Thiosemicarbazone derivatives: Evaluation as cruzipain inhibitors and molecular modeling study of complexes with cruzain.

The development of cruzipain inhibitors represents one of the most attractive challenges in the search for drugs for the treatment of Chagas disease. A recombinant form of this enzyme, cruzain, has been crystallized with numerous inhibitors, excluding thiosemicarbazones. These compounds have been established as potent inhibitors of cruzain, although there is very little data in the literature of thiosemicarbazones tested on cruzipain. In this work, we present the results of the evaluation of eleven thiosemicarbazones on cruzipain, isolated from T. cruzi epimastigotes, six of them previously evaluated on cruzain. For these latter, we studied through computational methods, the mode of interaction with the active site of cruzain and the contribution of geometric parameters to the possible mechanism of action involved in the observed inhibition. Finally, from some geometric parameters analyzed on modeled TSC-cruzain complexes, a semi-quantitative relationship was established that could explain the inhibitory activity of thiosemicarbazones on cruzipain, the enzyme actually present in the parasite.

Evaluation of quinoxaline compounds as ligands of a site adjacent to S2 (AS2) of cruzain.

The binding of ten quinoxaline compounds (1-10) to a site adjacent to S2 (AS2) of cruzain (CRZ) was evaluated by a protocol that include a first analysis through docking experiments followed by a second analysis using the Molecular Mechanics-Poisson-Boltzmann Surface Area method (MM-PBSA). Through them we demonstrated that quinoxaline compounds bearing substituents of different sizes at positions 3 or 4 of the heterocyclic ring might interact with the AS2, particularly interesting site for drug design. These compounds showed docking scores (ΔGdock) which were similar to those estimated for inhibitors that bind to the enzyme through non-covalent interactions. Nevertheless, the free binding energies (ΔG) values estimated by MM-PBSA indicated that the derivatives 8-10, which bear bulky substituents at position 3 of the heterocycle ring, became detached from the binding site under a dynamic study. Surprisingly, the evaluation of the inhibitory activity of cruzipain (CZ) of some derivatives showed that they increase the enzymatic activity. These results lead us to conclude about the relevance of AS2 as a pocket for compounds binding site, but not necessarily for the design of anti-chagasic compounds.

Synthesis of New Indanyl Nucleoside Analogues and their Biological Evaluation on Hepatitis C Virus (HCV) Replicon.

Here, we report a convenient synthetic procedure for the preparation of four novel indanyl carbanucleoside derivatives in the racemic form. The action of these compounds against hepatitis C virus was evaluated in vitro using the replicon cell line, Huh7.5 SG. Contrary to our expectations, all these compounds did not inhibit, but rather promoted HCV genotype 1b (HCVg1b) replication. Similar effects have been reported for morphine in the replicon cell lines, Huh7 and Huh8. Several biological experiments and computational studies were performed to elucidate the effect of these compounds on HCVg1b replication. Based on all the experiments performed, we propose that the increase in HCVg1b replication could be mediated, at least in part, by a similar mechanism to that of morphine on the enhancement of this replication. The presence of opioid receptors in Huh7.5 SG cells was indirectly determined for the first time in this work.

Screening assay for inhibitors of a recombinant Streptococcus pneumoniae UDP-glucose pyrophosphorylase.

The UDP-glucose pyrophosphorylase of Streptococcus pneumoniae (GalUSpn) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of pneumococcus. Since the eukaryotic enzymes are completely unrelated to their prokaryotic counterparts, we propose that the GalU enzyme is a critical target to fight the pneumococcal disease. A recombinant GalUSpn was overexpressed and purified. An enzymatic assay that is rapid, sensitive and easy to perform was developed. This assay was appropriate for screening chemical libraries for searching GalU inhibitors. This work represents a fundamental step in the exploration of novel antipneumococcal drugs.

Complexation of a 1-Indanone Thiosemicarbazone with Hydroxypropyl-ß-Cyclodextrin Enhances Its Activity Against a Hepatitis C Virus Surrogate Model.

The current standard of care of the infection by hepatitis C virus (HCV) is effective in a limited number of patients and the high cost hinders therapy affordability and compliance. In this context, the research of new direct-acting antiviral agents (DAAs) for a more effective and long-lasting therapy is an urgent need and an area of active investigation. In an effort to develop novel DAAs, a series of 1-indanone thiosemicarbazones (TSCs) was synthesized and fully characterized. However, the high self-aggregation tendency and extremely poor aqueous solubility of these antiviral candidates often preclude their reliable biological evaluation in vitro. To maintain constant TSC concentrations over the biological assays, different TSC/cyclodextrin complexes were produced. In the present work, we report for the first time the cytotoxicity and antiviral activity of 5,6-dimethoxy TSC inclusion complexes with hydroxypropyl-ß-cyclodextrin on bovine viral diarrhea virus (BVDV) as HCV surrogate model. Results showed a potent suppression of the virus replication, with greater activity for the inclusion complexes than the free compound.

Molecular study of endo and phytocannabinoids on lipid membranes of different composition.

The discovery of the endocannabinoid system (ECS) dates back only 30 years. Although many research groups have been elucidating its components, location, functions and metabolism, the peculiarities of the compounds considered "neurotransmitters" of ECS generate questions that have not yet been answered or controversies in the literature. In this context, we studied the molecular behaviour of the main endocannabinoid compounds and the main phytocannabinoids in eukaryotic outer and inner model membranes. The high lipophilicity of these compounds gives place to the hypothesis that cannabinoids may reach the molecular targets through the lipid bilayer. This consideration is not only for the cannabinoid receptors but also for other (many) targets that these bioactive molecules modulate (Watkins, 2019; Nelson et al., 2020; Jakowiecki and Filipek, 2016). Given the reported multitarget action of these compounds and the differential behaviour towards the different receptors, studying the properties and dynamics of these cannabinoids in POPC and POPE model membranes become relevant. In this regard, we have studied the differential modulation of the endocannabinoids anandamide and 2-arachidonoyl-glycerol and the phytocannabinoids cannabidiol and trans-Δ9-tetrahydrocannabinol to eukaryotic outer and inner model membranes. Results show that behaviours favour the mobility of the bioactive molecules studied by the external eukaryotic model membrane. As well as, the internal eukaryotic model membrane is less fluid, favouring the stabilisation of folded conformations or the positioning of the molecules in the centre of the bilayer. These results provide relevant evidence that contributes to a deep inside understanding of the behaviour of the primary endogenous ligands of ECS, together with the principal phytocannabinoids of C. sativa.

Design, synthesis and biological evaluation of novel thiosemicarbazones as cruzipain inhibitors.

Based on the activity of 23 TSCs on CZ taken from the literature, we have developed a QSAR model for predicting the activity of TSCs. New TSCs were designed and then tested against CZP, resulting in inhibitors with IC50 values in the nanomolar range. The modelling of the corresponding TSC-CZ complexes by molecular docking and QM/QM ONIOM refinement indicates a binding mode compatible with what was expected for active TSCs, according to a geometry-based theoretical model previously developed by our research group. Kinetic experiments on CZP suggest that the new TSCs act by a mechanism that involves the formation of a reversible covalent adduct with slow association and dissociation kinetics. These results demonstrate the strong inhibitory effect of the new TSCs and the benefit of the combined use of QSAR and molecular modelling techniques in the design of new and potent CZ/CZP inhibitors.

Inhibition of bovine viral diarrhea virus RNA synthesis by thiosemicarbazone derived from 5,6-dimethoxy-1-indanone.

In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV).

Novel 1-indanone Thiosemicarbazone Antiviral Candidates: Aqueous Solubilization and Physical Stabilization by Means of Cyclodextrins.

PURPOSE: To investigate cyclodextrin-mediated solubilization and physical stabilization of novel 1-indanone thiosemicarbazone (TSC) candidate drugs that display extremely high self-aggregation and precipitation tendency in water. METHODS: TSC/CD complexes were produced by co-solvent method, and TSC/CD phase-solubility diagrams were obtained by plotting TSC concentration as a function of increasing CD concentration. Size, size distribution, and zeta-potential of the different TSC/CD complexes and aggregates were fully characterized by dynamic light scattering. The morphology of the structures was visualized by atomic force microscopy. RESULTS: Results indicated the formation of Type A inclusion complexes; the solubility of different TSCs was enhanced up to 215 times. The study of physical stability revealed that, as opposed to free TSCs that self-aggregate, crystallize, and precipitate in water very rapidly, complexed TSCs remain in solution for at least 1 week. On the other hand, a gradual size growth was observed. This phenomenon stemmed from the self-aggregation of the TSC/CD complex. CONCLUSIONS: 1-indanone TSC/CD inclusion complexes improved aqueous solubility and physical stability of these new drug candidates and constitute a promising technological approach towards evaluation of their activity against the viruses hepatitis B and C.

Synthesis and biological evaluation of novel homochiral carbocyclic nucleosides from 1-amino-2-indanols.

New chiral purinyl and 8-azapurinyl carbanucleoside derivatives based on indanol were synthesized from commercial available (1S,2S)-trans-1-amino-2-indanol and (1R,2R)-trans-1-amino-2-indanol using a linear methodology. The antiviral activity and cytotoxicity of these compounds were evaluated against herpes simplex virus type 1 (HSV-1) in Vero cells, bovine viral diarrhea virus (BVDV) in Mardin-Darby bovine kidney (MDBK) cells and hepatitis B virus (HBV) in HepG2 2.2.15 cell line. Three compounds, showed an inhibition of the HBsAg levels similar to reference drug lamivudine. One chloropurinyl nucleoside, derived from the cis-1-amino-2-indanol, was cytotoxic on MDBK cells and it could be a lead for developing anticancer agents.

Structure-anti-leukemic activity relationship study of ortho-dihydroxycoumarins in U-937 cells: key role of the δ-lactone ring in determining differentiation-inducing potency and selective pro-apoptotic action.

Previous studies indicated the need of at least one phenolic hydroxyl group in the coumarin core for induction of cytotoxicity in different cell lines. Herein, we present an exhaustive structure-activity relationship study including ortho-dihydroxycoumarins (o-DHC) derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and 1,2-pyrones (representative of the δ-lactone ring of the coumarin core), carried out to further identify the structural features of o-DHC required to induce leukemic cell differentiation and apoptosis in U-937 cells. Our results show for the first time that the δ-lactone ring positively influences the aforementioned biological effects, by conferring greater potency to compounds with an intact coumarin nucleus. Most tellingly, we reveal herein the crucial role of this molecular portion in determining the selective toxicity that o-DHC show for leukemic cells over normal blood cells. From a pharmacological perspective, our findings point out that o-DHC may be useful prototypes for the development of novel chemotherapeutic agents.

In Vitro, In Vivo, and In Silico Studies of Cumanin Diacetate as a Potential Drug against Trypanosoma cruzi Infection.

The sesquiterpene lactones cumanin, helenalin, and hymenin and their semisynthetic derivatives were evaluated against Trypanosoma cruzi epimastigotes. The cytotoxicity of the compounds was evaluated on murine splenocytes. Cumanin diacetate was one of the most active and selective compounds [IC50 = 3.20 ± 0.52 µg/mL, selectivity index (SI) = 26.0]. This sesquiterpene lactone was selected for its evaluation on trypomastigote and amastigote forms of the parasite. The diacetylated derivative of cumanin showed moderate activity on trypomastigotes (IC50 = 32.4 ± 5.8 µg/mL). However, this compound was able to efficiently inhibit parasite replication with an IC50 value of 2.2 ± 0.05 µg/mL against the amastigote forms. Cumanin diacetate showed selectivity against the intracellular forms of Trypanosoma cruzi with an SI value of 52.7. This cumanin analogue was also active on an in vivo model of Chagas disease, leading to a reduction in the parasitemia levels in comparison with nontreated animals. Histopathological analysis of skeletal muscular tissues from treated mice showed only focal interstitial lymphocyte inflammatory infiltrates with slight myocyte necrosis; in contrast, nontreated animals showed severe lymphocyte inflammatory infiltrates with necrosis of the myocytes. A molecular docking study of cumanin and its derivatives on trypanothione reductase from T. cruzi (TcTR) was performed. The results of ΔG docking achieved let the identification of diacetylated and O-alkylated derivatives of cumanin as good inhibitors of TcTR. Cumanin diacetate could be considered a potential candidate for further studies for the development of new therapies against Chagas disease.

Thiosemicarbazones derived from 1-indanones as new anti-Trypanosoma cruzi agents.

In the present work, we synthesized a series of thiosemicarbazones derived from 1-indanones with good anti-Trypanosoma cruzi activity. Most of them displayed remarkable trypanosomicidal activity. All the compounds showed nonspecific cytotoxicity on human erythrocytes. The ability of the new compounds to inhibit cruzipain, the major cysteine protease of T. cruzi, was also explored. Thiosemicarbazones 12 and 24 inhibited this enzyme at the dose assayed. This interaction was also studied in terms of molecular docking.

