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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

To identify enantioselective nonsteroidal aromatase inhibitors, a multidisciplinary medicinal chemistry approach was pursued. First, our earlier CoMFA model [Bioorg. Med. Chem. 1998,6, 377-388] was extended taking purposely into account previously discovered enantioselective aromatase inhibitors. The 3D QSAR model was then exploited to design chiral ligands, whose configurational assignment was obtained, after HPLC separation, by means of a combination of circular dichroism measurements and time dependent density functional calculations. Finally, the new enantiomeric inhibitors were separately tested to ascertain both their potency against the cytochrome P450 aromatase (CYP19; EC 1.14.14.1), and their selectivity relative to another enzyme of the P450 family. A satisfactory agreement between experimental and predicted data allowed us to assert that a properly built "enantioselective CoMFA model" might constitute a useful tool for addressing enantioselective ligands design.

The binding of two cholecystographic agents to human serum albumin (HSA) was evaluated by means of two different complementary methodologies. In particular, the inhibition of drug HSA binding caused by iopanoic- and iophenoxic-acid was investigated by circular dichroism (CD) and resonant mirror (RM) optical biosensor techniques. The CD study allowed to obtain information both on the cholecystographic agent binding site and on the effect of the binding on the protein conformation. Iopanoic acid (IOP), a drug potentially useful for thyrotoxic disorders, resulted a direct competitor for ligands that selectively bind to site II, in agreement to literature data. No definite evidence was obtained for the highest affinity binding site of iophenoxic acid (IOPH), however, this diagnostic tool markedly affected the binding of ligands to the most characterized high affinity sites on HSA, namely sites I, II and III. Binding parameters were obtained by optical biosensor analysis: K D values were 3.6×10 −7 and 2.8×10 −8 M for IOP and IOPH, respectively.

A water soluble paclitaxel analogue, the 7-hemisuccinylpaclitaxel, was synthesised and its binding to human serum albumin (HSA) was characterised by difference circular dichroism and optical biosensor methodologies. The carboxylate group was introduced at paclitaxel C-7 position to improve the drug water solubility without significantly changing the biological activity. The paclitaxel analogue showed a relatively low affinity to HSA (3.5×104 M−1), while no significant interactions were evidenced with selective markers for the most characterised binding sites on the carrier, suggesting a non-selective binding to low affinity binding sites.

The aggregation of beta-amyloid (1-40) (Abeta) induced by human recombinant acetylcholinesterase (HuAChE) was studied by means of circular dichroism (CD) and by thioflavin T fluorescence spectroscopy. Abeta was incubated alone and with HuAChE. The kinetic of fibrils formation was followed for 48 hr. The increasing beta-conformation content induced by HuAChE, preliminary to the formation of Abeta fibrils, was determined by circular dichroism. This phenomenon was found to be related to the thioflavin T emission of fluorescence at 490 nm. Incubation experiments were performed in the presence of known AChE inhibitors (physostigmine, edrophonium, decamethonium, propidium) and drugs used for Alzheimer's disease (AD) (tacrine, donepezil), to test their capability of preventing the HuAChE-induced Abeta aggregation. The non-competitive or mixed mode of AChE inhibition was confirmed to be an essential feature. At 100 microM propidium, decamethonium, donepezil and physostigmine were found to inhibit the HuAChE-induced Abeta aggregation by 82, 25, 22 and 30%, respectively.

Human serum albumin (HSA) plays a fundamental role in the transport of drugs, metabolites, and endogenous ligands. Binding to HSA controls the free, active concentration of a drug, provides a reservoir for a long duration of action, and ultimately affects drug absorption, metabolism, distribution and excretion. The free concentration of a drug can also be affected by interaction with co-administered drugs or by pathological conditions that can modify to a significant extent the binding properties of the carrier, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This manuscript will review the physiological role of albumin in the human body and the pharmacological consequences of drug-albumin binding, and then focus on the structure and the properties of the protein binding sites, as studied by different methodologies. Among these, biochromatography on immobilized albumin has been shown to be a rapid and effective tool for the characterization of albumin binding sites and their enantioselectivity, and for the study of the changes in the binding properties of the protein arising by interaction between different ligands. We will discuss the potential offered by the combined use of circular dichroism on the same protein/drug system in solution, not only for the determination of binding parameters and the detection of displacement phenomena, but also for the identification of conformational features underlying binding stereoselectivity. In particular, the essential role of these methodologies in the study of the enantioselective phenomena occurring in the HSA binding of chiral drugs will be addressed. The effect of reversible or covalent binding of drugs will also be discussed and examples of physiological relevance reported.

The reversible binding of valproate to human serum albumin determines a decrease of the binding of ligands that selectively bind to site I, site II, and bilirubin binding site. The binding inhibition was followed by displacement chromatography methodology using increasing concentrations of the competitor, i.e. valproate, in the mobile phase. Significant binding inhibition was observed for drugs binding at site I and site II. The greater displacement was observed for the more retained enantiomer of benzodiazepines and profens. A reduction of the affinity was observed also in the case of phenol red, this compound being selected as representative of bilirubin binding site. Difference circular dichroism spectroscopy was also used to characterise the binding of valproate to human serum albumin. This antiepilectic drug was proved to affect the binding at site I, II, and bilirubin binding site. The data have physiological relevance because significant inhibition of the binding resulted at clinic concentrations of valproate.

HPLC on chiral stationary phases has been used for the enantioselective assay of propafenone (PPF), 5-hydroxypropafenone (PPF-50H) and N-despropylpropafenone (PPF-NOR) enantiomers. The results obtained on Chiralpak AD column showed that it is useful for the resolution of PPF and of its main metabolites, although the peaks obtained for PPF-NOR were not symmetrical under the conditions investigated. This column and circular dichroism-based detection system were used to determine the absolute configuration of the eluates. Furthermore, the influence of the mobile phase composition on the resolution of PPF and of its main metabolites was investigated on cellulose derivatives (Chiralcel OD-H and Chiralcel OD-R) and protein (Chiral AGP and Ultron ES-OVM)-based chiral stationary phases. The enantiomers of PPF were resolved on all the columns, except for the Ultron ES-OVM. This column, the Chiralpak AD and the Chiralcel OD-H columns were suitable for the resolution of the PPF-50H enantiomers. The PPF-NOR enantiomers were resolved on the Chiralpak AD, Chiral AGP and Chiralcel OD-R columns. Copyright © 2000 John Wiley & Sons, Ltd.

The binding of drugs known to interact with area I on human serum albumin (HSA) was investigated using a chiral stationary phase obtained by anchoring HSA to a silica matrix. In particular, this high-pressure affinity chromatography selector was employed to study the binding properties of the individual enantiomers of warfarin. The pH and composition of the mobile phase modulate the enantioselective binding of warfarin. Displacement chromatography experiments evidenced significant differences in the binding of the warfarin enantiomers to site I. The (S)-enantiomer was shown to be a direct competitor for (R)-warfarin, while (R)-warfarin was an indirect competitor for the (S)-enantiomer. Salicylate directly competed with (R)-warfarin and indirectly with (S)-warfarin. This behavior was confirmed by difference CD experiments, carried out with the same [HSA]/[drug] system in solution. Chirality 11:675–679, 1999. © 1999 Wiley-Liss, Inc.

The reversible binding of ethacrynic acid was characterized by a difference circular dichroism method. A 2/1 stoichiometry was determined for the [drug]/[HSA] (human serum albumin) complex. The reversible binding of ethacrynic acid to HSA determines direct competition with ligands that selectivity bind to site II and to the fatty acid site. Furthermore, indirect competition was shown for ligands for site I (anticooperative) and to site III (cooperative). Chirality 11:33–38, 1999. © 1999 Wiley-Liss, Inc.

The absolute stereochemistry of a series of (3R)-3-hydroxy-3-alkyl-β-lactams has been determined by circular dichroism and NMR spectroscopies. The sign of the circular dichroism band between 250 and 220 nm was related to the stereochemistry by applying the β-lactam sector rule. The NMR analysis unambiguously determines the relative configuration at C3 and C4 of the β-lactam ring. The reliability of the method has been proved by X-ray analysis of two of the examined compounds. The obtained results are in agreement with the mechanism proposed for the employed synthetic route.

HPLC resolution on chiral stationary phases has been successfully employed to obtain single enantiomers of C5 chiral 4,5-dihydro-1,4-benzodiazepines and to determine the enantiomeric composition of the collected stereoisomeric fractions. The absolute configuration of the prevailing enantiomer has been assigned on the basis of the circular dichroism spectra, as compared with that of the structural analogue (5R)- and (5S)-dihydrodiazepam. The single enantiomers, assayed for their binding to the central nervous system receptor, showed relatively low affinity but significant differences in displacing radioactively labelled flunitrazepam from specific benzodiazepine site. GABA shift experiments allowed the classification of these benzodiazepines as partial agonist or antagonist.

The chromatographic resolution of 7-chloro-1,3-dihydro-1-(1,1-dimethylethyl)-5- (2-fluorophenyl)-2H-1,4-benzodiazepin-2-on (7), the 2'-fluoro, N1-tertbutyl analogue of diazepam, was attained on both analytical and preparative (mgs) scales, by using several chiral stationary phases (CSPs). The stereochemistry of this compound was characterized by means of 1H-NMR Nuclear Overhauser Effect (NOE) analysis. The single enantiomers of 7 were tested for their configuration and stereochemical stability by circular dichroism (CD), and their interaction with the central nervous system (CNS) benzodiazepine receptor was assayed, showing a significant difference in their binding affinities. Protein binding studies with human serum albumin (HSA, the main benzodiazepine carrier in human plasma) immobilized on a silica stationary phase revealed that HSA also preferentially binds one stereoisomer of 7. However, both on line CD detection and stereospecific interaction with other common drugs clearly demonstrated that the stereoselectivity of immobilized HSA for 7 is opposite to that for all the other studied benzodiazepines. In addition, HSA stereoselectivity for 7 is opposite to CNS receptor binding stereoselectivity for the same compound. Such HSA anomalous stereoselectivity for 7 was also confirmed in aqueous buffer solution by competitive displacement studies. Compared to other chiral 1,4-benzodiazepines, compound 7 thus shows several anomalous binding properties: HSA and the CNS receptor demonstrated opposite enantioselective discrimination; HSA has reversed enantioselectivity for compound 7; and HSA stereospecifically binds the low-affinity enantiomer.

A new aryltetralin lignan derivative, 1 , was obtained by reacting dimethyl succinate and piperonal, furnishing the lactone 4-(3′,4′-methylenedioxybenzyl)-4,5-dihydro-2(3H)-furanone, which was reacted once again with piperonal and LDA to give the dibenzylbutirolactone 7-hydroxyhinokinin. The cyclization of 7-hydroxyhinokinin into polygamain occurred in the presence of trifluoroacetic acid. The reduction of the furanic ring of polygamain was done by its reaction with DIBAL in THF, furnishing the diol functionalized lignin derivative 1 as single diastereomer. The enantiomeric fractions of 1 were obtained by preparative enantioselective HPLC. The absolute stereochemistry was assigned by electronic circular dichroism (ECD) and nuclear magnetic resonance (NMR) spectroscopy. An all-trans relative configuration was determined by NMR on the bases of 1 H coupling constants and nuclear Overhauser effect (n.O.e.) experiments. The absolute configuration at C1 was assigned on the basis of the ECD sign at 296 nm by comparison to the ECD spectra of structural analogues with defined stereochemistry. The assignment of the absolute configuration was confirmed by applying the exciton chirality method to the well-defined ECD couplets at 285 and 200 nm allied to the two electronic transitions L b and B b of the aromatic moieties, respectively. Rac- 1 and its enantiomeric isomers were evaluated against important bacteria responsible for dental caries. The best results obtained for the (1R,2S,3S) isomer were against Streptococcus mutans (250 μM), Streptococcus salivarius (250 μM), Streptococcus sobrinus (280 μM) and Streptococcus mitis (280 μM). The (1S,2R,3R) isomer was active only against Streptococcus sanguinis (280 μM). The enantiomeric mixture was less active than the (1R,2S,3S) isomer.

Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2×6 mm i.d.), under a three-solvent gradient elution mode and UV detection at 256 nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples.

5-Fluorouracil (FU) is an important and widely used antineoplastic drug that is carried in the serum by plasma proteins. Protein binding studies of this drug to human serum albumin (HSA) have been carried out by several spectroscopic techniques. Difference circular dichroism and UV studies provided information on the class of binding sites involved in the interaction. In particular, displacement experiments showed that FU has at least one secondary binding site in the coumarin binding area, but does not interact with the benzodiazepine binding area. Binding was also investigated by difference 1H NMR and by measuring the increase in the 19F NMR signal of FU when bound to HSA. Finally, evidence was obtained that chemical acetylation of Lys199 results in a decreased apparent binding affinity constant (nK) for FU. Such a modification is induced under physiological conditions by aspirin.

The performance of four chiral liquid chromatographic columns (Chiralcel OA, OJ, OC, OD) was studied with respect to the enantioseparation of flavanones and some derivatives. The effect of mobile phase composition on the retention time and stereo-selectivity was studied. A good enantioseparation (alpha up to 1.45) was achieved for most of the racemates. Further, the elution order of the enantiomers from the chiral stationary phases has been determined by using a circular dichroism based detection system.

Accepted Manuscript version. The Published Journal Article is available on the Journal of Chromatography A, Volume 1232, pages 128–133 (DOI: https://doi.org/10.1016/j.chroma.2011.10.090). Supplementary Material available free of charge on the article webpage. © 2011. 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 Enantioselective high performance liquid chromatography (HPLC) coupled with a detection system based on the simultaneous measurement of UV absorption and electronic circular dichroism (ECD) allows a complete stereochemical characterization of chiral compounds, once the relationship between sign of the chiroptical properties and absolute configuration is determined. In the present communication, the development of enantioselective HPLC methods for the resolution of a series of fluorinated 2-phenanthrenylpropionic acids (1-6) is reported. Different chiral stationary phases (CSPs) were tested: Chiralcel® OJ, Chiralcel® OD, Chiralpak® AD, (S,S)-Whelk-O® 1, Chirobiotic™ T and α1-acid glycoprotein (AGP). The results allow the application of the methods to a reliable determination of the enantiomeric excess for all the examined compounds; the highest enantioselectivity values were obtained with the Hibar® [(S,S)-Whelk-O® 1] column for some of the examined compounds. In the case of rac-2-(6-fluorophenanthren-1-yl)propionic acid (1), the relationship between circular dichroism and absolute configuration of the enantiomeric fractions was determined by ECD analysis and time-dependent density functional theory (TD-DFT) calculations. The experimental ECD spectrum of the second-eluted fraction of 1 on the Hibar® [(S,S)-Whelk-O® 1] column was found to be in excellent agreement with the theoretical ECD spectrum of (S)-1; therefore, the absolute configuration of the first- and second-eluted enantiomers on the (S,S)-Whelk-O® 1 CSP was assessed as (R) and (S), respectively, and the elution orders of the enantiomeric forms of 1 were determined on all the different CSPs.

