Easy to Use DFT Approach for Computational pKa Determination of Carboxylic Acids.

In pKa computational determination, the challenge in exploring and fostering new methodologies and approaches goes in parallel with the amelioration of computational performances. In this paper a "ready to use methodology" has been compared to other strategies, such as the re-shaping in solvation cavity (Bondi radius re-shaping), wanting to assess its reliability in predicting the pKa of a broad list of carboxylic acids. Thus, the functionals B3LYP and CAM-B3LYP have been selected, using SMD as continuum solvation model. Exploiting our previous results, two water molecules were made explicit on the reaction centre. Data show that our model (CAM-B3LYP/2H2 O) is capable to accurately predict pKa, leading to mean absolute error (MAE) values lower than 0.5. Noteworthy, good results were achieved in computing the pKa of substituents bearing nitro and cyano groups. Focusing on B3LYP, eventually remarkable outputs were obtained only when Bondi correction was applied to the complex with two water molecules. Hence, massive outcomes were obtained in foreseeing the trichloro and trifluoro acetic acid pKa. These findings demonstrated that no complex level of theory nor external factor is required to accurately predict carboxylic acids pKa, with MAE well below 0.5 units.

New insights in the computational pKb determination of primary amines and anilines.

Extensive research has already provided reliable methods for the in-silico prediction of pKa, while a trustworthy strategy for pKb determination is still being sought. Indeed, the approaches previously exploited for computing pKa have shown their weakness in predicting pKb. In the light of the exceptional reliability demonstrated in the pKa calculation of a wide panel of organic acids, in this work, we exploited our "easy to use methodology", based on the direct approach, to predict the pKb of primary amines. Herein, CAM-B3LYP was compared to WB97XD and B3PW91, exploring the solvation model based on density (SMD) and the polarizable continuum model (PCM), in the presence of two explicit water molecules. Noteworthy, CAM-B3LYP and WB97XD returned completely different solvent accessible surfaces (SAS) and electron potential maps (EPM) for the bases and the conjugated acids, independently from the nature of the substituents. Once again, CAM-B3LYP/SMD/2H2O method confirmed its remarkable reliability, leading to a minimum average error (MAE) lower than 0.3. This outstanding result strengthens the trustworthiness of our method, already successfully applied to predict the pKa of different substituted phenols and carboxylic acids. Thus, our "easy-to-use" process can predict also the pKb of primary ammines and anilines, always ensuring consistent outputs.

Towards the "Eldorado" of pKa Determination: A Reliable and Rapid DFT Model.

The selection of a "perfect tool" for the theoretical determination of acid-base dissociation constants (Ka) is still puzzling. Recently, we developed a user-friendly model exploiting CAM-B3LYP for determining pKa with impressive reliability. Herein, a new challenge is faced, examining a panel of functionals belonging to different rungs of the "Jacob's ladder" organization, which classifies functionals according to their level of theory. Specifically, meta-generalized gradient approximations (GGAs), hybrid-GGAs, and the more complex range-separated hybrid (RSH)-GGAs were investigated in predicting the pKa of differently substituted carboxylic acids. Therefore, CAM-B3LYP, WB97XD, B3PW91, PBE1PBE, PBEPBE and TPSSTPSS were used, with 6-311G+(d,p) as the basis set and the solvation model based on density (SMD). CAM-B3LYP showed the lowest mean absolute error value (MAE = 0.23) with relatively high processing time. PBE1PBE and B3PW91 provided satisfactory predictions (MAE = 0.34 and 0.38, respectively) with moderate computational time cost, while PBEPBE, TPSSTPSS and WB97XD led to unreliable results (MAE > 1). These findings validate the reliability of our model in predicting carboxylic acids pKa, with MAE well below 0.5 units, using a simplistic theoretical level and a low-cost computational approach.

An Accurate Approach for Computational pKa Determination of Phenolic Compounds.

