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Supplementary Tables 1-3 from Paclitaxel Directly Binds to Bcl-2 and Functionally Mimics Activity of Nur77

We reported previously that Bcl-2 is paradoxically down-regulated in paclitaxel-resistant cancer cells. We reveal here that paclitaxel directly targets Bcl-2 in the loop domain, thereby facilitating the initiation of apoptosis. Molecular modeling revealed an extraordinary similarity between the paclitaxel binding sites in Bcl-2 and β-tubulin, leading us to speculate that paclitaxel could be mimetic of an endogenous peptide ligand, which binds both proteins. We tested the hypothesis that paclitaxel mimics Nur77, which, like paclitaxel, changes the function of Bcl-2. This premise was confirmed by Nur77 interacting with both paclitaxel targets (Bcl-2 and β-tubulin) and a peptide sequence mimicking the Nur77 structural region, thus reproducing the paclitaxel-like effects of tubulin polymerization and opening the permeability transition pore channel in mitochondria. This discovery could help in the development of novel anticancer agents with nontaxane skeleton as well as in identifying the clinical subsets responsive to paclitaxel-based therapy. [Cancer Res 2009;69(17):6906–14]

SET and MYND domain-containing protein 3 (SMYD3) is a lysine methyltransferase that plays a central role in a variety of cancer diseases, exerting its pro-oncogenic activity by methylation of key proteins, of both nuclear and cytoplasmic nature. However, the role of SMYD3 in the initiation and progression of cancer is not yet fully understood and further biochemical characterization is required to support the discovery of therapeutics targeting this enzyme. We have therefore developed robust protocols for production, handling, and crystallization of SMYD3 and biophysical and biochemical assays for clarification of SMYD3 biochemistry and identification of useful lead compounds. Specifically, a time-resolved biosensor assay was developed for kinetic characterization of SMYD3 interactions. Functional differences in SMYD3 interactions with its natural small molecule ligands SAM and SAH were revealed, with SAM forming a very stable complex. A variety of peptides mimicking putative substrates of SMYD3 were explored in order to expose structural features important for recognition. The interaction between SMYD3 and some peptides was influenced by SAM. A nonradioactive SMYD3 activity assay using liquid chromatography-mass spectrometry (LC-MS) analysis explored substrate features of importance also for methylation. Methylation was notable only toward MAP kinase kinase kinase 2 (MAP3K2_K-260)-mimicking peptides, although binary and tertiary complexes were detected also with other peptides. The analysis supported a random bi-bi mechanistic model for SMYD3 methyltransferase catalysis. Our work unveiled complexities in SMYD3 biochemistry and resulted in procedures suitable for further studies and identification of novel starting points for design of effective and specific leads for this potential oncology target.

The ADMET profile of drugs is strongly affected by human serum albumin (HSA), due to its leading role as carrier of poorly soluble compounds in plasma; a critical assessment of the binding capacity of HSA and the evaluation of binding competition between drugs are therefore pivotal for a reliable pharmacokinetic and pharmacodynamic characterization. In clinical practice, a potential source of impairment in the binding properties of HSA is the use of octanoate and N-acetyltryptophan as stabilizers during the production of pharmaceutical-grade HSA for infusion (i-HSA), which is currently administered in the treatment of a growing range of pathological conditions. The peculiar sensitivity of circular dichroism (CD) spectroscopy towards the stereochemical features of high-affinity binding events is herein exploited to achieve a site-specific assessment of the effect of stabilizers on the binding properties of i-HSA. The binding affinity and capacity of fatty-acid-free HSA towards site-selective induced circular dichroism (ICD) markers for the three high-affinity binding sites of HSA was compared to that of i-HSA submitted to ultrafiltration and dialysis to remove both stabilizers. Results showed a considerable impairment of the binding capacity of i-HSA at site II and a relatively lower influence on the binding properties of site I. Ultrafiltration proved to be ineffective in depleting octanoate, while the proposed dialysis protocol, which involves a pH-induced reversible unfolding of the protein, resulted in a total clearance of both stabilizers, confirmed by the full restoration of the binding properties of HSA at all binding sites. The outcomes of this study proved that CD spectroscopy is a suitable technique to evaluate the binding properties of i-HSA, ensuring an assessment of the availability of the binding sites and the possibility of monitoring the clearance of stabilizers. Eventually, the proposed method for their depletion might constitute a connection bridge between albumin in vitro studies and its clinical applications.

Ovarian cancer remains the leading cause of mortality among gynecological tumors. Estrogen receptor beta (ER&beta, ) expression has been suggested to act as a tumor suppressor in epithelial ovarian cancer by reducing both tumor growth and metastasis. ER&beta, expression abnormalities represent a critical step in the development and progression of ovarian cancer: for these reasons, its re-expression by genetic engineering, as well as the use of targeted ER&beta, therapies, still constitute an important therapeutic approach. 3-{[2-chloro-1-(4-chlorobenzyl)-5-methoxy-6-methyl-1H-indol-3-yl]methylene}-5-hydroxy-6-methyl-1,3-dihydro-2H-indol-2-one, referred to here as compound 3, has been shown to have cytostatic as well cytotoxic effects on various hormone-dependent cancer cell lines. However, the mechanism of its anti-carcinogenic activity is not well understood. Here, we offer a possible explanation of such an effect in the human ovarian cancer cell line IGROV1. Chromatin binding protein assay and liquid chromatography mass spectrometry were exploited to localize and quantify compound 3 in cells. Molecular docking was used to prove compound 3 binding to ER&beta, Mass spectrometry-based approaches were used to analyze histone post-translational modifications. Finally, gene expression analyses revealed a set of genes regulated by the ER&beta, /3 complex, namely CCND1, MYC, CDKN2A, and ESR2, providing possible molecular mechanisms that underline the observed antiproliferative effects.

Caffeic acid-derived polyethers are a class of natural products isolated from the root extracts of comfrey and bugloss, which are endowed with intriguing pharmacological properties as anticancer agents. The synthesis of new polyether derivatives is achieved through ring-opening polymerization of chiral 2,3-disubstituted oxiranes, whose absolute configurations define the overall stereochemistry of the produced polymer. The absolute stereochemistry of one of these building blocks, methyl trans-3-(3,4-dimethoxy-phenyl)glycidate (3), was therefore characterized by the combination of enantioselective high-performance liquid chromatography (HPLC), electronic circular dichroism (ECD) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. Initial efforts aiming at the isolation of enantiomers by means of a standard preparative HPLC protocol followed by offline ECD analysis failed due to unexpected degradation of the samples after collection. The stopped-flow HPLC-CD approach, by which the ECD spectra of enantiomers are measured online with the HPLC system, was applied to overcome this issue and allowed a fast, reliable, and chemical-saving analysis, while avoiding the risks of sample degradation during the collection and processing of enantiomeric fractions. Subsequent TD-DFT calculations identified ( as the first eluted enantiomeric fraction on the Lux Cellulose-2 column, therefore achieving a full stereochemical characterization of the chiral oxirane under investigation.

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings. Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC-MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity.

