High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

Antibacterial Properties of Mesoporous Silica Nanoparticles Modified with Fluoroquinolones and Copper or Silver Species.

Antibiotic resistance is a global problem and bacterial biofilms contribute to its development. In this context, this study aimed to perform the synthesis and characterization of seven materials based on silica mesoporous nanoparticles functionalized with three types of fluoroquinolones, along with Cu2+ or Ag+ species to evaluate the antibacterial properties against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa, including clinical and multi-drug-resistant strains of S. aureus and P. aeruginosa. In addition, in order to obtain an effective material to promote wound healing, a well-known proliferative agent, phenytoin sodium, was adsorbed onto one of the silver-functionalized materials. Furthermore, biofilm studies and the generation of reactive oxygen species (ROS) were also carried out to determine the antibacterial potential of the synthesized materials. In this sense, the Cu2+ materials showed antibacterial activity against S. aureus and E. coli, potentially due to increased ROS generation (up to 3 times), whereas the Ag+ materials exhibited a broader spectrum of activity, even inhibiting clinical strains of MRSA and P. aeruginosa. In particular, the Ag+ material with phenytoin sodium showed the ability to reduce biofilm development by up to 55% and inhibit bacterial growth in a "wound-like medium" by up to 89.33%.

Dual Anticancer and Antibacterial Properties of Silica-Based Theranostic Nanomaterials Functionalized with Coumarin343, Folic Acid and a Cytotoxic Organotin(IV) Metallodrug.

Five different silica nanoparticles functionalized with vitamin B12, a derivative of coumarin found in green plants and a minimum content of an organotin(IV) fragment (1-MSN-Sn, 2-MSN-Sn, 2-SBA-Sn, 2-FSPm-Sn and 2-FSPs-Sn), were identified as excellent anticancer agents against triple negative breast cancer, one of the most diagnosed and aggressive cancerous tumors, with very poor prognosis. Notably, compound 2-MSN-Sn shows selectivity for cancer cells and excellent luminescent properties detectable by imaging techniques once internalized. The same compound is also able to interact with and nearly eradicate biofilms of Staphylococcus aureus, the most common bacteria isolated from chronic wounds and burns, whose treatment is a clinical challenge. 2-MSN-Sn is efficiently internalized by bacteria in a biofilm state and destroys the latter through reactive oxygen species (ROS) generation. Its internalization by bacteria was also efficiently monitored by fluorescence imaging. Since silica nanoparticles are particularly suitable for oral or topical administration, and considering both its anticancer and antibacterial activity, 2-MSN-Sn represents a new dual-condition theranostic agent, based primarily on natural products or their derivatives and with only a minimum amount of a novel metallodrug.

Hybrid Nanosystems Based on Nicotinate-Functionalized Mesoporous Silica and Silver Chloride Nanoparticles Loaded with Phenytoin for Preventing Pseudomonas aeruginosa Biofilm Development.

Pseudomonas aeruginosa (PA) is one of the most common bacteria isolated from chronic wounds and burns. Its treatment is a challenge due to antimicrobial drug resistance and biofilm formation. In this context, this study aimed to perform the synthesis and full characterization of hybrid nanosystems based on mesoporous silica nanoparticles (MSNs) functionalized with a nicotinic ligand and silver chloride nanoparticles, both phenytoin sodium (Ph)-loaded and unloaded, to evaluate the antibacterial properties against three different strains of PA (including two clinical strains) in a planktonic state and as biofilms. Ph is a well-known proliferative agent, which was incorporated into the hybrid nanomaterials to obtain an effective material for tissue healing and prevention of infection caused by PA. The Ph-loaded materials promoted a quasi-complete inhibition of bacterial growth in wound-like medium and biofilm development, with values of 99% and 96%, respectively, with selectivity indices above the requirements for drugs to become promising agents for the topic preventive treatment of chronic wounds and burns.

Combined experimental and theoretical investigation on the magnetic properties derived from the coordination of 6-methyl-2-oxonicotinate to 3d-metal ions.

