Identification of small molecules affecting the interaction between human hemoglobin and Staphylococcus aureus IsdB hemophore.

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein-protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico/in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.

ITC study on the interaction of some bile salts with tragacanth, Arabic, and guar gums with potential cholesterol-lowering ability.

Introduction: The urge of designing new safe and natural functional foods to control blood lipids and dispensable without the need of physician supervision, has increased especially after the coming into effect of the recent EU Commission regulation 2022/860, that regulates the consumption of "red yeast rice," made by fermentation of rice with Monascus purpureus, and perceived as a natural functional food, due to a health risk for frail consumers. The results of the present work are a part of the systematic study we are carrying out of the binding ability of some soluble dietary fibers (SDF) from different natural sources toward selected bile salts (BS). Methods: Measurements were carried out by isothermal titration calorimetry (ITC) with the idea to shed light on the mechanism, if any, by which they show cholesterol-lowering activity. Results and discussion: Epidemiological studies are sometimes conflicting and offer only hypothesis about the mechanism of action, the most accredited being the reduction of reabsorption of BS in the gut. Previous measurements done on negatively charged pectin and alginate, showed specific binding interaction with monomer NaDC for pectin and no interaction at all for alginate. Chitosan, positively charged and soluble only at low pH, in 100 mM acetate buffer at pH = 3 shows strong exothermic interactions with NaTC and NaTDC. Here we considered two plant exudates (Arabic gum and tragacanth gum) and guar gum, extracted from guar beans, and their interaction with the same bile salts. ITC measurements do not evidence specific interactions between gums and the studied BS, so that their cholesterol lowering ability, if any, is due to a different mechanism very probably bound to the viscosity increase. Moreover, the addition of NaC, the most abundant BS in the bile, at very low concentration (under the cmc) causes a structural change of the solution. The obtained results seem to corroborate the hypothesis that the cholesterol lowering activity is related to the increase in viscosity of guar solution favored by NaC, the major component of the bile.

Chemico-Physical Properties of Some 1,1'-Bis-alkyl-2,2'-hexane-1,6-diyl-bispyridinium Chlorides Hydrogenated and Partially Fluorinated for Gene Delivery.

The development of very efficient and safe non-viral vectors, constituted mainly by cationic lipids bearing multiple charges, is a landmark for in vivo gene-based medicine. To understand the effect of the hydrophobic chain's length, we here report the synthesis, and the chemico-physical and biological characterization, of a new term of the homologous series of hydrogenated gemini bispyridinium surfactants, the 1,1'-bis-dodecyl-2,2'-hexane-1,6-diyl-bispyridinium chloride (GP12_6). Moreover, we have collected and compared the thermodynamic micellization parameters (cmc, changes in enthalpy, free energy, and entropy of micellization) obtained by isothermal titration calorimetry (ITC) experiments for hydrogenated surfactants GP12_6 and GP16_6, and for the partially fluorinated ones, FGPn (where n is the spacer length). The data obtained for GP12_6 by EMSA, MTT, transient transfection assays, and AFM imaging show that in this class of compounds, the gene delivery ability strictly depends on the spacer length but barely on the hydrophobic tail length. CD spectra have been shown to be a useful tool to verify the formation of lipoplexes due to the presence of a "tail" in the 288-320 nm region attributed to a chiroptical feature named ψ-phase. Ellipsometric measurements suggest that FGP6 and FGP8 (showing a very interesting gene delivery activity, when formulated with DOPE) act in a very similar way, and dissimilar from FGP4, exactly as in the case of transfection, and confirm the hypothesis suggested by previously obtained thermodynamic data about the requirement of a proper length of the spacer to allow the molecule to form a sort of molecular tong able to intercalate DNA.

On the mechanism of the cholesterol lowering ability of soluble dietary fibers: Interaction of some bile salts with pectin, alginate, and chitosan studied by isothermal titration calorimetry.