The binding of some novel bicalutamide analogues to human serum albumin (HSA) and rat serum albumin (RSA) was investigated by surface plasmon resonance (SPR) based optical biosensor technique. The serum protein binding of the bicalutamide analogues was determined and compared to that of the parent compound. Furthermore, HSA and RSA were used as target plasma proteins, in order to highlight possible differences among species when performing pharmacokinetic studies. HSA and RSA were covalently immobilized on carboxymethyl dextran matrixes, using an amine coupling procedure. The anchoring method was validated by determining the dissociation constant (KD) of a standard analyte to confirm that the binding properties of the proteins were maintained. The ranking of the bicalutamide analogues for their HSA and RSA bound fractions was used to compare the behaviour of the two albumins. Most of the bicalutamide analogues showed higher binding levels with respect to the lead compound, (R)-bicalutamide. Further, meaningful differences in the binding level to the two serum proteins were obtained. The dissociation constants (KD) of the interaction between the lead compound, (R)-bicalutamide, and the two proteins were calculated. As a result, the KD obtained with HSA was one order of magnitude higher than that obtained with RSA.The observed differences in the HSA and RSA bonding of the bicalutamide analogues increase the knowledge on the possible low reliability in extrapolating the distribution data obtained on animals to humans. This work demonstrates that SPR based optical biosensor technique is well suited for the medium-high throughput screening of compounds' ligand binding to serum albumins.

SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 and H4K5me levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.

Levetiracetam (LEV), an antiepileptic drug (AED) with favorable pharmacokinetic profile, is increasingly being used in clinical practice, although information on its metabolism and disposition are still being generated. Therefore a simple, robust and fast liquid-liquid extraction (LLE) followed by high-performance liquid chromatography method is described that could be used for both pharmacokinetic and therapeutic drug monitoring (TDM) purposes. Moreover, recovery rates of LEV in plasma were compared among LLE, stir bar-sorptive extraction (SBSE), and solid-phase extraction (SPE). Solvent extraction with dichloromethane yielded a plasma residue free from usual interferences such as commonly co-prescribed AEDs, and recoveries around 90% (LLE), 60% (SPE) and 10% (SBSE). Separation was obtained using reverse phase Select B column with ultraviolet detection (235 nm). Mobile phase consisted of methanol:sodium acetate buffer 0.125 M pH 4.4 (20:80, v/v). The method was linear over a range of 2.8-220.0 µg mL-1. The intra- and inter-assay precision and accuracy were studied at three concentrations; relative standard deviation was less than 10%. The limit of quantification was 2.8 µg mL-1. This robust method was successfully applied to analyze plasma samples from patients with epilepsy and therefore might be used for pharmacokinetic and TDM purposes. Levetiracetam, fármaco antiepiléptico com perfil farmacocinético favorável, tem sido cada vez mais utilizado na prática clínica, embora informações sobre seu metabolismo e disposição cinética ainda estejam sendo geradas. Um método simples, robusto e rápido de extração líquido-líquido seguido por análise por cromatografia líquida de alta eficiência é aqui descrito para servir tanto a investigações farmacocinéticas quanto à monitorização terapêutica. Além disso, as taxas de recuperação do levetiracetam em plasma foram comparadas entre a extração líquido-líquido, a extração sortiva em barra de agitação e a extração em fase sólida. Extração com o solvente diclorometano resultou em plasma livre de interferentes, tais como fármacos antiepilépticos co-prescritos, e apresentou taxas de recuperação em torno de 90% (extração líquido-líquido), 60% (extração em fase sólida) e 10% (extração sortiva em barra de agitação). A separação foi obtida utilizando-se coluna de fase reversa Select B e detecção ultravioleta (235 nm). A fase móvel foi composta por metanol:tampão acetato de sódio 0,125 M pH 4,4 (20:80, v/v). O método mostrou-se linear para o intervalo de 2,8 a 220,0 µg mL-1. Precisão intra- e interdias e a exatidão foram avaliadas em três concentrações; o desvio padrão relativo foi inferior a 10%. O limite de quantificação foi 2.8 µg mL-1. Este método foi aplicado para análise de amostras de plasma de pacientes com epilepsia e, desta forma, pode ser utilizado satisfatoriamente tanto para fins de farmacocinética quanto de monitorização terapêutica.

Several studies identified the Calvin–Benson cycle (CBC), the photosynthetic pathway responsible for carbon fixation (Figure 1), as a redox-regulated process. New biochemical and proteomic approaches suggest that all the CBC enzymes may withstand redox regulation through multiple redox post- translational modifications (PTMs), like disulfide bond formation, glutathionylation and nitrosylation 1 . This redox control is likely involved in the adaptative response to environmental conditions, including light/dark transitions and abiotic/biotic stress. This ongoing work aims at investigating the redox regulation of the chloroplastic CBC enzyme transketolase from the green microalga Chlamydomonas reinhardtii (CrTK). Recombinant CrTK was heterologously expressed in E. coli and purified to homogeneity through metal affinity chromatography. In vitro activity assays showed that the purified enzyme displays a redox-sensitivity being partially inhibited by oxidizing treatments. Moreover, the 3D-structure of the reduced protein (solved at 1.8 Å resolution) revealed the presence of several cysteine couples located at suitable distance for disulfide bond formation and in close proximity to catalytic residues. The influence of the redox state on the structural features of CrTK was further investigated by LC-ESI-MS/MS analysis, allowing the identification of the regulatory cysteines. Concomitantly, circular dichroism (CD) analysis in the far-UV spectral region allowed evaluating redox-dependent changes in the secondary structure of the enzyme. Furthermore, CD analysis in the near-UV region showed the onset of an induced circular dichroism (ICD) signal 3 arising from the interaction between CrTK and the cofactor thiamine pyrophosphate (TPP), which was exploited to monitor CrTK-TPP binding under different redox states of the enzyme.

Enantioselective high-performance liquid chromatography (eHPLC) using chiral stationary phases (CSPs) is surely the most used technique for the determination of the enantiomeric excess (e.e.) of chiral drugs, a fundamental parameter for reliable studies on the relationship between stereochemistry and pharmacological activity. A key aspect of this enantioseparation technique is the efficiency of the chiral selector, which can be optimized to obtain higher selectivity and a wider applicability. Thus, the determination of the mechanisms behind chiral recognition is very important to predict and improve the enantioselectivity of CSPs. The present poster reviews the use of human serum albumin (HSA) as a chiral selectors in eHPLC, with particular emphasis on the modulation of the chromatographic performance of CSPs based on HSA. HSA CSPs allow relatively easy predictions of the chiral discrimination mechanisms and the possibility to improve the selectivity of enantioresolution by modulating the binding process, using either reversible or covalent modifications of the protein. Significant improvements of the chromatographic parameters, such as reduction of analysis time and increase of enantioselectivity, have been obtained for selected analytes by using competitors for a particular binding site of HSA dissolved in the mobile phase or by selectively modifying the protein structure at single amino acid residues.

Accepted Manuscript version. The Published Journal Article is available on Biochimica et Biophysica Acta (BBA) – General Subjects, Volume 1861, Issue 8, Pages 2132–2145 (DOI: https://doi.org/10.1016/j.bbagen.2017.05.021). Supplementary Material available free of charge on the article webpage. © 2017. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT Background: In photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg2+. Methods: TK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography. Results: CrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg2+. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg2+-dependent and proceeds with a slow kinetics. The 3D-structure of CrTK without cofactors (CrTKapo) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg2+ participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation. Conclusion: These evidences indicate that Mg2+ is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg2+ is involved in the control of redox sensitivity of CrTK. General significance: The importance of Mg2+ in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg2+ in chloroplasts.

Beside the regulation of fluid distribution, human serum albumin (HSA) carries other activities, such as binding, transport, and detoxification of many molecules. In patients with cirrhosis, HSA exhibits posttranscriptional alterations that likely affect its functions. This study aimed at identifying the structural HSA alterations occurring in cirrhosis and determining their relationship with specific clinical complications and patient survival. One hundred sixty-eight patients with cirrhosis, 35 with stable conditions and 133 hospitalized for acute clinical complications, and 94 healthy controls were enrolled. Posttranscriptional HSA molecular changes were identified and quantified by using a high-performance liquid chromatography/electrospray ionization mass spectrometry technique. Clinical and biochemical parameters were also recorded and hospitalized patients were followed for up to 1 year. Seven HSA isoforms carrying one or more posttranscriptional changes were identified. Altered HSA isoforms were significantly more represented in patients than in healthy controls. Conversely, the native, unchanged HSA isoform was significantly reduced in cirrhosis. Native HSA and most altered isoforms correlated with both Child-Pugh and Model for End-Stage Liver Disease scores. In hospitalized patients, oxidized and N-terminal truncated isoforms were independently associated with ascites, renal impairment, and bacterial infection. Finally, the native HSA and cysteinylated/N-terminal truncated isoforms were predictors of 1-year survival, with greater prognostic accuracy than total serum albumin concentration. Conclusions: Extensive posttranscriptional changes of HSA, involving several molecular sites and increasing in parallel with disease severity, occur in patients with cirrhosis. Altered isoforms are independently associated with specific clinical complications, whereas the residual, native HSA isoform independently predicts patient survival. These findings support the concept of the “effective albumin concentration,” which implies that the global HSA function is related not only to its serum concentration, but also to the preservation of its structural integrity. (Hepatology 2014;60:1850–1859)

On the basis of a 3D-QSAR study, a new generation of tocainide analogues were designed and synthesized as voltage-gated skeletal muscle sodium channel blockers. Data obtained by screening new compounds by means of Hille-Campbell Vaseline gap voltage-clamp recordings showed that the elongation of the alkyl chain and the introduction of lipophilic and sterically hindered groups on the amino function enhance both potency and use-dependent block. The results provide additional indications about the structural requirement of pharmacophores for further increasing potency and state-dependent block and allowed us to identify a new tocainide analogue (6f) with a favorable pharmacodynamic profile to be proposed as a valid candidate for studies aimed at evaluating its usefulness in the treatment of myotonias.

A series of camptothecin open-ring lactone tripartate conjugates were synthesized, in which polyamine side chains with different architecture (ethane-1,2-diamine, spermidine, homospermidine, spermine, and 4,8,13,17-tetraza-icosane-1,20-diamine) are linked to the 21-carboxylic function through an amidic bond, while the 17-CH(2)OH is acetylated. The rationale for the synthesis of these compounds was to explore the influence of the polyamine architecture on the activity of these CPT conjugates into cells, since the positively charged ammonium cations would favor interaction through electrostatic binding to the negatively charged DNA backbone. Topoisomerase I-mediated DNA cleavage assay was used to investigate the ability of these compounds to stimulate the DNA damage. The cleavage pattern was found to be similar to that of SN38 for all the new CPTs. The CPT tripartates were tested for growth inhibition ability against the human non-small-cell lung cancer carcinoma NCI-H460 cell line. Although these compounds were less potent than topotecan, SN38, and CPT after 1 h of treatment, the antiproliferative effects greatly increased after 72 h of exposure. The growth inhibition potency during long-term exposure is correlated with the number of charges of the 21-amide substituent. Both cleavage assay and in vitro effects support the interpretation that the compounds may have inhibitory activity also in the open-ring form. The architecture of the polyamine moiety is important for antiproliferative activity, and a balance between the hydrophilic and lipophilic properties of the polyamine is critical for CPT potency.

Accepted Manuscript version. The Published Journal Article is available on the Journal of Pharmaceutical and Biomedical Analysis, Volume 113, pages 34–42 (DOI: https://doi.org/10.1016/j.jpba.2015.02.024). Supplementary Material available free of charge on the article webpage. © 2015. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT Induced circular dichroism (ICD) is a spectroscopic phenomenon that provides versatile and useful methods for characterizing the structural and dynamic properties of the binding of drugs to target proteins. The understanding of biorecognition processes at the molecular level is essential to discover and validate new pharmacological targets, and to design and develop new potent and selective drugs. The present article reviews the main applications of ICD to drug binding studies on serum carrier proteins, going from the classic approaches for the derivation of drug binding parameters and the identification of binding sites, to an overview of the emerging trends for the characterization of binding modes by means of quantum chemical (QC) techniques. The advantages and limits of the ICD methods for the determination of binding parameters are critically reviewed; the capability to investigate the binding interactions of drugs and metabolites to their target proteins is also underlined, as well as the possibility of characterizing the binding sites to obtain a complete picture of the binding mechanism and dynamics. The new applications of ICD methods to identify stereoselective binding modes of drug/protein complexes are then reviewed with relevant examples. The combined application of experimental ICD spectroscopy and QC calculations is shown to identify qualitatively the bound conformations of ligands to target proteins even in the absence of a detailed structure of the binding sites, either obtained from experimental X-ray crystallography and NMR measurements or from computational models of the complex.

When enantioselective high-performance liquid chromatography (eHPLC) is hyphenated with detection systems based on chiroptical properties, in particular circular dichroism (CD), the stereochemistry of a chiral analyte can be fully determined. Indeed, eHPLC-CD systems allow the simultaneous assessment of the absolute configuration of stereoisomers and the evaluation of the enantiomeric/diastereomeric composition of samples. These features are particularly important in pharmaceutical analysis, because the assignment of the absolute stereochemistry of drugs is essential to establish reliable structure–activity relationships [1]. An extremely useful application of the eHPLC-CD technique is given by the stopped-flow method: the chromatographic fractions of the analyzed chiral compound are trapped inside the cell of the CD detection system, allowing the measurement of full UV and CD spectra without time-consuming collections of the pure stereoisomers for standard CD spectroscopic analysis. We report the development and application of stopped-flow eHPLC-CD methods for the resolution of a series of inherently chiral O- substituted C-undecylresorcin[4]arenes, recently synthesized by weak-base-promoted O-alkylation [2], with the future aim of characterizing their stereochemistry by means of density functional theory (DFT) calculations. [1] C. Bertucci, D. Tedesco, Advantages of electronic circular dichroism detection for the stereochemical analysis and characterization of drugs and natural products by liquid chromatography, J. Chromatogr. A 1269 (2012) 69–81. [2] F. Farina, C. Talotta, C. Gaeta, P. Neri, Regioselective O-substitution of C-undecylresorcin[4]arene, Org. Lett. 13 (2011) 4842–4845.

Enantiomerically pure oxiranes are valuable electrophilic, chiral synthons [1] and have been introduced into pharmaceutical applications for the synthesis of biologically active polyethers. Stereochemical characterization plays an important role in medicinal chemistry, since chirality is fundamental in the definition of the activity of several biologically active compounds. Consequently, analytical techniques allowing for the stereochemistry to be fully characterized are receiving increasing attention. The absolute configuration of methyl trans-3-(3,4- dimethoxyphenyl)glycidate (trans-1), a recently synthesized building block for the synthesis of methylated analogues of biologically active polymers from different species of comfrey and bugloss [2], was investigated by means of enantioselective high-performance liquid chromatography hyphenated with a circular dichroism detection system (eHPLC-CD) [3]. A Lux Cellulose-4 and a Lux Cellulose-2 columns were successfully employed for the preparative and analytical enantioresolution of racemic trans-1 [4]. The CD spectra of the enantiomeric fractions of trans-1 were then measured both by off-line analysis after preparative chromatographic separation and by stopped-flow measurements during the eHPLC-CD analysis [3]. This last strategy resulted more reliable, because the risk of degradation of the analyte was drastically reduced. The absolute configuration of each enantiomeric fraction was finally determined by comparison of the experimental CD spectra with quantum mechanical (QM) calculations based on time-dependent density functional theory (TD-DFT). The conformationally-averaged theoretical CD spectrum of (2S,3R)-1 reproduced with a reasonable degree of accuracy the CD spectrum of the first-eluted fraction of trans-1 on the Lux Cellulose-2 column: consequently, a full stereochemical characterization of the enantiomers of trans-1 was achieved and the elution order on Lux Cellulose-2 was determined. [1] H.C. Kolb, M.S. VanNieuwenhze, K.B. Sharpless, Catalytic asymmetric dihydroxylation, Chem. Rev. 94 (1994) 2483–2547. [2] V. Barbakadze, L. Gogilashvili, L. Amiranashvili, M. Merlani, K. Mulkijanyan, Novel biologically active phenolic polymers from different species of genera Symphytum and Anchusa (Boraginaceae), J. Chem. Eng. Chem. Res. 1 (2014) 47–53. [3] C. Bertucci, D. Tedesco, Advantages of electronic circular dichroism detection for the stereochemical analysis and characterization of drugs and natural products by liquid chromatography, J. Chromatogr. A 1269 (2012) 69–81. [4] K. Lomsadze, M. Merlani, V. Barbakadze, T. Farkas, B. Chankvetadze, Enantioseparation of chiral epoxides with polysaccharide-based chiral columns in HPLC, Chromatographia 75 (2012) 839–845.

The stereospecificity of high-affinity biorecognition phenomena at the basis of the activity of drugs is an important topic of active research in medicinal chemistry, contributing to the design and development of new potent and selective drugs. The binding of drugs to their targets may lead to the onset of an induced circular dichroism (ICD) signal, which can be detected experimentally (Figure 1). ICD spectroscopy, therefore, provides versatile and useful methods for characterizing the structural and dynamic properties of the binding of drugs to target proteins. [1] Within this framework, the combined application of experimental ICD spectroscopy and quantum chemical (QC) calculations based on density functional theory (DFT) and its time-dependent formulation (TD-DFT) is a recently emerging approach which allows to identify the conformational features of ligands bound to target proteins even in the absence of a detailed structure of the binding sites, either obtained from experimental X-ray crystallography and NMR measurements or from computational models of the complex. [2] QC methods can be used to determine the theoretical chiroptical responses of all the possible conformations of drugs bound to their hosts and compare them with the experimental ICD spectra of drug–host complexes. For instance, the peculiar species-dependent ICD spectra observed for the binding of (S)-ketoprofen to different serum albumins could be explained by the selection of different mutual arrangements of the phenyl moieties inside the binding pocket, contributing to a better understanding of the changes in the pharmacokinetic and pharmacodynamic profiles of drugs among different species. [3] Further applications of this combined ICD/QC approach to the characterization of the binding modes of known ligands for the high-affinity binding sites of human serum albumin (HSA) will be reported, namely for pyrazolidine-3,5-diones and benzodiazepines, which are frequently used as ICD binding markers for Sudlow’s site I and site II, respectively. [1] 1. Tedesco, D.; Bertucci, C. Induced circular dichroism as a tool to investigate the binding of drugs to carrier proteins: Classic approaches and new trends. J. Pharm. Biomed. Anal. 2015, in press, doi: 10.1016/j.jpba.2015.02.024. 2. Ionescu, S.; Matei, I.; Tablet, C.; Hillebrand, M. Theoretical ECD calculations – a useful tool for estimating the conformational change of a ligand in the binding pocket of proteins. Phys. Chem. Chem. Phys. 2013, 15, 11604-14. 3. Tedesco, D.; Pistolozzi, M.; Zanasi, R.; Bertucci, C. Characterization of the species-dependent ketoprofen/albumin binding modes by induced CD spectroscopy and TD-DFT calculations. J. Pharm. Biomed. Anal. 2015, in press, doi: 10.1016/j.jpba.2014.11.029.

The description of solvation effects on the chiroptical properties of chiral molecules is still a difficult challenge in the field of computational spectroscopy; this issue is critical in stereochemical characterization, since a reliable assessment of absolute configuration requires high accuracy. The present case study reports the huge effect of solvation on the chiroptical properties of austdiol, a fungal metabolite of known stereochemistry. Standard protocols based on time-dependent density functional theory calculations failed to reproduce its experimental chiroptical properties in methanol. When short-range solvation effects are explicitly considered by means of ab initio molecular dynamics, the correlation between calculated and experimental data is greatly improved because of a better description of the chiral environment around the ketone chromophore, showing that the modeling of subtle solvent-induced perturbations may require the most accurate computational methods.

Accepted Manuscript version. The Published Journal Article is available on the Journal of Pharmaceutical and Biomedical Analysis, Volume 112, pages 176–180 (DOI: https://doi.org/10.1016/j.jpba.2014.11.029). Supplementary Material available free of charge on the article webpage. © 2014. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT The stereospecificity of high-affinity biorecognition phenomena at the basis of the activity of drugs is an important topic of active research in medicinal chemistry. The binding of drugs to their targets or to carrier proteins may lead to the onset of an induced circular dichroism (ICD) signal, which can be detected experimentally. Quantum mechanical (QM) calculations based on density functional theory (DFT) and its time-dependent formulation (TD-DFT) can be used to determine the theoretical chiroptical response of all the possible conformations of drugs bound to their hosts; by comparison with the experimental ICD spectra of drug-host complexes, this approach can lead to the identification of possible binding modes in the absence of X-ray crystallography or NMR data. The present article reports the application of experimental electronic circular dichroism (ECD) spectroscopy, DFT conformational analysis and TD-DFT calculations to the investigation of the binding modes of (S)-ketoprofen to serum albumins. The peculiar species-dependent ICD spectra observed for the binding of (S)-ketoprofen to different serum albumins can be explained by the selection of different mutual arrangements of the phenyl moieties inside the binding pocket. Such structural elucidations contribute to a better understanding of the changes in the pharmacokinetic and pharmacodynamic profiles of drugs among different species.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Organic Chemistry, 82, 202–210 (DOI: 10.1021/acs.joc.6b02349), © 2016 American Chemical Society, after peer review and technical editing by the publisher. To access the final edited and published work, see http://pubs.acs.org/articlesonrequest/AOR-qqdQ8QSnr8M5MmPPRw2V This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT Racemates of 5 chiral resorcin[4]arenes, 4 tetra O substituted and 1 hepta O substituted, have been resolved by enantioselective HPLC and their ECD spectra have been recorded on line by stopped flow measurements. The absolute configuration has been assigned by comparison of the experimental ECD spectra with DFT and semiempirical calculations. For the 4 tetra O substituted resorcin[4]arenes, the ECD exciton couplet at longer wavelength depends on the chirality induced in the arene scaffold by the substituents, rather than on the precise nature of the substituents themselves. Accordingly, the exciton chirality model with excitons localized on the arene scaffold, here generalized to Cn symmetry, accurately describes the relationship between stereochemistry and chiroptical properties for this couplet, while its application at shorter wavelengths is unsafe. For the significantly larger hepta O substituted system the assignment has particularly benefit from the use of the semiempirical ZINDO method.

Accepted Manuscript version. The Published Journal Article is available on theJournal of ChromatographyA, Volume 1363, pages 150-154 (DOI: https://doi.org/10.1016/j.chroma.2014.07.069). SupplementaryMaterial available free of charge on the article webpage. © 2014. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license.https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT Levamisole, an anthelmintic drug, has been increasingly employed as an adulterant of illicit street cocaine over the last decade; recently, the use of tetramisole, the racemic mixture of levamisole and its enantiomer dexamisole, was also occasionally observed. A new enantioselective high-performance liquid chromatography (HPLC) method, performed on cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases in normal-phase mode, was validated and applied to determine the enantiomeric composition of tetramisole enantiomers in seized cocaine samples. Furthermore, the hyphenation of the validated HPLC method with a circular dichroism (CD) detection system allowed the direct determination of elution order and a selective monitoring of levamisole and dexamisole in the presence of possible interferences. The method was applied to the identification and quantitation of the two enantiomers of tetramisole in seized street cocaine samples.

Accepted manuscript version made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ The published manuscript (Current Medicinal Chemistry 2017, 24(8), 743–757) is available at EurekaSelect via https://www.eurekaselect.com/openurl/content.php?genre=article&doi=10.2174/0929867324666161118115711. © 2017 Bentham Science Publishers ABSTRACT Enantioselective high-performance liquid chromatography (eHPLC) using chiral stationary phases (CSPs) is surely the most used technique for the determination of the enantiomeric excess (e.e.) of chiral drugs, a fundamental parameter for reliable studies on the relationship between stereochemistry and pharmacological activity. A key aspect of this enantioseparation technique is the efficiency of the chiral selector, which can be optimized to obtain higher selectivity and a wider applicability. Thus, the determination of the mechanisms behind chiral recognition is very important to predict and improve the enantioselectivity of CSPs. The present review deals with the preparation and use of CSPs for eHPLC with human serum albumin (HSA) as chiral selector, with particular emphasis on the modulation of the chromatographic performance. HSA-based CSPs allow a relatively easy prediction of the binding sites involved in the retention of analytes and the possibility to improve the selectivity of enantioresolution by modulating the binding process, using either reversible or covalent modifications of the protein. Significant improvements of the chromatographic parameters, such as reduction of analysis time and increase of enantioselectivity, have been obtained for selected analytes by using competitors for a particular binding site of HSA dissolved in the mobile phase or by selectively modifying the protein structure at single amino acid residues.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of Natural Products, 77, 70–78 (DOI: 10.1021/np4006822), © 2014 American Chemical Society, after peer review and technical editing by the publisher. To access the final edited and published work, see http://pubs.acs.org/articlesonrequest/AOR-sFHBNNK8A6rMfuWb5v8t This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT Three new azaphilones with an unusual methylene bridge, named mycoleptones A, B and C (2, 4 and 5), were isolated from cultures of Mycoleptodiscus indicus, a fungus associated with the South American medicinal plant Borreria verticillata (Rubiaceae). Additionally, four known polyketides, austdiol (1), eugenitin (3), 6-methoxyeugenin (6) and 9-hydroxyeugenin (7), were also isolated. The structural characterization of compounds was carried out by nuclear magnetic resonance (NMR) spectroscopy, high-resolution mass spectrometry (HRMS), electronic circular dichroism (ECD) spectroscopy, time-dependent density functional theory (TD-DFT) calculations and X-ray crystallography. Compounds 1–9 were weakly active when tested in anti-leishmanial and cytotoxicity assays.

In the past few years, our two groups of research have been collaborating on a number of projects dealing with the assignment of the absolute configuration (AC) of complex chiral molecules of phar- macological interest in the disordered phase. [1] These studies have been mainly carried out by means of electronic circular dichroism (ECD) spectroscopy and quantum mechanical (QM) calculations at the den- sity functional theory (DFT) level of approximation. Particular attention has been payed to non-trivial chiroptical responses due to conformational flexibility and solvation. Beside these shared activities, some more specific research fields have been investigated too. In par- ticular, the Bologna group has performed studies concerning the hyphenation of enantioselective HPLC methods with detection systems based on ECD [2] and the characterization of biomolecular recognition phenomena between drugs and target or carrier macromolecules. [3] The Salerno group has developed some skills in vibrational circular dichroism (VCD) [4] and chiral NMR [5] spectroscopies, thanks to the availability of a VCD spectrometer and previous studies concerning the non-linear response of molecules exposed to radiation. [1] (a) C. Bertucci, M. Pistolozzi, D. Tedesco, R. Zanasi, R. Ruzziconi, A. M. Di Pietra, J. Chromatogr. A 2012, 1232, 128–133; (b) D. Tedesco, R. Zanasi, A. Guerrini, C. Bertucci, Chirality 2012, 24, 741–750; (c) F. Dong, J. Li, B. Chankvetadze, Y. Cheng, J. Xu, X. Liu, Y. Li, X. Chen, C. Bertucci, D. Tedesco, R. Zanasi, Y. Zheng, Environ. Sci. Technol. 2013, 47, 3386–3394; (d) W. J. Andrioli, R. Conti, M. J. Araújo, R. Zanasi, B. C. Cavalcanti, V. Manfrim, J. S. Toledo, D. Tedesco, M. O. de Moraes, C. Pessoa, A. K. Cruz, C. Bertucci, J. Sabino, D. N. P. Nanayakkara, M. T. Pupo, J. K. Bastos, J. Nat. Prod. 2014, 77, 70–78; (e) D. Tedesco, R. Zanasi, I. W. Wainer, C. Bertucci, J. Pharm. Biomed. Anal. 2014, 91, 92–96. [2] (a) C. Bertucci, D. Tedesco, J. Chromatogr. A 2012, 1269, 69–81; (b) D. Tedesco, A. M. Di Pietra, F. Rossi, M. Garagnani, E. Del Borrello, C. Bertucci, V. Andrisano, J. Pharm. Biomed. Anal. 2013, 81-82, 76–79. [3] (a) G. A. Ascoli, E. Domenici, C. Bertucci, Chirality 2006, 18, 667–679; (b) M. Pistolozzi, C. Bertucci, Chirality 2008, 20, 552–558; [4] (a) A. Lattanzi, A. Russo, P. Rizzo, G. Monaco, R. Zanasi, Chirality 2010, 22, E130–E135; (b) A. Massa, P. Rizzo, G. Monaco, R. Zanasi, Tetrahed. Lett. 2013, 54, 6242–6246. [5] (a) S. Pelloni, P. Lazzeretti, R. Zanasi, J. Chem. Theory Comput. 2007, 3, 1691–1698; (b) G. Monaco, R. Zanasi, Chirality 2011, 23, 752–755.

This study highlights the great importance of even small perturbations in molecular geometry for the definition of chiroptical properties, and therefore for the stereochemical characterization, of chiral drugs. A thorough DFT conformational analysis and TD-DFT calculations were needed for a reliable assessment of the absolute configuration of a (3R)-3-hydroxy-4-thienyl-β-lactam derivative, for which semi-empirical β-lactam sector rules failed to predict the correct stereochemistry. [Chirality 2012, 24, 741]

Accepted Manuscript version. The Published Journal Article is available on the Journal of Chromatography A, Volume 1363, pages 162–168 (DOI: https://doi.org/10.1016/j.chroma.2014.06.020). Supplementary Material available free of charge on the article webpage. © 2014. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT A number of large-conductance calcium-activated potassium (BK) channel openers based on the 2-aryl-1,4-benzothiazine scaffold were previously synthesized, and 2-(5-bromo-2-methoxyphenyl)-6-trifluoromethyl-2H-1,4-benzothiazin-3(4H)-one (1) was identified as the most active compound. Since a stereoselective activation of BK channels was demonstrated for arylindolone derivatives, the effect of the absolute configuration at the C-2 position on the vasorelaxing potency of 2-aryl-1,4-benzothiazines is investigated in this article. Compound 1 was initially evaluated as a racemate: subsequently, the “racemic approach” was used to isolate its enantiomers. The excellent enantioresolution obtained using the Sepapak-4 column (CSP 4, cellulose tris(4-chloro-3-metylphenylcarbamate); RS = 8.36; α = 2.03) allowed to collect highly pure enantiomeric fractions, with enantiomeric excess (e.e.) values higher than 97% and 98% for the first- and second-eluted enantiomer, respectively. Electronic circular dichroism (ECD) studies on the two isolated enantiomers, combined with time-dependent density functional theory (TD-DFT) calculations allowed to characterize the configuration of the enantiomers and determine a (R), (S) elution order. Results from biological assays indicated that the racemate and the isolated enantiomers are endowed with comparable vasorelaxing potency.

Fenoterol and its derivatives are selective β2-adrenergic receptor (β2-AR) agonists whose stereoselective biological activities have been extensively investigated in the past decade; a complete stereochemical characterization of fenoterol derivatives is therefore crucial for a better understanding of the effects of stereochemistry on β2-AR binding. In the present project, the relationship between chiroptical properties and absolute stereochemistry of the stereoisomers of fenoterol (1) was investigated by experimental ECD spectroscopy and time-dependent density functional theory (TD-DFT). DFT geometry optimizations were carried out at the RI-B97D/TZVP/IEFPCM(MeOH) level and subsequent TD-DFT calculations were performed using the PBE0 hybrid functional. Despite the large pool of equilibrium conformers found for the investigated compounds and the known limitations of the level of theory employed, the computational protocol was able to reproduce the experimental ECD spectra of the stereoisomers of 1. The main contribution to the overall chiroptical properties was found to arise from the absolute configuration of the chiral center in α-position to the resorcinol moiety. Based on this evidence, a thorough conformational analysis was performed on the optimized DFT conformers, which revealed the occurrence of a different equilibrium between conformational patterns for the diastereomers of fenoterol: the (R,R')/(S,S') enantiomeric pair showed a higher population of folded conformations than the (R,S')/(S,R') pair.

Human serum albumin (HSA) is the most abundant plasma protein, endowed with several biological properties unrelated to its oncotic power, such as antioxidant and free-radicals scavenging activities, binding and transport of many endogenous and exogenous substances, and regulation of endothelial function and inflammatory response. These non-oncotic activities are closely connected to the peculiarly dynamic structure of the albumin molecule. HSA undergoes spontaneous structural modifications, mainly by reaction with oxidants and saccharides; however, patients with cirrhosis show extensive post-transcriptional changes at several molecular sites of HSA, the degree of which parallels the severity of the disease. The present work reports the development and application of an innovative LC-MS analytical method for a rapid and reproducible determination of the relative abundance of HSA isoforms in plasma samples from alcoholic hepatitis (AH) patients. A condition of severe oxidative stress, similar to that observed in AH patients, is associated with profound changes in circulating HSA microheterogeneity. More interestingly, the high resolution provided by the analytical platform allowed the monitoring of novel oxidative products of HSA never reported before.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal ofMedicinal Chemistry, 59, 2612–2632 (DOI: 10.1021/acs.jmedchem.5b01812), © 2016 American ChemicalSociety, after peer review and technical editing by the publisher. To access the final edited and published work,see http://pubsdc3.acs.org/articlesonrequest/AOR-teeM6WpzZVZ52Aqz9AMW This Manuscript version is made available under the CC-BY-NC-ND 4.0license.https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT We report the discovery of compound4a, a potent β-lactam-based monoacylglycerol lipase (MGL) inhibitor characterized by an irreversible and stereoselective mechanism of action, high membrane permeability, high brain penetration evaluated using a human in vitro blood–brain barrier model, high selectivity in binding and affinity-based proteomic profiling assays, and low in vitro toxicity. Mode-of-action studies demonstrate that4a, by blocking MGL, increases 2-arachidonoylglycerol and behaves as a cannabinoid (CB1/CB2) receptor indirect agonist. Administration of4ain mice suffering from experimental autoimmune encephalitis ameliorates the severity of the clinical symptoms in a CB1/CB2-dependent manner. Moreover,4aproduced analgesic effects in a rodent model of acute inflammatory pain, which was antagonized by CB1 and CB2 receptor antagonists/inverse agonists.4aalso relieves the neuropathic hypersensitivity induced by oxaliplatin. Given these evidence,4a, as MGL selective inhibitor, could represent a valuable lead for the future development of therapeutic options for multiple sclerosis and chronic pain.

NikR is a transcription factor that regulates the expression of Ni(II)-dependent enzymes and other proteins involved in nickel trafficking. In the human pathogenic bacterium Helicobacter pylori, NikR (HpNikR) controls, among others, the expression of the Ni(II) enzyme urease by binding the double-strand DNA (dsDNA) operator region of the urease promoter (OPureA) in a Ni(II)-dependent mode. This article describes the complementary use of surface plasmon resonance (SPR) spectroscopy and isothermal titration calorimetry (ITC) to carry out a mechanistic characterization of the HpNikR–OPureA interaction. An active surface was prepared by affinity capture of OPureA and validated for the recognition process in the SPR experiments. Subsequently, the Ni(II)-dependent affinity of the transcription factor for its operator region was assessed through kinetic evaluation of the binding process at variable Ni(II) concentrations. The kinetic data are consistent with a two-step binding mode involving an initial encounter between the two interactants, followed by a conformational rearrangement of the HpNikR–OPureA complex, leading to high affinity binding. This conformational change is only observed in the presence of the full set of four Ni(II) ions bound to the protein. The SPR assay developed and validated in this study constitutes a suitable method to screen potential drug lead candidates acting as inhibitors of this protein–dsDNA interaction. [Figure not available: see fulltext.]

Decompensated cirrhosis is associated to extensive post-transcriptional changes of human albumin (HA). This study aims to characterize the occurrence of HA homodimerization in a large cohort of patients with decompensated cirrhosis and to evaluate its association with clinical features and prognosis. HA monomeric and dimeric isoforms were identified in peripheral blood by using a HPLC-ESI-MS technique in 123 cirrhotic patients hospitalized for acute decompensation and 50 age- and sex-comparable healthy controls. Clinical and biochemical parameters were recorded and patients followed up to one year. Among the monomeric isoforms identified, the N- and C-terminal truncated and the native HA underwent homodimerization. All three homodimers were significantly more abundant in patients with cirrhosis, acute-on-chronic liver failure and correlate with the prognostic scores. The homodimeric N-terminal truncated isoform was independently associated to disease complications and was able to stratify 1-year survival. As a result of all these changes, the monomeric native HA was significantly decreased in patients with cirrhosis, being also associated with a poorer prognosis. In conclusion homodimerization is a novel described structural alteration of the HA molecule in decompensated cirrhosis and contributes to the progressive reduction of the monomeric native HA, the only isoform provided of structural and functional integrity.

Combined results of theoretical molecular dynamic simulations and in vitro spectroscopic (circular dichroism and fluorescence) studies are presented, providing the atomistic and secondary structure details of the process by which a selected small molecule may destabilize the beta-sheet ordered "amyloid" oligomers formed by the model undecapeptide of amyloid beta-peptide 25-35 [Abeta(25-35)]. Abeta(25-35) was chosen because it is the shortest fragment capable of forming large beta-sheet fibrils and retaining the toxicity of the full length Abeta(1-40/42) peptides. The conformational transition, that leads to the formation of beta-sheet fibrils from soluble unordered structures, was found to depend on the environmental conditions, whereas the presence of myricetin destabilizes the self-assembly and antagonizes this conformational shift. In parallel, we analyzed several molecular dynamics trajectories describing the evolution of five monomer fragments, without inhibitor as well as in the presence of myricetin. Other well-known inhibitors (curcumin and (-)-tetracycline), found to be stronger and weaker Abeta(1-42) aggregation inhibitors, respectively, were also studied. The combined in vitro and theoretical studies of the Abeta(25-35) self-assembly and its inhibition contribute to understanding the mechanism of action of well-known inhibitors and the peptide amino acid residues involved in the interaction leading to a rational drug design of more potent new molecules able to antagonize the self-assembly process

The effect of conformational flexibility on the chiroptical properties of a series of synthetic (3R)-3-hydroxy-4-aryl-β-lactams of known stereochemistry (1–6) was investigated by means of electronic circular dichroism (ECD) measurements and time-dependent density functional theory (TD-DFT) calculations. The application of the β-lactam sector rules allowed a correct stereochemical characterization of these compounds, with the exception of a thienyl-substituted derivative (cis-6). TD-DFT calculations yielded accurate predictions of experimental ECD spectra and [α]D values, allowing us to assign the correct absolute configuration to all the investigated compounds. A detailed analysis of the β-lactam ring equilibrium geometry on optimized conformers identified regular patterns for the arrangement of atoms around the amide chromophore, confirming the validity of the β-lactam sector rules. However, relevant variations in theoretical chiroptical properties were found for compounds bearing a heterocyclic substituent at C4 or a phenyl substituent at C3, whose conformers deviate from these regular geometric patterns. This behavior explains the failure of the β-lactam sector rules in cis-6. This study showed the importance of conformational flexibility for the determination of chiroptical properties and highlighted the strengths and weaknesses of the different methods for the stereochemical characterization of chiral molecules in solution. Chirality 24:741-750, 2012. © 2012 Wiley Periodicals, Inc.

Lipocrine has been selected as an effective candidate for in vivo investigation because of its multiple biological properties, namely inhibition of AChE and BChE activities, inhibition of AChE-induced Aβ aggregation, and ability to protect cells against reactive oxygen species. To evaluate the possibility for lipocrine to become a lead and to be developed as a multipotent drug for the treatment of Alzheimer's disease, ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters need to be determined. Among ADMET parameters, distribution plays a key role in determining the lead drugability, and the drug binding to plasma proteins greatly influences the drug distribution. Here, the human serum albumin (HSA) binding of lipocrine has been studied by circular dichroism (CD) spectroscopy. The reversible binding of lipocrine is stereoselective as shown by the well-defined induced CD spectrum in its binding to HSA. The intensity of the CD signal changes upon changing the [drug]/[HSA] molar ratio, showing a different behavior for a [drug]/[HSA] up to 2/1 or over this molar ratio, suggesting a binding to multiple sites. Competition experiments show that lipocrine interacts significantly with all the main binding sites on the serum carrier. A direct competition has been monitored for site II and bilirubin-binding site, whereas a noncooperative binding should better describe the displacement observed at site I. Rac-lipocrine and its enantiomers are characterized by two different binding modes. Almost the same induced CD spectra were obtained for both (R)- and (S)-lipocrine complexed to HSA, suggesting a similar stereochemistry for the bound enantiomers. Chirality, 2011. © 2011 Wiley-Liss, Inc.

Adverse effects have limited the clinical potential of 20-(S)-camptothecin (CPT) and led to a growing interest in the development of CPT analogues that exhibit less severe drawbacks, while maintaining their therapeutic activity. Recently, a thiopyridone isostere of CPT, 20-(S)-thiocamptothecin (TCPT), was developed that resulted more potent than the parent compound in H460, HT29 and IGROV-1 cell lines. The improved activity of TCPT over CPT might be due to the greater stability of the lactone ring. Here, reversible hydrolysis to the ring-open carboxylate forms of CPT and TCPT was studied by HPLC, both in the presence and absence of human serum albumin (HSA). The amount of TCPT that exists in the lactone form at equilibrium in buffer solution (24 h) was found to be significantly higher than CPT, and the same trend was observed in the presence of HSA. Specifically, HSA caused a shift in the hydrolysis equilibrium of TCPT towards the carboxylate form, but the proportion of lactone form remained higher than that observed for CPT under the same conditions, and also in the presence of a higher excess of the protein. The role of the sulfur atom in the stability of the open and closed lactone derivatives was investigated by theoretical calculations using stabilization energies and comparison between experimental and calculated absorption spectra. Our results suggest that, in aqueous solution, more ionic species (anionic and enolic forms) are present for TCPT. This study provides further insights into the effects of oxygen/sulfur replacement in the CPT pyridone ring.

Chirality plays a fundamental role in determining the pharmacodynamic and pharmacokinetic properties of drugs, and contributes significantly to our understanding of the mechanisms that lie behind biorecognition phenomena. Circular dichroism spectroscopy is the technique of choice for determining the stereochemistry of chiral drugs and proteins, and for monitoring and characterizing molecular recognition phenomena in solution. The role of chirality in our understanding of recognition phenomena at the molecular level is discussed here via several selected systems of interest in the drug discovery and development area. The examples were selected in order to underline the utility of circular dichroism in emerging studies of protein-protein interactions in biological context. In particular, the following aspects are discussed here: the relationship between stereochemistry and pharmacological activity--stereochemical characterization of new leads and drugs; stereoselective binding of leads and drugs to target proteins--the binding of drugs to serum albumins; conformational transitions of peptides and proteins of physiological relevance, and the stereochemical characterization of therapeutic peptides.

A sensitive and reproducible stir bar-sorptive extraction and high performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of rifampicin in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. This miniaturized method can result in faster analysis, higher sample throughput, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity. Important factors in the optimization of SBSE efficiency such as pH, temperature, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) were optimized recoveries ranging from 75 to 80%. Separation was obtained using a reverse phase C 8 column with UV detection (254 nm). The mobile phase consisted of methanol:0.25 N sodium acetate buffer, pH 5.0 (58:42, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.125–50.0 μg mL −1 . The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.25, 6.25 and 25.0 μg mL −1 ). The intra-assay coefficients of variation (CVs) for all compounds were less than 10% and all inter-CVs were less than 10%. Limits of quantification were 0.125 μg mL −1 . Stability studies showed rifampicin was stable in plasma for 12 h after thawing; the samples were also stable for 24 h after preparation. Based on the figures of merit results, the SBSE/HPLC-UV proved to be adequate to the rifampicin analyses from therapeutic to toxic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method.