Five new compounds are reported herein starting from 2-hydroxy-6-methylnicotinic acid (H2h6mnic) and first-row transition metal ions, although H2h6mnic shows a prototropy in solution to lead to the 6-methyl-2-oxonicotinate (6m2onic) ligand that is the molecule eventually present in the compounds. The structural and chemical characterization reveals the following chemical formulae: {[MnNa(µ3-6m2onic)2(µ-6m2onic)(MeOH)]·H2O·MeOH}n (1Mn), {[M2Na2(µ3-6m2onic)2(µ-6m2onic)2(µ-H2O)(H2O)6](NO3)2}n [MII = Co (2Co) and Ni (3Ni)], 2[Cu2(6m2onic)3(µ-6m2onic)(MeOH)]·[Cu2(6m2onic)2(µ-6m2onic)2]·2[Cu(6m2onic)2(MeOH)]·32H2O (4Cu) and {[Cu(µ-6m2onic)2]·6H2O}n (5Cu) (where 6m2onic = 6-methyl-2-oxonicotinate). An unusual structural diversity is observed for the compounds, ranging from isolated complexes (in 4Cu), 1D arrays (in 1Mn and 5Cu) and 3D frameworks (in 2Co and 3Ni). Magnetic properties have been studied for all compounds. Analysis of the magnetic dc susceptibility and magnetization data for 4Cu and 5Cu suggests the occurrence of ferromagnetic exchange, which is well explained by broken-symmetry and CASSCF calculations. The sizeable easy-plane magnetic anisotropy present in compound 2Co allows for a field-induced magnet behaviour with an experimental effective energy barrier of 16.2 cm-1, although the slow relaxation seems to be best described through Raman and direct processes in agreement with the results of ab initio calculations.

Rational Design and Experimental Analysis of Short-Oligonucleotide Substrate Specificity for Targeting Bacterial Nucleases.

An undecamer oligonucleotide probe based on a pair of deoxythymidines flanked by several modified nucleotides is a specific and highly efficient biosensor for micrococcal nuclease (MNase), an endonuclease produced by Staphylococcus aureus. Herein, the interaction mode and cleavage process on such oligonucleotide probes are identified and described for the first time. Also, we designed truncated pentamer probes as the minimum-length substrates required for specific and efficient biosensing. By means of computational (virtual docking) and experimental (ultra-performance liquid chromatography-mass spectrometry and matrix-assisted laser desorption ionization time-of-flight) techniques, we perform a sequence/structure-activity relationship analysis, propose a catalytically active substrate-enzyme complex, and elucidate a novel two-step phosphodiester bond hydrolysis mechanism, identifying the cleavage sites and detecting and quantifying the resulting probe fragments. Our results unravel a picture of both the enzyme-biosensor complex and a two-step cleavage/biosensing mechanism, key to the rational oligonucleotide design process.

An Ideal Spin Filter: Long-Range, High-Spin Selectivity in Chiral Helicoidal 3-Dimensional Metal Organic Frameworks.

An enantiopure, conductive, and paramagnetic crystalline 3-D metal-organic framework (MOF), based on Dy(III) and the l-tartrate chiral ligand, is proved to behave as an almost ideal electron spin filtering material at room temperature, transmitting one spin component only, leading to a spin polarization (SP) power close to 100% in the ±2 V range, which is conserved over a long spatial range, larger than 1 µm in some cases. This impressive spin polarization capacity of this class of nanostructured materials is measured by means of magnetically polarized conductive atomic force microscopy and is attributed to the Chirality-Induced Spin Selectivity (CISS) effect of the material arising from a multidimensional helicity pattern, the inherited chirality of the organic motive, and the enhancing influence of Dy(III) ions on the CISS effect, with large spin-orbit coupling values. Our results represent the first example of a MOF-based and CISS-effect-mediated spin filtering material that shows a nearly perfect SP. These striking results obtained with our robust and easy-to-synthesize chiral MOFs constitute an important step forward in to improve the performance of spin filtering materials for spintronic device fabrication.

Enantiospecific Response in Cross-Polarization Solid-State Nuclear Magnetic Resonance of Optically Active Metal Organic Frameworks.