Reducing high blood cholesterol is an important strategy to decrease the chances of a cardiovascular disease occurrence, the main cause of mortality in western developed countries. Therefore, the search for an alternative therapeutic or preventive approach being natural, biocompatible, and not toxic is still more relevant than ever. This need is particularly felt in Pediatrics for treating childhood hypercholesterolemia, due to statins interference in the production of steroid hormones in prepuberal children. Notwithstanding the general acceptance of the healthy role of the fibers in the diet, the mechanism underlying the cholesterol-lowering ability of soluble fibers is still under discussion. Therefore, we started a systematic study of the binding ability of some soluble dietary fibers (SDF) originated from different natural sources toward selected bile salts (BS) by isothermal titration calorimetry (ITC). Here we report the results of our ITC studies on the interaction of alginate, pectin and chitosan with sodium cholate (NaC), sodium deoxycholate (NaDC), sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC). Thermodynamic data on the micelle formation process of the above bile salts, as a premise to the study of their binding ability to the SDF, are also reported. Alginate does not show specific binding interaction with BS, while pectin shows a strong exothermic bond with NaDC in monomeric form. Chitosan, positively charged and soluble only at low pH, shows strong exothermic interactions with NaTC and NaTDC (soluble at pH = 3 in acetate buffer) with precipitate formation. For NaTC, the exothermic peak starts at about 5 mM. At this concentration NaTC bound on the fiber reaches locally the cmc value and micelles start forming on the fiber inducing its conformational change. For NaTDC the same process occurs at much lower concentrations, due to lower cmc, and with a greater quantity of heat involved. The first set of results here presented shows that for some SDF the binding of BS could be an important mechanism in cholesterol lowering but not the only one. The information here presented could be a starting point for the design of optimized functional foods with high cholesterol lowering ability.

Human Serine Racemase Weakly Binds the Third PDZ Domain of PSD-95.

Human serine racemase (hSR) is a pyridoxal-5'-phosphate (PLP)-dependent dimer that catalyzes the formation of D-serine from L-serine, as well as the dehydration of both L- and D-serine to pyruvate and ammonia. As D-serine is a co-agonist of N-methyl-D-aspartate receptors (NMDARs), hSR is a key enzyme in glutamatergic neurotransmission. hSR activity is finely regulated by Mg2+, ATP, post-translational modifications, and the interaction with protein partners. In particular, the C-terminus of murine SR binds the third PDZ domain (PDZ3) of postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase (MAGUK) family involved in the trafficking and localization of glutamate receptors. The structural details of the interaction and the stability of the complex have not been elucidated yet. We evaluated the binding of recombinant human PSD-95 PDZ3 to hSR by glutaraldehyde cross-linking, pull-down assays, isothermal titration calorimetry, nuclear magnetic resonance, and enzymatic assays. Overall, a weak interaction was observed, confirming the binding for the human orthologs but supporting the hypothesis that a third protein partner (i.e., stargazin) is required for the regulation of hSR activity by PSD-95 and to stabilize their interaction.

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

Statistical Inference for Ergodic Algorithmic Model (EAM), Applied to Hydrophobic Hydration Processes.

The thermodynamic properties of hydrophobic hydration processes can be represented in probability space by a Dual-Structure Partition Function {DS-PF} = {M-PF} · {T-PF}, which is the product of a Motive Partition Function {M-PF} multiplied by a Thermal Partition Function {T-PF}. By development of {DS-PF}, parabolic binding potential functions α) RlnKdual = (-ΔG°dual/T) ={f(1/T)*g(T)} and ß) RTlnKdual = (-ΔG°dual) = {f(T)*g(lnT)} have been calculated. The resulting binding functions are "convoluted" functions dependent on the reciprocal interactions between the primary function f(1/T) or f(T) with the secondary function g(T) or g(lnT), respectively. The binding potential functions carry the essential thermodynamic information elements of each system. The analysis of the binding potential functions experimentally determined at different temperatures by means of the Thermal Equivalent Dilution (TED) principle has made possible the evaluation, for each compound, of the pseudo-stoichiometric coefficient ±ξw, from the curvature of the binding potential functions. The positive value indicates convex binding functions (Class A), whereas the negative value indicates concave binding function (Class B). All the information elements concern sets of compounds that are very different from one set to another, in molecular dimension, in chemical function, and in aggregation state. Notwithstanding the differences between, surprising equal unitary values of niche (cavity) formation in Class A <Δhfor>A = -22.7 ± 0.7 kJ·mol-1·ξw-1 sets with standard deviation σ = ±3.1% and <Δsfor>A = -445 ± 3J·K-1·mol-1·ξw-1J·K-1·mol-1·ξw-1 with standard deviation σ = ±0.7%. Other surprising similarities have been found, demonstrating that all the data analyzed belong to the same normal statistical population. The Ergodic Algorithmic Model (EAM) has been applied to the analysis of important classes of reactions, such as thermal and chemical denaturation, denaturation of proteins, iceberg formation or reduction, hydrophobic bonding, and null thermal free energy. The statistical analysis of errors has shown that EAM has a general validity, well beyond the limits of our experiments. Specifically, the properties of hydrophobic hydration processes as biphasic systems generating convoluted binding potential functions, with water as the implicit solvent, hold for all biochemical and biological solutions, on the ground that they also are necessarily diluted solutions, statistically validated.