Drug binding to albumins from different mammalian species was investigated to disclose evidence of species-dependent stereoselectivity in drug-binding processes and affinities. This aspect is important for evaluating the reliability of extrapolating distribution data among species. The circular dichroism (CD) signal induced by drug binding to the albumins [human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), and dog serum albumin (DSA)] were measured and analyzed. The binding of selected drugs and metabolites to HSA significantly differed from the binding to the other albumins in terms of affinity and conformation of the bound ligands. In particular, phenylbutazone, a marker of site one on HSA, showed a higher affinity for binding to BSA with respect to RSA, HSA, and DSA, respectively. In the case of diazepam, a marker of site two on HSA, the affinity decreased in order from HSA to DSA, RSA, and BSA. The induced CD spectra were similar in terms of energy and band signs, suggesting almost the same conformation for the bound drug to the different albumins. Stereoselectivity was high for the binding of ketoprofen to HSA and RSA. A different sign was observed for the CD spectra induced by the drug to the two albumins because of the prevalence of a different conformation of the bound drug. Interestingly, the same induced CD spectra were obtained using either the racemic form or the (S)-enantiomer. Finally, significant differences were observed in the affinity of bilirubin, being highest for BSA, then decreasing for RSA, HSA, and DSA. A more complex conformational equilibrium was observed for bound bilirubin.

The secondary structure of two synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H was determined by circular dichroism. In particular, the propensity of these peptides to assume an ordered structure was investigated upon by changing the solvent's polarity and the temperature. A reduction of solvent polarity led to a significant increase in the alpha-helix content in the case of HR1, whereas only a slight change in the secondary structure was observed in the case of HR2. In both cases the conformational change followed a two-state transition model. The interaction of the peptides was monitored by the conformational change in the mixture with respect to the single peptides. However, formation of the complex did not significantly enhance thermal stability. A reliable estimation of the secondary structure was obtained by optimising the experimental conditions to collect CD data down to 180 nm, and by comparing the structure data yielded by different software packages.

Accepted Manuscript version. The Published Journal Article is available on the Journal of Pharmaceutical and Biomedical Analysis, Volume 122, pages 166–172 (DOI: https://doi.org/10.1016/j.jpba.2016.01.051). Supplementary Material available free of charge on the article webpage. © 2016. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT Cucurbitacins are a group of tetracyclic triterpenoids, known for centuries for their anti-cancer and anti-inflammatory properties, which are being actively investigated over the past decades in order to elucidate their mechanism of action. In perspective of being used as therapeutic molecules, a pharmacokinetic characterization is crucial to assess the affinity towards blood carrier proteins and extrapolate distribution volumes. Usually, pharmacokinetic data are first collected on animal models and later translated to humans; therefore, an early characterization of the interaction with carrier proteins from different species is highly desirable. In the present study, the interactions of cucurbitacins E and I with human and rat serum albumins (HSA and RSA) were investigated by means of surface plasmon resonance (SPR)-based optical biosensing and circular dichroism (CD) spectroscopy. Active HSA and RSA sensor chip surfaces were prepared through an amine coupling reaction protocol, and the equilibrium dissociation constants (Kd) for the different cucurbitacins-serum albumins complexes were then determined by SPR analysis. Further information on the binding of cucurbitacins to serum albumins was obtained by CD competition experiments with biliverdin, a specific marker binding to subdomain IB of HSA. SPR data unveiled a previously unreported binding event between CucI and HSA; the determined binding affinities of both compounds were slightly higher for RSA with respect to HSA, even though all the compounds can be ranked as high-affinity binders for both carriers. CD analysis showed that the two cucurbitacins modify the binding of biliverdin to serum albumins through opposite allosteric modulation (positive for HSA, negative for RSA), confirming the need for caution in the translation of pharmacokinetic data across species.

Accepted Manuscript version. The Published Journal Article is available on theJournal of Pharmaceutical andBiomedical Analysis, Volume 81-82, pages 76-79 (DOI: https://doi.org/10.1016/j.jpba.2013.03.024).SupplementaryMaterial available free of charge on the article webpage. © 2013. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license.https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT A new enantioselective HPLC method was developed for the resolution and determination of the enantiomers of methorphan, dextromethorphan (DXM) and levomethorphan (LVM), on a Chiralcel® OJ column (250mm×4.6mm I.D.). The resolution of DXM and LVM was obtained using a mobile phase consisting of (n-hexane)-(2-propanol)-diethylamine (70:30:0.1, v/v/v) at a flow rate of 0.5 mL/min. The enantioselective method was found to be selective (α=1.92) and sensitive (LOD=2.8 μg/mL for both DXM and LVM). The method was coupled with electronic circular dichroism (CD), allowing the determination of the elution order based on the sign of CD signals of the single enantiomers at 285nm (positive for DXM, negative for LVM). Under the optimized conditions, the validated method was applied to the identification and quantitation of the enantiomers of methorphan in samples of different sources of illicit drugs of abuse (heroin). DXM was found in eight seized samples of street heroin; two of these samples, for which the DXM content was found to be over 5% (w/w) and exceeding a 10% (w/w) ratio with respect to diacetylmorphine, were the cause of two deaths for overdose due to acute narcotism.

The mechanism of action underlying β-secretase 1 (BACE-1) inhibition was characterized by a surface plasmon resonance (SPR) method using primary amino groups to immobilize OM99-2, a well-known highly potent peptidic BACE-1 inhibitor, on the carboxyl groups of the dextran layer of a sensor chip. The diluted BACE-1 was mixed with buffer or the test compound and the mixture was flushed through the chip. BACE-1 binding to the immobilized peptide inhibitor was quantified. This SPR method was used to identify BACE-1 inhibitor binding sites and the mechanism of action (competitive/noncompetitive) and to validate findings of fluorescence resonance energy transfer (FRET) inhibition studies. To support this, a multimethodological approach (circular dichroism and fluorescence spectroscopy) was applied in parallel to FRET inhibition studies to characterize the binding modes of peptidic and nonpeptidic BACE-1 inhibitors. Circular dichroism spectroscopy served to correlate the conformation of BACE-1 with enzymatic activity and to monitor secondary structure changes upon ligand binding. In a complementary approach, direct fluorescence spectroscopy was used to characterize different BACE-1 inhibitor binding sites. The influence of pH and inhibitors on BACE-1 secondary structure was also elucidated. This multimethodological approach was applied to identify binding modes of bis(7)-tacrine and myricetin in comparison with well-known peptidic inhibitors.

Background & Aims Patients with cirrhosis present structural changes of human serum albumin (HSA) affecting non-oncotic functions. Ischaemia-modified albumin (IMA), which reflects the capacity to bind cobalt, has been associated to patient mortality during acute-on-chronic liver failure. This study aimed to assess whether circulating IMA is elevated in advanced cirrhosis and its relationship with severity of cirrhosis and specific complications. Methods A total of 127 cirrhotic patients hospitalized for an acute complication of the disease and 44 healthy controls were enrolled. Plasma IMA and IMA to albumin ratio (IMAr) were measured with a cobalt-binding assay. HSA isoforms carrying post-transcriptional molecular changes were assessed with HPLC-ESI-MS. The effect of endotoxemia on IMA was evaluated in rats with CCl4-cirrhosis. Results IMA/IMAr is significantly higher in cirrhotic patients than in controls, but no correlations were found with prognostic scores. IMA did not correlate with the altered HSA isoforms. Ascites, renal impairment and hepatic encephalopathy did not influence IMA/IMAr levels. In contrast, IMA/IMAr is significantly higher in infected than non-infected patients. ROC curves showed that IMA/IMAr had similar discriminating performances for bacterial infection as C-reactive protein (CRP). Moreover, CRP and IMA were independently associated with bacterial infection. Consistently, endotoxin injection significantly increased IMA in cirrhotic, but not in healthy rats. Conclusions IMA is elevated in patients with advanced cirrhosis. The IMA level does not correlate with disease severity scores, but it is specifically associated to bacterial infection, showing a discriminating performance similar to CRP. Further investigations to assess IMA as a novel diagnostic test for bacterial infection are advocated.