5-Imino-1,2,4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3β (GSK-3β) and phosphodiesterase 7 (PDE7) inhibitors were characterized for their ability to pass the blood-brain barrier (BBB) together with their human serum albumin (HSA) binding using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). To study the blood-brain barrier penetration, a parallel artificial membrane permeability assay (PAMPA) using a porcine brain lipid was employed. For the HPLAC investigation, HSA was previously covalently immobilized to the silica matrix of the HPLC column. This HSA-based column was used to characterize the high affinity binding sites of 5-imino-1,2,4-thiadiazoles and quinazolines derivatives to HSA. Displacement experiments in the presence of increasing concentrations of competitors known to bind selectively to the main binding sites of HSA were carried out to determine their possible binding site. The same drug-protein system was studied by CD. The analysed compounds were able to pass BBB, they present good drug-like properties and they showed a high affinity to HSA. Competition experiments showed an anticooperative interaction at sites I and II, and an independent binding at bilirubin binding site on HSA. © 2012 Elsevier B.V. All rights reserved.

Most of the protein therapeutics are now produced by recombinant DNA technology. The advantages of recombinant proteins are related to their higher specificity and to their safety as exposure to animal or human diseases. However, several problems are still present in development of recombinant proteins as therapeutics, such as low bioavailability, short serum half-life, and immune response. Their successful application hinges on the protein stereochemical stability, and on the folding and the tendency to aggregate induced by purification steps and storage. All these aspects determine the failure of many potential protein therapies, and limitations in the development of the formulation. The application of multiple analytical techniques is important in order to obtain a detailed product profile and to understand how manufacturing can influence product structure and activity. Surely the protein conformation is a key aspect to be assessed, because a specific conformation is often essential for the biological function of the protein. Thus, there is a growing need to perform structural studies under the conditions in which the proteins operate, and to monitor the structural changes of the protein. Circular dichroism has been increasingly recognised as a valuable and reliable technique to get this information. In particular, examples will be here reported on the use of circular dichroism spectroscopy in the structural characterization of free and formulated recombinant proteins, looking at the prediction of the secondary structure, propensity to conformational changes, stability, and tendency to aggregate.

Thioridazine (THD) is a commonly prescribed phenotiazine neuroleptic drug, which is extensively biotransformed in the organism producing as main metabolites sulfoxides and a sulfone by sulfur oxidation. Significant differences have been observed in the activity of the THD enantiomers as well as for its main metabolites, and enantioselectivity phenomena have been proved in the metabolic pathway. Here the assignment of the absolute configuration at the sulfur atom of enantiomeric THD-2-sulfoxide (THD-2-SO) has been carried out by circular dichroism (CD) spectroscopy. The stereoisomers were separated by HPLC on Chiralpak AS column, recording the CD spectra for the two collected enantiomeric fractions. The theoretical electronic CD spectrum has been obtained by the TDDFT/B3LYP/6-31G*, as Boltzmann averaging of the contributions calculated for the most stable conformations of the drug. The comparison of the simulated and experimental spectra allowed the absolute configuration at the sulfur atom of the four THD-2-SO stereoisomers to be assigned. The developed method should be useful for a reliable correlation between stereochemistry and activity and/or toxicity.

A series of synthesised tocainide analogues were characterized for their human serum albumin (HSA) binding, using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). The synthesis and physico-chemical characterization of compounds 7a-7d is reported here. For the HPLAC investigation HSA was covalently immobilized to the silica matrix of the HPLC column, using an anchoring procedure, which allows the binding properties of the protein to be maintained. The HSA-based column was used for getting information on the high affinity binding sites of the tocainide analogues to HSA. According to the displacement chromatography approach, the retentions of the analytes were determined in the absence and in the presence of increasing concentrations of competitors known to bind to specific binding sites on the protein. The same system, drug/protein, was investigated in solution by CD. The analysed compounds, proved active as sodium channel blockers, showed a much higher affinity to the serum carrier with respect to the parent compound, tocainide. Further, a non-cooperative interaction at sites I and II, and an almost independent binding at the bilirubin binding site on HSA were hypothesised on the bases of the competition experiments.