Hydrophobic Hydration Processes: Intensity Entropy and Null Thermal Free Energy and Density Entropy and Motive Free Energy.

The processes at the molecule level, which are the source of the ergodic properties of thermodynamic systems, are analyzed with special reference to entropy. The entropy change produced by increasing the temperature T depends on the increase of velocity of the particles with a decrease of the squared mean sojourn time (τm 2) and gradual loss of instant energy intensity. The diminution, which is due to dilution, of the number of terms in the summation of cumulative sojourn time (τi 2)Σ produces loss of energy density, thus generating a gradual increase of density entropy, dS Dens. The ergodic property of thermodynamic systems consists of the equivalence of density entropy (dependent on dilution) with intensity entropy (dependent on temperature). This equivalence has been experimentally verified in every hydrophobic hydration process as thermal equivalent dilution. An ergodic dual-structure partition function {DS-PF} represents the state probability of every hydrophobic hydration process, corresponding to the biphasic composition of these systems. The dual-structure partition function {DS-PF} (K mot·Î¶th) is the product of a motive partition function {M-PF} (K mot) multiplied by a thermal partition function {T-PF} (ζth = 1). {M-PF} gives rise to changes of density entropy, whereas {T-PF} gives rise to changes of intensity entropy. {M-PF} is referred to a reacting mole ensemble (reacting solute) composed of few elements (moles), ruled by binomial distribution, whereas {T-PF} is referred to a nonreacting molecule ensemble (NoremE) (nonreacting solvent), which is composed of a very large population of elements (molecules), ruled by Boltzmann statistics. Statistical thermodynamic methods cannot be applied to {M-PF} that can be calculated by numerical methods from the experimental titration data. By development of the dual-structure partition function {DS-PF}, parabolic convoluted binding functions are obtained. The tangents to the binding functions represent the dual enthalpy, -ΔH dual = (-ΔH mot - ΔH th), and the dual entropy, ΔS dual = (ΔS mot + ΔS th). The connections between canonical and grand-canonical partition functions of statistical thermodynamics with thermal and motive partition functions of chemical thermodynamics, respectively, are discussed. Special attention has been devoted to the equality ΔH th/T + ΔS th = 0, typical of NoremEs, as an entropy-enthalpy compensation with ΔG th/T = 0. The thermodynamic potential change Δµ, as proposed by potential distribution theorem (PDT) for iceberg formation from {T-PF} of the solvent, is nonexistent because the excess solvent is at a constant potential (Δµsolv = 0). The information level offered by the ergodic algorithmic model (EAM) is more complete and correct than that offered by the potential distribution theorem (PDT): the stoichiometry of the water reaction in hydrophobic hydration processes is determined by the EAM as the function of the number ±ξw. Quasi-chemical approximation, renamed the chemical molecule/mole scaling function (Che. m/M. sF), is a fundamental breakthrough in the application of statistical thermodynamics to chemical reactions. Boltzmann statistical molecule distribution of the thermal partition function {T-PF} is scaled with binomial mole distribution of the motive partition function {M-PF}. For computer-assisted drug design, the alternative calculation procedure of Talhout, based on the previous experimental determination of binding functions, is recommended. The ergodic algorithmic model (EAM), applied to the experimental convoluted binding functions, can recover the distinct terms of intensity entropy (ΔH mot/T) and density entropy (ΔS mot), together with other essential information elements, lost by computer simulations.

Hydrophobic Hydration Processes. I: Dual-Structure Partition Function for Biphasic Aqueous Systems.

The thermodynamic properties of hydrophobic hydration processes have been analyzed and assessed. The thermodynamic binding functions result to be related to each other by the mathematical relationships of an ergodic algorithmic model (EAM). The active dilution d A of species A in solution is expressed as d A = 1/(Φ·x A) with thermal factor Φ = T -(C p,A/R) and (1/x A) = d id(A), where d id(A) = ideal dilution. Entropy function is set as S = f(d id(A),T). Thermal change of entropy (i.e., entropy intensity change) is represented by the equation (dS) d = C p dln T. Configuration change of entropy (i.e., entropy density change) is represented by the equation (dS)T = (-R dln x A)T = (R dln d id(A)) T . Because every logarithmic function in thermodynamic space corresponds to an exponential function in probability space, the sum functions ΔH dual = (ΔH mot + ΔH th) and ΔS dual = (ΔS mot + ΔS th) of the thermodynamic space give birth, in exponential probability space, to a dual-structure partition function { DS-PF }: exp(-ΔG dual/RT) = K dual = (K mot·Î¶th) = {(exp(-ΔH mot/RT))(exp(ΔS mot/R))}·{exp(-ΔH th/RT) exp(ΔS th/R)}. Every hydrophobic hydration process can be represented by { DS-PF } = { M-PF }·{ T-PF }, indicating biphasic systems. { M-PF } = f(T,d id(A)), concerning the solute, is monocentric and produces changes of entropy density, contributing to free energy -ΔG mot, whereas { T-PF } = g(T), concerning the solvent, produces changes of entropy intensity, not contributing to free energy. Entropy density and entropy intensity are equivalent and summed with each other (i.e., they are ergodic). From the dual-structure partition function { DS-PF }, the ergodic algorithmic model (EAM) can be developed. The model EAM consists of a set of mathematical relationships, generating parabolic convoluted binding functions R ln K dual = -ΔG dual/T = {f(1/T)*g(T)} and RT ln K dual = -ΔG dual = {f(T)*g(ln T)}. The first function in each convoluted couple f(1/T) or f(T) is generated by { M-PF }, whereas the second function, g(T) or g(ln T), respectively, is generated by { T-PF }. The mathematical properties of the thermodynamic functions of hydrophobic hydration processes, experimentally determined, correspond to the geometrical properties of parabolas, with constant curvature amplitude C ampl = 0.7071/ΔC p,hydr. The dual structure of the partition function conforms to the biphasic composition of every hydrophobic hydration solution, consisting of a diluted solution, with solvent in excess at constant potential.

Nonviral gene-delivery by highly fluorinated gemini bispyridinium surfactant-based DNA nanoparticles.

Biological and thermodynamic properties of a new homologous series of highly fluorinated bispyridinium cationic gemini surfactants, differing in the length of the spacer bridging the pyridinium polar heads in 1,1' position, are reported for the first time. Interestingly, gene delivery ability is closely associated with the spacer length due to a structural change of the molecule in solution. This conformation change is allowed when the spacer reaches the right length, and it is suggested by the trends of the apparent and partial molar enthalpies vs molality. To assess the compounds' biological activity, they were tested with an agarose gel electrophoresis mobility shift assay (EMSA), MTT proliferation assay and Transient Transfection assays on a human rhabdomyosarcoma cell line. Data from atomic force microscopy (AFM) allow for morphological characterization of DNA nanoparticles. Dilution enthalpies, measured at 298K, enabled the determination of apparent and partial molar enthalpies vs molality. All tested compounds (except that with the longest spacer), at different levels, can deliver the plasmid when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE). The compound with a spacer formed by eight carbon atoms gives rise to a gene delivery ability that is comparable to that of the commercial reagent. The compound with the longest spacer compacts DNA in loosely condensed structures by forming bows, which are not suitable for transfection. Regarding the compounds' hydrogenated counterparts, the tight relationship between the solution thermodynamics data and their biological performance is amazing, making "old" methods the foundation to deeply understanding "new" applications.

Synthesis, Physicochemical Characterization, and Interaction with DNA of Long-Alkyl-Chain Gemini Pyridinium Surfactants.

Pyridinium gemini surfactants with hexadecyl chains linked to nitrogen atoms and a tuned aliphatic spacer that bridges the two pyridinium polar heads in 2,2'-positions have been synthesized and characterized. A multitechnique approach allowed us to study the aggregation behavior, using conductivity, surface tension, and fluorescence. Graphs of the specific conductivity (κ) versus the surfactant molar concentration (C), and graphs of the molar conductivity (Λ) versus C0.5 suggest pre-aggregation phenomena of these amphiphiles at very low concentration. The trends of Amin as a function of the spacer length confirm the hypothesis of a conformational change of the molecule with four methylene groups as spacer owing to stacking interactions between the two pyridinium rings mediated by the counterion. Moreover, the trends of Amin and counterion binding (ß) suggest that the spacer must be longer than eight carbon atoms to fold efficiently toward the micellar core. The opportunity to tune the surfactant structure and aggregation properties make those surfactants-particularly the long-chain ones for which the DNA complexing ability was shown by means of atomic force microscopy (AFM) imaging-desirable candidates for gene-delivery experiments.

Nonviral gene delivery: gemini bispyridinium surfactant-based DNA nanoparticles.

The interaction with a model membrane, the formation of DNA nanoparticles, and the transfection ability of a homologous series of bispyridinium dihexadecyl cationic gemini surfactants, differing in the length of the alkyl spacer bridging the two pyridinium polar heads in the 1 and 1' positions (P16-n with n = 3, 4, 8, 12), have been studied by means of differential scanning calorimetry (DSC), atomic force microscopy, electrophoresis mobility shift assay, and transient transfection assay measurements. The results presented here show that their performance in gene delivery is strictly related to their structure in solution. For the first time the different transfection activities of the compounds can be explained by referring to their thermodynamic properties in solution, previously studied. The compound with a spacer formed by four carbon atoms, showing unexpected enthalpic properties vs concentration in solution, is the only one giving rise to a transfection activity comparable to that of the commercial reagent, when formulated with L-α-dioleoylphosphatidylethanolamine. We suggest that P16-4 behaves like molecular tongs able to grip basic groups near each other, allowing the formation of compact and nearly spherical DNA particles. The compound with the longest spacer gives rise to loosely condensed structures by forming a sort of bow, not able to give rise to transfection notwithstanding the double positive charge of the molecule. On the other hand, DSC measurements on synthetic membranes show that the compounds with the shortest spacers (three and four methylene groups) practically do not interact with the 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine membrane, while compounds P16-8 and, particularly, P16-12 induce the formation of surfactant-rich and surfactant-poor domains in the membrane, without showing any peculiarity for compound P16-4. This could suggest that the mechanisms involved in the interaction with the model membrane and in gene delivery are substantially different and could strike a blow for an endocytosis mechanism for the internalization in the cell of the DNA nanoparticles.

Antiretroviral activity of metal-chelating HIV-1 integrase inhibitors.

Data regarding the activity of metal complexes against HIV virus in cell are surprisingly scarce. In this study, we present the antiviral activity against HIV-infected cells of different types of chelating ligands and of their metal complexes. In particular, the carboxamide chelating scaffold and the corresponding coordination compounds demonstrated an interesting antiviral profile in the nanomolar range. These molecules inhibit not only HIV integrase catalytic activity, but they also interfere with the function of the RNase H component of the HIV reverse transcriptase. Here we also discuss the thermodynamic characterization in solution of the metal complexes of the most active ligands, affording to the best of our knowledge for the first time this type of data for complexes with anti-HIV activity.

Diketoacid chelating ligands as dual inhibitors of HIV-1 integration process.

HIV-1 Integrase (IN) represents a very attractive pharmacological target for the development of new and more efficient drugs. Recently, an allosteric inhibitory approach also emerged, that targets the interaction between IN and cellular cofactors, such as LEDGF/p75. Small molecules based on the diketoacid pharmachophore were studied for their ability to inhibit at the same time integration and IN-LEDGF/p75 interaction (dual inhibitors): in this study, we evaluated three indole diketoacid derivatives and their magnesium(II) complexes for their ability to act as dual inhibitors. Effectively, the metal complexes exhibited IN inhibition potency in low nanomolar/micromolar concentration range; both the complexes and the free ligands are also able to inhibit the IN-LEDGF/p75 interaction at low µM values. Moreover, these magnesium compounds showed good antiviral activity, suggesting the possibility to exploit metal coordination for the design of new antivirals.

Metal-chelating 2-hydroxyphenyl amide pharmacophore for inhibition of influenza virus endonuclease.

The influenza virus PA endonuclease is an attractive target for development of novel anti-influenza virus therapeutics. Reported PA inhibitors chelate the divalent metal ion(s) in the enzyme's catalytic site, which is located in the N-terminal part of PA (PA-Nter). In this work, a series of 2-hydroxybenzamide-based compounds have been synthesized and biologically evaluated in order to identify the essential pharmacophoric motif, which could be involved in functional sequestration of the metal ions (probably Mg(2+)) in the catalytic site of PA. By using HL(1), H2L(2), and HL(3) as model ligands with Mg(2+) ions, we isolated and fully characterized a series of complexes and tested them for inhibitory activity toward PA-Nter endonuclease. H2L(2) and the corresponding Mg(2+) complex showed an interesting inhibition of the endonuclease activity. The crystal structures of the uncomplexed HL(1) and H2L(2) and of the isolated magnesium complex [Mg(L(3))2(MeOH)2]·2MeOH were solved by X-ray diffraction analysis. Furthermore, the speciation models for HL(1), H2L(2), and HL(3) with Mg(2+) were obtained, and the formation constants of the complexes were measured. Preliminary docking calculations were conducted to investigate the interactions of the title compounds with essential amino acids in the PA-Nter active site. These findings supported the "two-metal" coordination of divalent ions by a donor triad atoms chemotype as a powerful strategy to develop more potent PA endonuclease inhibitors.

Investigating the role of metal chelation in HIV-1 integrase strand transfer inhibitors.

HIV-1 integrase (IN) has been validated as an attractive target for the treatment of HIV/AIDS. Several studies have confirmed that the metal binding function is a crucial feature in many of the reported IN inhibitors. To provide new insights on the metal chelating mechanism of IN inhibitors, we prepared a series of metal complexes of two ligands (HL1 and HL2), designed as representative models of the clinically used compounds raltegravir and elvitegravir. Potentiometric measurements were conducted for HL2 in the presence of Mg(II), Mn(II), Co(II), and Zn(II) in order to delineate a metal speciation model. We also determined the X-ray structures of both of the ligands and of three representative metal complexes. Our results support the hypothesis that several selective strand transfer inhibitors preferentially chelate one cation in solution and that the metal complexes can interact with the active site of the enzyme.

HIV-1 IN strand transfer chelating inhibitors: a focus on metal binding.

Most active and selective strand transfer HIV-1 integrase (IN) inhibitors contain chelating functional groups that are crucial feature for the inhibition of the catalytic activities of the enzyme. In particular, diketo acids and their derivatives can coordinate one or two metal ions within the catalytic core of the enzyme. The present work is intended as a contribution to elucidate the mechanism of action of the HIV-IN inhibitors by studying the coordinative features of H2L¹ (L-708,906), an important member of the diketo acids family of inhibitors, and H2L2, a model for S-1360, another potent IN inhibitor. Magnesium(II) and manganese(II) complexes of H2L¹ and H2L² were isolated and fully characterized in solution and in the solid state. The crystal structures of the manganese complex [Mn(HL2)2(CH3OH)2]·2CH3OH were solved by X-ray diffraction analysis. Moreover, the speciation models for H2L2 with magnesium(II) and manganese(II) ions were performed and the formation constants of the complexes were measured. M(HL2)2 (M = Mg²+, Mn²+) was the most abundant species in solution at physiological pH. All the synthesized compounds were tested for their anti-IN activity, showing good results both for the ligand and the corresponding complexes. From analysis of the speciation models and of the biological data we can conclude that coordination of both metal cofactors could not be strictly necessary and that inhibitors can act as complexes and not only as free ligands.

