Synthesis and Biological Evaluation of Small Molecules as Potential Anticancer Multitarget Agents.

Twenty-six triazole-based derivatives were designed for targeting both PD-L1 (programmed death receptor ligand 1) and VEGFR-2 (vascular endothelial growth factor receptor 2). These compounds were synthetized and biologically evaluated as multitarget inhibitors of VEGFR-2, PD-L1 and c-Myc proteins. The antiproliferative activity of these molecules on several tumor cell lines (HT-29, A-549, and MCF-7) and on the non-tumor cell line HEK-293 was determined. The effects on the abovementioned biological targets were evaluated for some selected compounds. Compound 23, bearing a p-chlorophenyl group, showed better results than sorafenib in regard to the downregulation of VEGFR-2 and a similar effect to BMS-8 on both PD-L1 and c-Myc proteins. The antiangiogenic and antivascular activities of chloro derivatives were also established by endothelial microtube formation assay on Matrigel®.

Monitoring the evolution of SARS-CoV-2 on a Spanish university campus through wastewater analysis: A pilot project for the reopening strategy.

Wastewater surveillance is a fast and cost-effective tool that enables tracing of both symptomatic and asymptomatic transmission of SARS-CoV-2. In this paper, a pilot program carried out at the University Jaume I for monitoring the trends of the presence of SARS-CoV-2 in wastewater. To the best of our knowledge, this is the first such project conducted on a university campus in Spain. Wastewater samples (n = 838) were collected when students returned to campus, from October 2020 until August 2021, at a confluence sewer point and at the building level including different academic departments and services, the library, administration offices and the university student residence. It has been observed that the probability of SARS-CoV-2 RNA detection in wastewater depended on COVID-19 incidence on campus and visitors/occupants of the buildings i.e., high-, or low-traffic buildings with high or low frequency of potential contacts. Moreover, the third wave in Spain (after Christmas 2020) and an outbreak that occurred at the university student's residence could be carefully followed, allowing confirmation of the end of the outbreak. In addition, viral variants (i.e., mutations and linages) from selected time points were detected by sequencing and gave an indication of the evolution of the virus over time. The results illustrate the potential of wastewater-based epidemiology to provide an early warning for SARS-CoV-2 within the university, especially in buildings with low traffic and more defined populations, like the student residence. The strategy and experience gathered in this study will allow for implementation of improvements for reliable monitoring in the future.

Sizing Down a Supramolecular Gel into Micro- and Nanoparticles.

A low molecular weight gelator with a fluorescent 1,8-naphthalimide unit forms micro- and nanoparticles in aqueous media. Slow addition of a DMSO solution of the gelator into water affords either a self-assembled fibrillar network, sheaf-like microparticles, or nanoparticles depending to the concentration used in the experiment. The micro- and nanoparticles were characterized by dynamic light scattering (DLS), electron microscopy, and fluorescence measurements. In an initial assay of particle loading, Rose Bengal and Rhodamine 123 were shown to be incorporated in the particles. Light-promoted singlet oxygen generation capabilities of Rose Bengal were modulated by its incorporation in the particles. Additionally, the particles were found to promote the transport of Rhodamine 123 into human lung carcinoma live cells. These results indicate that nanoparticles arising from low molecular weight gelators may represent a new type of nanocarriers, being a potential alternative to polymeric nanogels used in nanomedicine.

Direct anodic hydrochloric acid and cathodic caustic production during water electrolysis.

Hydrochloric acid (HCl) and caustic (NaOH) are among the most widely used chemicals by the water industry. Direct anodic electrochemical HCl production by water electrolysis has not been successful as current commercially available electrodes are prone to chlorine formation. This study presents an innovative technology simultaneously generating HCl and NaOH from NaCl using a Mn0.84Mo0.16O2.23 oxygen evolution electrode during water electrolysis. The results showed that protons could be anodically generated at a high Coulombic efficiency (i.e. ≥ 95%) with chlorine formation accounting for 3 ~ 5% of the charge supplied. HCl was anodically produced at moderate strengths at a CE of 65 ± 4% together with a CE of 89 ± 1% for cathodic caustic production. The reduction in CE for HCl generation was caused by proton cross-over from the anode to the middle compartment. Overall, this study showed the potential of simultaneous HCl and NaOH generation from NaCl and represents a major step forward for the water industry towards on-site production of HCl and NaOH. In this study, artificial brine was used as a source of sodium and chloride ions. In theory, artificial brine could be replaced by saline waste streams such as Reverse Osmosis Concentrate (ROC), turning ROC into a valuable resource.

Inhibition of VEGF expression in cancer cells and endothelial cell differentiation by synthetic stilbene derivatives.

We here report the synthesis of several stilbene derivatives. They show a measurable inhibitory effect on angiogenesis, some of them to a higher degree than resveratrol. Test methods included cell proliferation and tube formation assays using bovine aorta endothelial cells. In addition, it has been confirmed through the reverse transcriptase/polymerase chain reaction experiment that these stilbene derivatives downregulate the expression of the gene related to the production of the angiogenesis factor VEGF in cancer cells.

Caspase-3 cleavage of DUSP6/MKP3 at the interdomain region generates active MKP3 fragments that regulate ERK1/2 subcellular localization and function.