Computational chemistry is a valuable tool, as it allows for in silico prediction of key parameters of novel compounds, such as pKa. In the framework of computational pKa determination, the literature offers several approaches based on different level of theories, functionals and continuum solvation models. However, correction factors are often used to provide reliable models that adequately predict pKa. In this work, an accurate protocol based on a direct approach is proposed for computing phenols pKa. Importantly, this methodology does not require the use of correction factors or mathematical fitting, making it highly practical, easy to use and fast. Above all, DFT calculations performed in the presence two explicit water molecules using CAM-B3LYP functional with 6-311G+dp basis set and a solvation model based on density (SMD) led to accurate pKa values. In particular, calculations performed on a series of 13 differently substituted phenols provided reliable results, with a mean absolute error of 0.3. Furthermore, the model achieves accurate results with -CN and -NO2 substituents, which are usually excluded from computational pKa studies, enabling easy and reliable pKa determination in a wide range of phenols.

A paper-based electrochemical sensor for H2O2 detection in aerosol phase: Measure of H2O2 nebulized by a reconverted ultrasonic aroma diffuser as a case of study.

The outbreak of COVID-19 is caused by high contagiousness and rapid spread of SARS-CoV-2 virus between people when an infected person is in close contact with another one. In this overall scenario, the disinfection processes have been largely improved. For instance, some countries have approved no-touch technologies by vaporizing disinfectants such as hydrogen peroxide, with the overriding goal to boost the safety of the places. In the era of sustainability, we designed an electrochemical paper-based device for the assessment of hydrogen peroxide nebulized by a cost-effective ultrasonic aroma diffuser. The paper-based sensor was fabricated by modifying via drop-casting a filter paper-based screen-printed electrode with a dispersion of carbon black-Prussian Blue nanocomposite, to assess the detection of hydrogen peroxide at -0.05 V vs Ag/AgCl. The use of paper-based modified screen-printed electrode loaded with phosphate buffer allowed for monitoring the concentration of hydrogen peroxide in aerosol, without any additional sampling instrument to capture the nebulized solution of hydrogen peroxide at a concentration up to 7% w/w. Hydrogen peroxide, a reconverted ultrasonic aroma diffuser, and the paper-based electrochemical sensor assisted by smartphone have demonstrated how different low-cost technologies are able to supply an useful and cost-effective solution for disinfection procedures.

KuQuinones Equilibria Assessment for Biomedical Applications.

A small library of pentacyclic quinoid compounds, called KuQuinones (KuQs), has been prepared through a one-pot reaction. KuQuinones complex structure is made up by two naphthoquinone units connected by a five-membered ring. Due to KuQs structural features, keto-enol tautomerization in solution likely occurs, leading to the generation of four different species, i.e., the enol, the enolate, the external enol and the diquinoid species. The interchange among KuQ tautomers leads to substantial spectral variations of the dye depending on the experimental conditions used. The comprehension of tautomeric equilibria of this new class of quinoid compounds is strongly required in order to explain their behavior in solution and in biological environment. UV-vis, 1H NMR spectroscopies, and DFT calculations resulted appropriate tools to understand the nature of the prevalent KuQuinone species in solution. Moreover, due to the structural similarity of KuQuinones with camptothecin (CPT), a largely used anticancer agent, KuQs have been tested against Cisplatin-resistant SKOV3 and SW480 cancer cell lines. Results highlighted that KuQs are highly active toward the analyzed cell lines and almost nontoxic for healthy cell, indicating a high specific activity.

Selective inhibition of miR-92 in hippocampal neurons alters contextual fear memory.

Post-transcriptional gene regulation mediated by microRNAs (miRNAs) is implicated in memory formation; however, the function of miR-92 in this regulation is uncharacterized. The present study shows that training mice in contextual fear conditioning produces a transient increase in miR-92 levels in the hippocampus and decreases several miR-92 gene targets, including: (i) the neuronal Cl(-) extruding K(+) Cl(-) co-transporter 2 (KCC2) protein; (ii) the cytoplasmic polyadenylation protein (CPEB3), an RNA-binding protein regulator of protein synthesis in neurons; and (iii) the transcription factor myocyte enhancer factor 2D (MEF2D), one of the MEF2 genes which negatively regulates memory-induced structural plasticity. Selective inhibition of endogenous miR-92 in CA1 hippocampal neurons, by a sponge lentiviral vector expressing multiple sequences imperfectly complementary to mature miR-92 under the control of the neuronal specific synapsin promoter, leads to up-regulation of KCC2, CPEB3 and MEF2D, impairs contextual fear conditioning, and prevents a memory-induced increase in the spine density. Taken together, the results indicate that neuronal-expressed miR-92 is an endogenous fine regulator of contextual fear memory in mice.

Role of glutathione transferases in the mechanism of brostallicin activation.

Brostallicin is a novel and unique glutathione transferase-activated pro-drug with promising anticancer activity, currently in phase I and II clinical evaluation. In this work, we show that, in comparison with the parental cell line showing low GST levels, the cytotoxic activity of brostallicin is significantly enhanced in the human breast carcinoma MCF-7 cell line, transfected with either human GST-pi or GST-mu. Moreover, we describe in detail the interaction of brostallicin with GSH in the presence of GSTP1-1 and GSTM2-2, the predominant GST isoenzymes found within tumor cells. The experiments reported here indicate that brostallicin binds reversibly to both isoenzymes with K(d) values in the micromolar range (the affinity being higher for GSTM2-2). Direct evidence that both GSTP1-1 and GSTM2-2 isoenzymes catalyze the Michael addition reaction of GSH to brostallicin has been obtained both by an HPLC-MS technique and by a new fluorometric assay. We also saw the rapid formation of an intermediate reactive species, which is slowly converted into the final products. This intermediate, identified as the alpha-chloroamido derivative of the GSH-brostallicin adduct, is able to alkylate DNA in a sequence-specific manner and appears to be the active form of the drug. The kinetic behavior of the reaction between brostallicin and GSH, catalyzed by GSTP1-1, has been studied in detail, and a minimum kinetic scheme that suitably describes the experimental data is provided. Overall, these data fully support and extend the findings that brostallicin could be indicated for the treatment of tumor overexpressing the pi or mu class GST.

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, a specific glutathione S-transferase inhibitor, overcomes the multidrug resistance (MDR)-associated protein 1-mediated MDR in small cell lung cancer.

In the present work, we have investigated the antitumor activity of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on aggressive small cell lung cancer. NBDHEX not only is cytotoxic toward the parental small cell lung cancer H69 cell line (LC(50) of 2.3 +/- 0.6 micromol/L) but also overcomes the multidrug resistance of its variant, H69AR, which overexpresses the ATP-binding cassette transporter multidrug resistance-associated protein 1 (MRP1; LC(50) of 4.5 +/- 0.9 micromol/L). Drug efflux experiments, done in the presence of a specific inhibitor of MRP1, confirmed that NBDHEX is not a substrate for this export pump. Interestingly, NBDHEX triggers two different types of cell death: a caspase-dependent apoptosis in the H69AR cells and a necrotic phenotype in the parental H69 cells. The apoptotic pathway triggered by NBDHEX in H69AR cells is associated with c-Jun NH(2)-terminal kinase and c-Jun activation, whereas glutathione oxidation and activation of p38(MAPK) is observed in the NBDHEX-treated H69 cells. In contrast to the parental cells, the higher propensity to die through apoptosis of the H69AR cell line may be related to the lower expression of the antiapoptotic protein Bcl-2. Therefore, down-regulation of a factor crucial for cell survival makes H69AR cells more sensitive to the cytotoxic action of NBDHEX, which is not a MRP1 substrate. We have previously shown that NBDHEX is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines. Therefore, NBDHEX seems a very promising compound in the search for new molecules able to overcome the ATP-binding cassette family of proteins, one of the major mechanisms of multidrug resistance in cancer cells.

Synthesis and structure--activity relationship of new cytotoxic agents targeting human glutathione-S-transferases.

The 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX, 1), a "suicide inhibitor" of the glutathione-S-transferase GSTP1-1, showed pro-apoptotic properties in tumor cells, but in vivo studies were limited by poor bioavailability and high affinity towards GSTM2-2, expressed in many non-cancerous tissues. Here we describe the synthesis and biological characterization of new 1 analogs (2-40), in which the hydroxyhexyl portion at the C4-sulfur atom has been replaced with phenyl-containing moieties as well as substituted alkyl chains. Some of the new compounds displayed 10-100 times increased water-solubility (8, 11, 17, 26-28, 34, 35), and most of them showed higher GSTP1-1 selectivity (2-20, 23-26, 31-33, 35) than 1. The presence of a phenyl ring with polar substituents is in general associated, with some exceptions (23, 24) to low cytotoxicity in osteosarcoma U-2OS cells. Differently, some alkyl derivatives possess cytotoxicity comparable (26, 34, 35) or higher (30, 32) than 1. Among the novel compounds, selected ones (26, 27, 34, and 35) deserve further investigation for their anticancer potential.