Synthesis and properties of cationic surfactants with tuned hydrophylicity.

A series of pyridinium-based cationic surfactants has been synthesised and their amphiphilic properties have been studied by conductivity and surface tension measurements. The modification of the substitution pattern on the pyridinium ring by hydrophobic moieties (methyl vs. hydrogen and presence or not of condensed benzene ring) gave the opportunity to investigate structure-activity relationships. Characterization by conductivity and surface tension measurements shed light on the behaviour at the air/water interface and in the micellar environment. In particular, the tendency to form ion pairs at very low concentration was evidenced for all the surfactants substituted on the ring, but not for the simple pyridinium ones. The formation of ion pairs affects both the conductivity and the surface tension plots, showing that a series of steps is involved during the adsorption to the air/water surface. An attempt was made to qualify the single steps in the adsorption at the surface layer. Those steps were attributed to different chemical species (free surfactant ions or ion pairs) and to different arrangements of the surfactant. This work also represents a contribution of investigation at very low surfactant concentrations and high surface tension values.

From ligand to complexes. Part 2. Remarks on human immunodeficiency virus type 1 integrase inhibition by beta-diketo acid metal complexes.

Previously, we synthesized a series of beta-diketo acid metal complexes as novel HIV-1 integrase (IN) inhibitors (J. Med. Chem. 2006, 46, 4248-4260). Herein, a further extension of this study is reported. First, detailed docking studies were performed in order to investigate the mode of binding in the active site of the free ligands and of their metal complexes. Second, a series of potentiometric measurements were conducted for two diketo acids chosen as model ligands, with Mn(2+) and Ca(2+), in order to outline a speciation model. Third, we designed and synthesized a new set of complexes with different stoichiometries and tested them in an in vitro assay specific for IN. Finally, we obtained the first X-ray structure of a metal complex with HIV-1 IN inhibition activity. Analysis of these results supports the hypothesis that the diketo acids could act as complexes and form complexes with the metal ions on the active site of the enzyme.

Unusual behavior of the aqueous solutions of gemini bispyridinium surfactants: apparent and partial molar enthalpies of the dimethanesulfonates.

Apparent and partial molar enthalpies at 298 K of the aqueous solutions of cationic gemini surfactants 1,1'-didodecyl-2,2'-dimethylenebispyridinium dimethanesulfonate (12-Py(2)-2-(2)Py-12 MS); 1,1'-didodecyl-2,2'-trimethylenebispyridinium dimethanesulfonate (12-Py(2)-3-(2)Py-12 MS); 1,1'-didodecyl-2,2'-tetramethylenebispyridinium dimethanesulfonate (12-Py(2)-4-(2)Py-12 MS); 1,1'-didodecyl-2,2'-octamethylenebispyridinium dimethanesulfonate (12-Py(2)-8-(2)Py-12 MS); 1,1'-didodecyl-2,2'-dodecamethylenebispyridinium dimethanesulfonate (12-Py(2)-12-(2)Py-12 MS) were measured as a function of concentration and are here reported for the first time. They show a very peculiar behavior as a function of the spacer length, not allowing for the determination of a -CH 2- group contribution when this group is added to the spacer. The curve of the compound with a four-carbon-atom-long spacer lies between those of the compound with a spacer of 2 and 3 carbon atoms, instead of that below the latter, as expected. This surprising behavior, never found before in the literature and different from that found for the more popular m- s- m-type bisquaternary ammonium gemini surfactants, could be explained by a conformation change of the molecule, caused by stacking interactions between the two pyridinium rings, mediated by the counterion and appearing at an optimum length of the spacer. The hypothesis is also supported by the data obtained from the surface tension vs log c curves, showing that A min, the minimum area taken at the air-water interface by the molecule, is significantly lower for 12-Py(2)-4-(2)Py-12 MS than that of the other compounds of the same homologous series, and that the same compound has a greater tendency to form micelles instead of adsorbing at the air/water interface. The evaluation of the micellization enthalpies, by means of a pseudophase transition model, agrees with the exposed trends. These results confirm the great crop of information that can be derived from the study of the solution thermodynamics of aggregate systems and in particular from the curves of apparent and molar enthalpies vs concentration.