A common polymorphism at codon 72 of human TP53 gene determines a proline to arginine aminoacidic substitution within the proline-rich domain of p53 protein. The two resulting isoforms (p53P(72) and p53R(72)) are different from a biochemical and biological point of view and many reports suggest that they can modulate individual cancer susceptibility and overall survival. In the attempt to explain the observed biological differences, we characterized the two isoforms by mass spectrometry and circular dichroism (CD) to evaluate the possible alteration in the secondary structure of p53 introduced by this polymorphism. Recombinant human p53R(72) and p53P(72) were produced by using E. coli expression system then purified by chromatography (affinity chromatography and RP-HPLC), and the whole proteins identified by HPLC-ESI-IT and MALDI-TOF analysis. A bottom-up approach, using both MALDI-TOF and HPLC-ESI-QTOF analysis, was then adopted to obtain the sequence information on the two p53 isoforms. To this purpose, peptide maps were obtained by trypsin proteolysis on the two p53 isoforms. The two isoforms proteolytic digests were separated by LC and subsequent mass spectrometry analysis of both entire and fragmented peptides was performed. In particular, precursor peptide ions obtained by ESI were subjected to collision by the triple quadrupole and TOF separation, allowing us to determine the isoforms aminoacidic peptide sequence by peptide ladder sequencing. Because of the presence of arginine, a selective trypsin proteolytic cleavage at R(72), giving rise to two selective shorter peptides, occurred in p53R(72), but was missing in the case of p53P(72) trypsin digest, in which an uncleaved longer peptide was instead identified. Upon primary structure confirmation, the two p53 isoforms were studied by CD in order to investigate the experimental variables, which affect ordered secondary structure adoption. CD analysis indicated that the two isoforms are not structurally different, thus allowing us to exclude that the observed biological differences can be due to a different conformation of the two isoforms introduced by this polymorphism. Furthermore, these studies establish a mass spectrometry method to identify the two isoforms that can be useful for future interactome studies and cancer drug discovery.

The binding of HIV protease inhibitors, drugs important for anti-HIV chemotherapy, to human serum albumin (HSA) was examined by high-performance affinity chromatography. Frontal analysis was first used to determine the amount of anchored protein and the binding capacity for selected markers on this column. Zonal elution experiments then ranked the HSA bound fraction of the examined compounds. Information on the binding region was obtained by competitive zonal elution experiments using probe compounds with known sites on HSA. An allosteric competition between HIV protease inhibitors (PIs) and valproate (a probe for the bilirubin site) was detected, consistent with a non-cooperative binding mechanism. No significant competition was observed between the examined compounds and salicylate or ibuprofen, probes for sites I and II, respectively. The observations were confirmed by circular dichroism spectroscopy, based on the change in the induced circular dichroism signals of selected markers for the main binding sites of HSA when ritonavir was added as the competitor. These results were in good agreement with previous literature reports and provide more details on how PIs are transported in plasma and how they may compete with other drugs in the body.

A circular dichroism-based detection system presents several advantages in the HPLC analysis of chiral compounds because of the selective monitoring of optically active molecules. Its use allows reliable determination of enantiomeric excesses and elution order. To this end, the application of empirical, semiempirical, and nonempirical methods to get stereochemical information from the CD signal is reported. Furthermore, recording the CD spectra on line and evaluation of the dissymetry factor make the CD detection very powerful in characterizing the stereochemistry of chiral eluates.

The stereochemistry of an achiral (Diazepam) and two chiral (3-methyl and 3-succinyloxy substituted) 1,4-benzodiazepin-2-ones interacting with human serum albumin (HSA) has been investigated by making use of difference absorption (UV) and circular dichroism (CD) spectroscopies. Evidence is obtained for a higher affinity with HSA for one of the two possible conformations of the seven-membered benzodiazepine ring. The red shift revealed by the absorption difference spectrum between the free and the bound drug accounts for the CD difference spectra observed.