A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons.

Neurodegeneration associated with amyloid ß (Aß) peptide accumulation, synaptic loss, and memory impairment are pathophysiological features of Alzheimer's disease (AD). Numerous microRNAs regulate amyloid precursor protein (APP) expression and metabolism. We previously reported that miR-101 is a negative regulator of APP expression in cultured hippocampal neurons. In this study, a search for predicted APP metabolism-associated miR-101 targets led to the identification of a conserved miR-101 binding site within the 3' untranslated region (UTR) of the mRNA encoding Ran-binding protein 9 (RanBP9). RanBP9 increases APP processing by ß-amyloid converting enzyme 1 (BACE1), secretion of soluble APPß (sAPPß), and generation of Aß. MiR-101 significantly reduced reporter gene expression when co-transfected with a RanBP9 3'-UTR reporter construct, while site-directed mutagenesis of the predicted miR-101 target site eliminated the reporter response. To investigate the effect of stable inhibition of miR-101 both in vitro and in vivo, a microRNA sponge was developed to bind miR-101 and derepress its targets. Four tandem bulged miR-101 responsive elements (REs), located downstream of the enhanced green fluorescence protein (EGFP) open reading frame and driven by the synapsin promoter, were placed in a lentiviral vector to create the pLSyn-miR-101 sponge. Delivery of the sponge to primary hippocampal neurons significantly increased both APP and RanBP9 expression, as well as sAPPß levels in the conditioned medium. Importantly, silencing of endogenous RanBP9 reduced sAPPß levels in miR-101 sponge-containing hippocampal cultures, indicating that miR-101 inhibition may increase amyloidogenic processing of APP by RanBP9. Lastly, the impact of miR-101 on its targets was demonstrated in vivo by intrahippocampal injection of the pLSyn-miR-101 sponge into C57BL6 mice. This study thus provides the basis for studying the consequences of long-term miR-101 inhibition on the pathology of AD.

Targeting GSTP1-1 induces JNK activation and leads to apoptosis in cisplatin-sensitive and -resistant human osteosarcoma cell lines.

The effect of the glutathione transferase P1-1 (GSTP1-1) targeting has been investigated in both sensitive (U-2OS) and cisplatin-resistant (U-2OS/CDDP4 µg) human osteosarcoma cell lines. Despite the different enzyme's content, inhibition of GSTP1-1 by 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) causes the activation of c-Jun N-terminal kinase (JNK) and apoptosis in both cell lines. However, different time courses of JNK activation and cell responses are observed. Whereas in the U-2OS/CDDP4 µg cell line drug treatment results in an early increase of caspase activity and secondary necrosis, in the U-2OS cells it mainly causes an early cell cycle arrest followed by apoptosis. In order to elucidate the action mechanism of NBDHEX we performed a proteomic investigation by label-free nLC-MS(E). The high-throughput analysis associated with a bioinformatic tool suggested the involvement of the TNF receptor associated factor (TRAF) family in the cellular response to the drug treatment. We report experimental evidence of the interaction between GSTP1-1 and TRAF2 and we demonstrate that NBDHEX is able to dissociate the GSTP1-1 : TRAF2 complex. This restores the TRAF2 : ASK1 signaling, thereby leading to the simultaneous and prolonged activation of JNK and p38. These mitogen-activated protein kinases (MAPKs) mediate different effects: JNK is crucial for apoptosis, whereas p38 causes an increase in the p21 level and a concomitant cell cycle arrest. Our study shows that GSTP1-1 plays an important regulatory role in TRAF signaling of osteosarcoma and discloses new features of the action mechanism of NBDHEX that suggest potentially practical consequences of these findings.

The glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol overcomes the MDR1-P-glycoprotein and MRP1-mediated multidrug resistance in acute myeloid leukemia cells.