T908 Polymeric Micelles Improved the Uptake of Sgc8-c Aptamer Probe in Tumor-Bearing Mice: A Co-Association Study between the Probe and Preformed Nanostructures.

Aptamers are oligonucleotides that have the characteristic of recognizing a target with high affinity and specificity. Based on our previous studies, the aptamer probe Sgc8-c-Alexa647 is a promising tool for molecular imaging of PTK7, which is an interesting biomarker in cancer. In order to improve the delivery of this probe as well as create a novel drug delivery nanosystem targeted to the PTK7 receptor, we evaluate the co-association between the probe and preformed nanostructures. In this work, preformed pegylated liposomes (PPL) and linear and branched pristine polymeric micelles (PMs), based on PEO-PPO-PEO triblock copolymers were used: poloxamer F127® and poloxamines T1307® and T908®. For it, Sgc8-c-Alexa647 and its co-association with the different nanostructures was exhaustively analyzed. DLS analysis showed nanometric sizes, and TEM and AFM showed notable differences between free- and co-associated probe. Likewise, all nanosystems were evaluated on A20 lymphoma cell line overexpressing PTK7, and the confocal microscopy images showed distinctness in cellular uptake. Finally, the biodistribution in BALB/c mice bearing lymphoma-tumor and pharmacokinetic study revealed an encouraging profile for T908-probe. All data obtained from this work suggested that PMs and, more specifically T908 ones, are good candidates to improve the pharmacokinetics and the tumor uptake of aptamer-based probes.

Design, synthesis and biological evaluation of quinoxaline compounds as anti-HIV agents targeting reverse transcriptase enzyme.

Infection by human immunodeficiency virus still represents a continuous serious concern and a global threat to human health. Due to appearance of multi-resistant virus strains and the serious adverse side effects of the antiretroviral therapy administered, there is an urgent need for the development of new treatment agents, more active, less toxic and with increased tolerability to mutations. Quinoxaline derivatives are an emergent class of heterocyclic compounds with a wide spectrum of biological activities and therapeutic applications. These types of compounds have also shown high potency in the inhibition of HIV reverse transcriptase and HIV replication in cell culture. For these reasons we propose, in this work, the design, synthesis and biological evaluation of quinoxaline derivatives targeting HIV reverse transcriptase enzyme. For this, we first carried out a structure-based development of target-specific compound virtual chemical library of quinoxaline derivatives. The rational construction of the virtual chemical library was based on previously assigned pharmacophore features. This library was processed by a virtual screening protocol employing molecular docking and 3D-QSAR. Twenty-five quinoxaline compounds were selected for synthesis in the basis of their docking and 3D-QSAR scores and chemical synthetic simplicity. They were evaluated as inhibitors of the recombinant wild-type reverse transcriptase enzyme. Finally, the anti-HIV activity and cytotoxicity of the synthesized quinoxaline compounds with highest reverse transcriptase inhibitory capabilities was evaluated. This simple screening strategy led to the discovery of two selective and potent quinoxaline reverse transcriptase inhibitors with high selectivity index.

Combining nuclear magnetic resonance with molecular dynamics simulations to address sumatriptan interaction with model membranes.

The goal of this work is to obtain a complete map on the interactions between sumatriptan, an amphiphilic ionizable anti-migraine drug, with lipid bilayers. To this end, we combined two physico-chemical techniques - nuclear magnetic resonance and molecular dynamics simulations - to obtain a detailed picture at different pH values. Both approaches were used considering the strength and constraints of each one. NMR experiments were performed at pH 7.4 where at least 95% of the drug molecules are in their protonated state. From NMR, sumatriptan shows partition on the interfacial region of model membranes (near the head groups and intercalating between adjacent lipids), inducing changes in chemical environment and affecting lipid dynamics of liposomes, in a dose dependent manner. Due to the experimental instability of lipid bilayers at high pH, we took advantage of the molecular dynamics power to emulate different pH values, to simulate sumatriptan in bilayers including at fully uncharged state. Simulations show that the neutral species have preferential orientation within the bilayer interface while the distribution of protonated drugs is independent on the initial conditions. In summary, several properties depicted the interfacial partition of the anti-migraine drug at the water-lipid interface at different conditions. Both techniques were found complementary to shed light on the structural and dynamics of sumatriptan-lipid bilayer interactions.

Biochemical mechanisms underlying the pro-apoptotic activity of 7,8-dihydroxy-4-methylcoumarin in human leukemic cells.