We report herein on a NMR-based enantiospecific response for a family of optically active metal-organic frameworks. Cross-polarization of the 1H-13C couple was performed, and the intensities of the 13C nuclei NMR signals were measured to be different for the two enantiomers. In a direct-pulse experiment, which prevents cross-polarization, the intensity difference of the 13C NMR signals of the two nanostructured enantiomers vanished. This result is due to changes of the nuclear spin relaxation times due to the electron spin spatial asymmetry induced by chemical bond polarization involving a chiral center. These experiments put forward on firm ground that the chiral-induced spin selectivity effect, which induces chemical bond polarization in the J-coupling, is the mechanism responsible for the enantiospecific response. The implications of this finding for the theory of this molecular electron spin polarization effect and the development of quantum biosensing and quantum storage devices are discussed.

Modulating Magnetic and Photoluminescence Properties in 2-Aminonicotinate-Based Bifunctional Coordination Polymers by Merging 3d Metal Ions.

Herein, the synthesis and study of bifunctional coordination polymers (CPs) with both magnetic and photoluminescence properties, derived from a heterometallic environment, are reported. As a starting point, three isostructural monometallic CPs with the formula [M(µ-2ani)2 ]n (MII =Mn (1Mn ), Co (3Co ) and Ni (4Ni ); 2ani=2-aminonicotinate), crystallise as chiral 2D-layered structures stacked by means of supramolecular interactions. These compounds show high thermal stability in the solid state (above 350 °C), despite which, in aqueous solution, compound 1Mn is shown to partially transform into a novel 1D chain CP with the formula [Mn(2ani)2 (µ-H2 O)2 ]n (2Mn ). A study of the direct current (dc) magnetic properties of 1Mn , 3Co and 4Ni reveals a spin-canted structure derived from antisymmetric antiferromagnetic weak exchanges along the chiral network (as confirmed by DFT calculations) and magnetic anisotropy of the ions, in such a way that long-range ordering is observed with variable magnitude for the spin carriers. Moreover, compounds 3Co and 4Ni show no frequency-dependent alternating current (ac) susceptibility curves under zero dc field; this is characteristic behaviour of a glassy state that may be partially supressed for 3Co by applying an external dc field. To overcome long-range magnetic ordering, CoII ions are diluted in a diamagnetic ZnII -based matrix, which enables single-molecule magnet behaviour. Interestingly, this strategy allows a bifunctional Cox Zn1-x 2ani material, which is imbued with a strong photoluminescent emitting capacity, as characterised by an intense blue light followed by a green afterglow, to be obtained.

(Diphenylphosphino)alkylaldehyde affords hydride- or alkyl-[(diphenylphosphino)alkylacyl]rhodium(iii) or (diphenylphosphino)alkylester complexes: theoretical and experimental diastereoselectivity.

The reaction of [RhCl(COD)]2 (COD = 1,5-cyclooctadiene) with racemic PPh2(CH(Ph)CH2CHO) and pyridine (py) led to the oxidative addition of the aldehyde, and a single geometric isomer of [RhHCl(PPh2(CH(Ph)CH2CO))(py)2] (1), with hydride trans to chloride, was obtained as a mixture of two diastereomers in a 95 : 5 ratio; this was in agreement with density functional theory (DFT) calculations. In a chloroform solution, the exchange of hydride by chloride yielded [RhCl2(PPh2(CH(Ph)CH2CO))(py)2] (2) as a mixture of a kinetically preferred species, trans-py-2a, and two diastereomers, cis-Cl-2b' and cis-Cl-2b, with cis pyridines and a chloride trans to acyl; as predicted by the DFT calculations, the latter was the major species. Complex 1 reacted with racemic PPh2(CH(Ph)CH2CHO) or PPh2(o-C6H4CHO) to afford [RhHCl(PPh2(CH(Ph)CH2CO))(κ1-PPh2(CH(Ph)CH2CHO))(py)] (3) or [RhHCl(PPh2(o-C6H4CO))(κ1-PPh2(CH(Ph)CH2CHO))(py)] (4), respectively, both with a dangling alkylaldehyde. Diastereomeric mixtures with the ratios 3a/3a' = 80 : 20 and 4a/4a' = 50 : 50 were obtained. Complex 4 reacted with N-donors to afford cationic [RhH(NN)(PPh2(o-C6H4CO))(κ1-PPh2(CH(Ph)CH2CHO))]BPh4 (NN = 1,10-phenanthroline, 5; 2,2'-bipyridine, 6) or with 8-aminoquinoline (aqui) or 2-(aminomethyl)pyridine to yield imination products with terdentate ligands: [RhH(PPh2(o-C6H4CO))(κ3-PNN)]BF4 (PNN = PPh2(CH(Ph)CH2CNC9H6N), 7 and PPh2(CH(Ph)CH2CNCH2C5H4N), 8, respectively. Compounds 5-8 were obtained as equimolar a/a' mixtures of diastereomers. Moreover, 5a and 5a' could be separated. [RhCl(NBD)]2 reacted with racemic PPh2(CH(Ph)CH2CHO) and N-donors to provide nortricyclyl (Ntyl) derivatives [RhCl(NN)(Ntyl)(PPh2CH(Ph)CH2CO)] (NN = phen, 9 and bipy, 10) as an a/a' = 75 : 25 mixture of diastereomers. By reacting [RhCl(NBD)]2 with PPh2(CH(Ph)CH2CHO) and quinoline-8-carbaldehyde in methanol, the phosphino-ester complex [RhCl(Ntyl)(C9H6NCO)(κ2-PPh2CH(Ph)CH2CO(OCH3)] 11 was obtained. The initial equimolar mixture of two diastereomers readily transformed into a single diastereomer, which was found to be thermodynamically most stable by the DFT calculations. Furthermore, single crystal X-ray diffraction analysis of cis-Cl-2b, 5a, 7a, 10a and 11 is reported.

A Potassium Metal-Organic Framework based on Perylene-3,4,9,10-tetracarboxylate as Sensing Layer for Humidity Actuators.

We have synthesized a novel three-dimensional metal-organic-framework (MOF) based on the perylene-3,4,9,10-tetracarboxylate linker and potassium as metallic centre. We report the formation of this K-based MOF using conventional routes with water as solvent. This material displays intense green photoluminescence at room temperature, and displays an aggregation dependent quenching. Correlation of the optical properties with the crystalline packing was confirmed by DFT calculations. We also demonstrate its potential to build humidity actuators with a reversible and reproducible response, with a change of 5 orders of magnitudes in its impedance at about 40% relative humidity (RH). This 3D-MOF is based on an interesting perylene derivative octadentate ligand, a moiety with interesting fluorescent properties and known component in organic semiconductors. To the best of our knowledge, this is the first time to build such a printed and flexible actuator towards humidity with a reversible response, enabling precise humidity threshold monitoring.

Chiral coordination polymers based on d10 metals and 2-aminonicotinate with blue fluorescent/green phosphorescent anisotropic emissions.

Herein, we report the detailed structural characterization and photoluminescence (PL) properties of two new isostructural coordination polymers (CPs) based on 2-aminonicotinate (2ani) ligand and zinc(ii) or cadmium(ii) metal ions, namely, [M(µ-2ani)2]n [MII = Zn(1) and Cd(2)]. These compounds are characterized as chiral 2D-layered structures, resulting from the bridging of the 2ani ligands in the MN2O4 coordination shell. Also, strong supramolecular interactions are found to sustain the overall crystal structure, which endows these materials with high stability. The single crystals of 1 exhibit waveguiding behaviour along some preferred orientations. The examination of the polycrystalline samples of both compounds under UV light also shows that the samples exhibit remarkably strong PL emissions, which consist of blue fluorescence and green phosphorescence involving long-lasting phosphorescence (LLP) at low temperature. Fitting of the decay curves confirms the occurrence of short (in the order of nanoseconds) and very large lifetimes (τ ≈ 320 and 560 ms for 1 and 2, respectively) of the wavelengths responsible for their blue and green emissions, respectively, which have been studied using DFT calculations.

Experimental and Theoretical Study of a Cadmium Coordination Polymer Based on Aminonicotinate with Second-Timescale Blue/Green Photoluminescent Emission.

A new cadmium/6-aminonicotinate-based coordination polymer (CP) with an unprecedented multicolored and long-lasting emission is reported. This material shows a blue fluorescence which rapidly turns to green persistent phosphorescence with a lifetime of nearly 1 s. Time-dependent density functional theory calculations revealed that electronic transitions arising from both first excited singlet and triplet states involving ligand-centered and ligand-to-metal charge-transfer mechanisms are responsible for such behavior.