The initial transition of amyloid beta (1-42) (Abeta42) soluble monomers/small oligomers from unordered/alpha-helix to a beta-sheet-rich conformation represents a suitable target to design new potent inhibitors and to obtain effective therapeutics for Alzheimer's disease. Under optimized conditions, this reliable and reproducible CD kinetic study showed a three-step sigmoid profile that was characterized by a lag phase (prevailing unordered/alpha-helix conformation), an exponential growth phase (increasing beta-sheet secondary structure) and a plateau phase (prevailing beta-sheet secondary structure). This kinetic analysis brought insight into the inhibitors' mechanism of action. In fact, an increase in the duration of the lag phase can be related to the formation of an inhibitor-Abeta complex, in which the non-amyloidogenic conformation is stabilized. When the exponential rate is affected exclusively, such as in the case of Congo red and tetracycline, then the inhibitor affinity might be higher for the pleated beta-sheet structure. Finally, by adding the inhibitor at the end of the exponential phase, the soluble protofibrils can be disrupted and the Abeta amyloidogenic structure can revert into monomers/small oligomers. Congo red and tetracycline preferentially bind to amyloid in the beta-sheet conformation because both decreased the slope of the exponential growth, even if to a different extent, whereas no effect was observed for tacrine and galantamine. Some very preliminary indications can be derived about the structural requirements for binding to nonamyloidogenic or beta-sheet amyloid secondary structure for the development of potent antiaggregating agents. On these premises, memoquin, a multifunctional molecule that was designed to become a drug candidate for the treatment of Alzheimer's disease, was investigated under the reported circular dichroism assay and its anti-amyloidogenic mechanism of action was elucidated.

Background: rac-Fenoterol is a β2-adrenoceptor agonist (β2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R′- and S,S′-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the β2-AR. Methods: R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. β2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human β2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. Results: Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound β2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 ± 55 nM (R-F) and 109,000 ± 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 ± 11.6)% to (306 ± 11.8)% of resting cell length (P < 0.05) and reduced EC50 from −7.0 ± 0.270 to −7.1 ± 0.2 log[M] (P < 0.05), while S-F had no significant effect. Discussion: Previous studies have shown that rac-fenoterol acts as an apparent β2-AR/Gs selective agonist and fully restores diminished β2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for β2-AR/Gs activation and if it can be used in the treatment of congestive heart failure. Chirality, 2006. Published 2006 Wiley-Liss, Inc.

The drug binding to plasma and tissue proteins are fundamental factors in determining the overall pharmacological activity of a drug. Human serum albumin (HSA), together with alpha1-acid glycoprotein (AGP), are the most important plasma proteins, which act as drug carriers, with drug pharmacokinetic implications, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This review focuses on the combination of biochromatography and circular dichroism as an effective approach for the characterization of albumin binding sites and their enantioselectivity. Furthermore, their applications to the study of changes in the binding properties of the protein arising by the reversible or covalent binding of drugs are discussed, and examples of physiological relevance reported. Perspectives of these studies reside in supporting the development of new drugs, which require miniaturization to facilitate the screening of classes of compounds for their binding to the target protein, and a deeper characterization of the mechanisms involved in the molecular recognition processes.

The binding to human serum albumin (HSA) of the antitumoural drug paclitaxel and of several structural analogues has been characterized by bioaffinity chromatography and circular dichroism. A ranking of the taxanes was obtained for their affinity to the protein by measuring their retention times on an albumin chromatographic column. This also allowed the calculation of the drug bound percentage. Affinity resulted significantly affected by the nature of the isoserinic side chain, the presence of the 1,14-carbonate moiety and the substituent at C-7, showing that the hydrophobicity of the drug is fundamental in the binding process. The analysis demonstrated that the organic solvent highly alters the interaction mechanism of taxanes to the protein and so the affinity results. Circular dichroism experiments supported this hypothesis. Furthermore, taxanes binding to the serum carrier was characterized by displacement chromatography, by adding into the mobile phase selected competitors, (S)-ibuprofen and valproic acid, that are known to bind to specific binding sites on HSA. These experiments established a non-cooperative binding mechanism.