The search for new drugs requires a deep understanding of the molecular basis of drug action, being necessary the elucidation of the mechanism of action with the understanding of the relationship between structure and activity. In the present study, we evaluated the pro-apoptotic activity of 7,8-dihydroxy-4-methylcoumarin (DHMC) and its underlying mechanisms in human leukemic cells. Here, we present evidence that DHMC induced selective and concentration-dependent apoptosis in human leukemic cells. The pro-apoptotic effect of DHMC was mediated by activation of the JNKs and inhibition of the ERK1/2 and PI3K/Akt pathways, with no participation of the p38 cascade after 24h of treatment. Indeed, down-regulation of the proto-oncogene c-myc as well as induction of the cell cycle inhibitor p21(WAF1/CIP1) through a p53 independent mechanism were observed in U-937 cells. These findings suggest that DHCM may have a potential therapeutic role in the future treatment of hematological malignancies.

Structural insights into hydroxycoumarin-induced apoptosis in U-937 cells.

In the present study, we sought to establish the effect of diverse structural-related hydroxycoumarins on the proliferation, cytotoxicity, and induction of apoptosis in promonocytic leukemic cells (U-937). The dihydroxylated coumarins, 7,8-dihydroxy-coumarin and esculetin, induced DNA fragmentation as well as characteristic morphological changes of programmed cell death in U-937 cells. With the aim to perform a structure-activity relationship study, the correlation between the physicochemical properties of the molecules and their pro-apoptotic activity was carried out. Results showed that the presence of two adjacent phenolic hydroxyl groups was the most important factor in terms of the SAR. The exposure of leukemic cells to 7,8-dihydroxy-coumarin evoked a phenoxyl radical generation that was detected by electron spin resonance spectroscopy. The present study suggests that reactive oxygen species generation plays a critical role in dihydroxycoumarin-induced apoptosis in U-937 cells. These findings further suggest that these compounds may have a potential therapeutic role in the treatment of hematological malignancies.

New 1-indanone thiosemicarbazone derivatives active against BVDV.

Identification of new therapeutic agents for the treatment of viral diseases represents an area of active investigation. In an effort to develop new antiviral compounds, a series of 1-indanone thiosemicarbazone derivatives were synthesized. These derivatives were structurally characterized using several spectroscopic techniques and evaluated against bovine viral diarrhoea virus as a surrogate model for hepatitis C virus. Thiosemicarbazone 2m showed potent anti-bovine viral diarrhoea virus activity with a higher selectivity index (SI=80.29) than that of ribavirin (SI=11.64). This result determines the potentiality of these thiosemicarbazones as antiviral agents for the treatment of infections caused by other highly related members of Flaviviridae family, as hepatitis C virus.

Chitosan-g-oligo(epsilon-caprolactone) polymeric micelles: microwave-assisted synthesis and physicochemical and cytocompatibility characterization.

With the aim to produce mucoadhesive polymeric micelles for drug administration by mucosal routes, chitosan-g-oligo(epsilon-caprolactone) copolymers were synthesized by the microwave-assisted ring-opening polymerization of epsilon-caprolactone using chitosan as the macroinitiator and methanesulfonic acid as the solvent, catalyst and protecting group of the amine moieties. The reaction was conducted under very mild conditions and was completed within 10 min with a monomer conversion above 90%. The grafting of oligo(epsilon-caprolactone) blocks to the free hydroxyl groups of chitosan was confirmed by ATR/FT-IR, 1H- and 13C-NMR, WAXD and thermal analysis (TGA/DSC). The molecular weight of the synthetic hybrid copolymers was determined by GPC and MALDI-ToF mass spectrometry. Polymeric micelles obtained by the solvent diffusion/evaporation method showed a spherical shape (TEM and AFM) with sizes between 111 and 154 nm and highly positive zeta potential (>+50 mV) (DLS). In addition, they displayed good cell compatibility in the human lung adenocarcinoma epithelial line, A549, and were readily up-taken by the cervical cancer cell line, HeLa. Results from the encapsulation of the antituberculosis drug, rifampicin, showed that the micelles had better performance than other nanocarriers previously investigated (e.g., cyclodextrins). Moreover, the micelles conserved the mucoadhesiveness displayed by pristine chitosan and are expected to transiently open tight cell junctions and lead to more prolonged residence times in mucosal tissues and greater drug bioavailability.