Irida-ß-ketoimines Derived from Hydrazines To Afford Metallapyrazoles or N-N Bond Cleavage: A Missing Metallacycle Disclosed by a Theoretical and Experimental Study.

Unprecedented metallapyrazoles [IrH2{Ph2P(o-C6H4)CNNHC(o-C6H4)PPh2}] (3) and [IrHCl{Ph2P(o-C6H4)CNNHC(o-C6H4)PPh2}] (4) were obtained by the reaction of the irida-ß-ketoimine [IrHCl{(PPh2(o-C6H4CO))(PPh2(o-C6H4CNNH2))H}] (2) in MeOH heated at reflux in the presence and absence of KOH, respectively. In solution, iridapyrazole 3 undergoes a dynamic process due to prototropic tautomerism with an experimental barrier for the exchange of ΔGcoal⧧ = 53.7 kJ mol-1. DFT calculations agreed with an intrapyrazole proton transfer process assisted by two water molecules (ΔG = 63.1 kJ mol-1). An X-ray diffraction study on 4 indicated electron delocalization in the iridapyrazole ring. The reaction of the irida-ß-diketone [IrHCl{(PPh2(o-C6H4CO))2H}] (1) with H2NNRR' in aprotic solvents gave irida-ß-ketoimines [IrHCl{(PPh2(o-C6H4CO))(PPh2(o-C6H4CNNRR'))H}] (R = R' = Me (5); R = H, R' = Ph (8)), which can undergo N-N bond cleavage to afford the acyl-amide complex [IrHCl(PPh2(o-C6H4CO))(PPh2(o-C6H4C(O)N(CH3)2))-κP,κO] (6) or [IrHCl(PPh2(o-C6H4CO))(PPh2(o-C6H4CN)-κP)(NH2NHPh-κNH2)] (9) containing o-(diphenylphosphine)benzonitrile and phenylhydrazine, respectively. From a CH2Cl2/CH3OH solution of 9 kept at -18 °C, single crystals of [IrHCl(PPh2(o-C6H4CO))(PPh2(o-C6H4CN)-κP))(HN═NPh-κNH)] (10) containing o-(diphenylphosphine)benzonitrile and phenyldiazene were formed, as shown by X-ray diffraction. The reaction of 1 with methylhydrazine in methanol gave the hydrazine complex [IrCl(PPh2(o-C6H4CO))2(NH2NH(CH3)-κNH2)] (7). Single-crystal X-ray diffraction analysis was performed on 6 and 7.

New Insights into the Reactivity of Cisplatin with Free and Restrained Nucleophiles: Microsolvation Effects and Base Selectivity in Cisplatin-DNA Interactions.

The reactivity of cisplatin towards different nucleophiles has been studied by using density functional theory (DFT). Water was considered first to analyze the factors that govern the transformation of cisplatin into more electrophilic aquated species by using an activation-strain model. It was found that the selectivity and reactivity of cisplatin is a delicate trade-off between strain and interaction energies and that the second chloride is a worse leaving group than the first. When similar studies were carried out with imidazole, guanine (G), and adenine (A), it was found that in general the second nucleophilic substitution reactions have lower activation barriers than the first ones. Finally, simulations of the structural restrictions imposed by the DNA scaffold in intra- and interstrand processes showed that the geometries of the reaction products are nonoptimal with respect to the unrestrained A and G nucleophiles, although the energetic cost is not considerable under physiological conditions, which thus permits nucleophilic substitution reactions that lead to highly distorted DNA.

Stereoselective formation and catalytic activity of hydrido(acylphosphane)(chlorido)(pyrazole)rhodium(III) complexes. Experimental and DFT studies.