MAPK (MAP kinase) phosphatase 3 (DUSP6/MKP3) is a cytosolic MKP (MAPK phosphatase) that regulates negatively ERK1/2 downstream to growth factor or apoptotic signaling. Transcription of DUSP6 gene is activated through the ERK1/2 pathway, which constitutes a feedback regulatory loop of ERK1/2 activation. However, the regulation of the function of the DUSP6/MKP3 protein is poorly known. MKP3 possesses a linker region between its N-terminal MAPK-binding domain and its C-terminal catalytic domain, which is conserved in the related MKPs DUSP7/MKPX and DUSP9/MKP4. In MKP3, the interdomain linker region contains a secondary ERK1/2 binding motif and an active nuclear export sequence. Here, we report that MKP3 protein levels are decreased in cells upon apoptotic stimulation in a caspase-dependent manner, and we identify a novel MKP3 regulatory mechanism mediated by the pro-apoptotic protease caspase-3, which involves the MKP3 interdomain linker region. Active caspase-3 targeted the linker region of MKP3 at several residues, rendering N-terminal and C-terminal MKP3 fragments that contain specific arrangements of nuclear export sequence and ERK1/2 interaction motifs. MKP3 caspase-3-generated fragments displayed differential properties to regulate ERK1/2 nuclear/cytosolic localization and activity. Our results indicate that caspase-3 cleavage of MKP3 down-regulates MKP3 full length and renders active MKP3 fragments, which may participate in novel regulatory pathways controlling the subcellular localization and activation of ERK1/2 during apoptosis.

Phosphorylation of the kinase interaction motif in mitogen-activated protein (MAP) kinase phosphatase-4 mediates cross-talk between protein kinase A and MAP kinase signaling pathways.

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.

Dual-specificity MAP kinase phosphatases as targets of cancer treatment.

The protein tyrosine phosphatase family (PTP) contains a group of dual-specificity phosphatases (DUSPs) that regulate the activivity of MAP kinases (MAPKs), which are key effectors in the control of cell growth and survival in physiological and pathological processes, including cancer. These phosphatases, named as MKP-DUSPs, include the MAPK phosphatases (MKPs) as well as a group of small-size atypical DUSPs structurally and functionally related to the MKPs. MKP-DUSPs, in most of the cases, are direct inactivators of MAPKs by dephosphorylation of both the Thr and the Tyr regulatory residues at the MAPKs catalytic loop. In some other cases, MKP-DUSPs regulate the activity of MAPKs indirectly, acting through upstream MAPK pathways components. The active involvement of MKP-DUSPs in oncogenesis or resistance to cancer therapies is now well documented, making the search and validation of MKP-DUSPs inhibitors a prominent area in clinical cancer research. Here, we review the current knowledge on the role of MKP-DUSPs in human cancer, the status of the preclinical development and validation of specific MKP-DUSP inhibitors, and the potential of MKP-DUSPs as targets for anti-cancer drugs.

Studying the regulation of MAP Kinase by MAP Kinase phosphatases in vitro and in cell systems.

Signaling through MAPK pathways involves a network of activating kinases and inactivating phosphatases. While single MAPK kinases account for specific activation of the distinct MAPKs, inactivation of MAPKs by phosphatases involves a wider spectrum of enzymes, with phosphatases from distinct families displaying specificity toward MAPKs. The dual-specificity family of MAPK phosphatases, MKPs, constitutes the major group of MAPK inactivating phosphatases. MKPs are widely expressed, in a tissue- and development-regulated manner, and the control of their expression and function is crucial for the regulation of MAPK signaling. Here, we present three methods to analyze the regulation of MAPKs by MKPs, using transient and stable-inducible MKP overexpression cell systems and in vitro phosphatase experiments.

Differential up-regulation of MAP kinase phosphatases MKP3/DUSP6 and DUSP5 by Ets2 and c-Jun converge in the control of the growth arrest versus proliferation response of MCF-7 breast cancer cells to phorbol ester.

Different levels of regulation account for the inactivation of MAP kinases by MAPK phosphatases (MKPs), in a cell type- and stimuli-dependent manner. MCF-7 human breast carcinoma cells treated with the phorbol 12-myristate 13-acetate (PMA) suffer growth arrest and show morphological alterations, which depend on the activation of the ERK1/2 MAP kinases. MKP3/DUSP6 and DUSP5 MAP kinase phosphatases, two negative regulators of ERK1/2, were specifically up-regulated in MCF-7 and SKBR3 cells in response to PMA. MKP3 and DUSP5 up-regulation required the prolonged activation of the ERK1/2 pathway, and correlated with the shutdown of this route. MKP3 induction relied on the activation of the Ets2 transcription factor, whereas DUSP5 induction depended on the activation of c-Jun. Diminishing the expression of MKP3 and DUSP5 raised the activation of ERK1/2, and accelerated growth arrest of PMA-treated MCF-7 cells. Conversely, MCF-7 cell lines expressing high levels of MKP3 or DUSP5 did not undergo PMA-triggered growth arrest, displayed a migratory phenotype, and formed colonies in soft agar. We propose that the differential up-regulation of MKP3 by Ets2 and of DUSP5 by c-Jun may converge in similar functional roles for these MAP kinase phosphatases in the growth arrest versus proliferation decisions of breast cancer cells.

ERK2 shows a restrictive and locally selective mechanism of recognition by its tyrosine phosphatase inactivators not shared by its activator MEK1.

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors.