid Sulfate from the Vanuatu Marine Sponge Crella sp.

A new nonsymmetric dimeric steroid, crellastatin A (1), was isolated from the Vanuatu Island marine sponge Crella sp. Structural assignment was accomplished through extensive 2D NMR spectroscopy. The stereochemistry of 1 was established from an analysis of ROESY experiments and from molecular mechanics and dynamics calculations. Crellastatin A (1), which possesses an unprecedented connection through the side chains, exhibits in vitro cytotoxic activity against NSCLC-N6 cells (IC(50) of 1.5 &mgr;g/mL).