PURPOSE: There has been an ever growing interest in the search for new anti-tumor compounds that do not interact with MDR1-Pgp and MRP1 drug transporters and so circumvent the effect of these proteins conferring multidrug resistance (MDR) and poor prognosis in AML patients. We have investigated the cytotoxic activity of the strong glutathione S-transferase (GST) inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on AML (HL60) cell lines. METHODS: Functional drug efflux studies and cell proliferation assays were performed on both sensitive and MDR AML (HL60) cells after incubation with NBDHEX. Moreover, the mode of cell death (apoptosis vs. necrosis) as well as the correlation between NBDHEX susceptibility and GST activity or Bcl-2 expression was investigated. RESULTS: NBDHEX is not a substrate of either MDR1-Pgp or MRP1 efflux pumps; in fact, it is not only cytotoxic toward the parental HL60 cell line, but also overcomes the MDR phenotype of its HL60/DNR and HL60/ADR variants. CONCLUSIONS: The data herein reported show that NBDHEX mediates efficient killing of both MDR1-Pgp and MRP1 over-expressing AML cells. Therefore, this drug can potentially be used as an effective agent for treating MDR in AML patients.

Structural basis for the binding of the anticancer compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol to human glutathione s-transferases.

Glutathione S-transferases (GST) constitute a superfamily of enzymes with diversified functions including detoxification from xenobiotics. In many human cancers, Pi class GST (GSTP1-1) is overexpressed and contributes to multidrug resistance by conjugating chemotherapeutics. In addition, GSTP1-1 displays antiapoptotic activity by interacting with c-Jun NH(2)-terminal kinase, a key regulator of apoptosis. Therefore, GSTP1-1 is considered a promising target for pharmaceutical treatment. Recently, a potent inhibitor of GSTs, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), was identified and tested on several tumor cell lines demonstrating high antiproliferative activity. To establish the structural basis of NBDHEX activity, we determined the crystal structure of NBDHEX bound to either GSTP1-1 or GSTM2-2 (mu class). NBDHEX in both cases binds to the H-site but occupies different positions. Furthermore, the compound is covalently attached to the GSH sulfur in the GSTM2-2 crystal, forming a sigma-complex, although it is bound but not conjugated in the GSTP1-1 crystal. Several differences in the H-sites of the two isozymes determine the higher affinity of NBDHEX for GSTM2-2 with respect to GSTP1-1. One such difference is the presence of Ile(104) in GSTP1-1 close to the bound NBDHEX, whereas the corresponding position is occupied by an alanine in GSTM2-2. Mutation of Ile(104) into valine is a frequent GSTP1-1 polymorphism and we show here that the Ile(104)Val and Ile(104)Ala variants display a 4-fold higher affinity for the compound. Remarkably, the GSTP1-1/Ile(104)Ala structure in complex with NBDHEX shows a considerable shift of the compound inside the H-site. These data might be useful for the development of new anticancer compounds.

In vitro and in vivo efficacy of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on human melanoma.

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is a powerful inhibitor of the glutathione transferase P1-1 (GSTP1-1) and causes the disruption of the complex between GSTP1-1 and c-Jun N-terminal Kinase (JNK). This induces JNK activation and apoptosis in tumour cells. In the present work we assess the in vitro and in vivo effectiveness of NBDHEX on two human melanoma cell lines, Me501 and A375. NBDHEX shows IC(50) values in the low micromolar range (IC(50) of 1.2+/-0.1microM and 2.0+/-0.2 microM for Me501 and A375, respectively) and is over 100 times more cytotoxic to these cell lines than temozolomide. Apoptosis is observed in Me501 cells within 3h of the addition of NBDHEX, while in A375 cells the apoptotic event is rather late, and is preceded by a G2/M phase arrest. In both melanoma cell lines, JNK activity is required for the ability of NBDHEX to trigger apoptosis, confirming that the JNK pathway is an important therapeutic target for this tumour. NBDHEX is also both effective and well tolerated in in vivo tumour models. A tumour inhibition of 70% is observed in vivo against Me501 human melanoma and a similar result is obtained on A375 model, with 63% of tumour inhibition. These findings indicate that the activation of the JNK pathway, through a selective GSTP1-1 targeting, could prove to be a promising new strategy for treating melanoma, which responds poorly to conventional therapies.