The reaction of [{RhCl(COD)}2] (COD = 1,5-cyclooctadiene) with L = pyrazole (Hpz), 3(5)-methylpyrazole (Hmpz) or 3,5-dimethylpyrazole (Hdmpz) and PPh2(o-C6H4CHO) (Rh : L : P = 1 : 2 : 1) gives hydridoacyl complexes [RhHCl{PPh2(o-C6H4CO)}(L)2] (). Stereoselective formation of and with pyrazoles trans to hydrido and phosphorus and hydrogen bond formation with O-acyl and chlorido occur. is a mixture of two linkage isomers in a 9 : 1 ratio, with two 5-methylpyrazole ligands or with one 3- and one 5-methylpyrazole ligand, respectively. Fluxional undergoes metallotropic tautomerization and is a mixture of equal amounts of and , with hydrido trans to pyrazole or chlorido, respectively. Complexes readily exchange hydrido by chlorido to afford [RhCl2{PPh2(o-C6H4CO)}(L)2] (, and ) as single isomers with cis chloridos and two N-HCl hydrogen bonds. The reaction of with PPh3 or PPh2OH affords static [RhHCl{PPh2(o-C6H4CO)}(PPh3)L] () or [RhHCl{PPh2(o-C6H4CO)}(PPh2OH)L] () respectively with trans P-atoms and pyrazoles forming N-HCl hydrogen bonds. and contain single species with hydrido cis to chlorido, while is a mixture of equal amounts of and . Complexes , with an additional O-HO hydrogen bond, selectively contain only the cis-H,Cl species with all the three ligands. The reaction of [{RhCl(COD)}2] with L and PPh2(o-C6H4CHO) (Rh : L : P = 1 : 1 : 2) led to complexes with trans P-atoms, [RhHCl{PPh2(o-C6H4CO)}{PPh2(o-C6H4CHO)-κP}L] (, and ), at room temperature, and to [RhCl{PPh2(o-C6H4CO)}{PPh2(o-C6H4CHOH)}(Hmpz)] () or [RhCl{PPh2(o-C6H4CO)}2L] () with hydrogen evolution in refluxing benzene. DFT calculations were used to predict the correct isomers, their ratios and the particular intramolecular hydrogen bonds in these complexes. Single crystal X-ray diffraction analysis was performed on , and . Complexes are efficient homogeneous catalysts (0.5 mol% loading) in the hydrolysis of amine- or ammonia-borane (AB) to generate up to 3 equivalents of hydrogen in the presence of air.

Development and validation of a LC-MS assay for the quantification of ikh12 a novel anti-tumor candidate in rat plasma and tissues and its application in a pharmacokinetic study.

IKH12 is a novel histone deacetylase 6 selective inhibitor. A rapid and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of IKH12 in rat plasma and tissue with kendine 91 as internal standard (IS). The samples were prepared by liquid-liquid extraction with tert-butyl methyl ether. The chromatographic separation was accomplished by using a Zorbax Extend C18 4.6 × 150 mm, 5 µm column, with a mobile phase consisting of methanol and 0.1% formic acid (75:25 v/v). Multiple reaction monitoring, using electrospray ionization in positive ion mode, was employed to quantitatively detect IKH12 and IS. The monitored transitions were set at m/z 418 → 252 and 444 → 169 for IKH12 and kendine 91, respectively. The calibration curve was linear over the concentration range 2-1000 ng mL(-1) . The intra- and inter-assay precision and accuracy of the quality controls and the limit of quantification were satisfactory in all cases (according to European Medicines Agency guidelines). Stability studies showed that plasma samples were stable in the chromatography rack for 24 h and at -80°C for 2 months and also after three freeze-thaw cycles. This method was successfully applied to a pharmacokinetic study of IKH12 in rat.

Design, synthesis, and functional evaluation of leukocyte function associated antigen-1 antagonists in early and late stages of cancer development.

The integrin leukocyte function associated antigen 1 (LFA-1) binds the intercellular adhesion molecule 1 (ICAM-1) by its α(L)-chain inserted domain (I-domain). This interaction plays a key role in cancer and other diseases. We report the structure-based design, small-scale synthesis, and biological activity evaluation of a novel family of LFA-1 antagonists. The design led to the synthesis of a family of highly substituted homochiral pyrrolidines with antiproliferative and antimetastatic activity in a murine model of colon carcinoma, as well as potent antiadhesive properties in several cancer cell lines in the low micromolar range. NMR analysis of their binding to the isolated I-domain shows that they bind to the I-domain allosteric site (IDAS), the binding site of other allosteric LFA-1 inhibitors. These results provide evidence of the potential therapeutic value of a new set of LFA-1 inhibitors, whose further development is facilitated by a synthetic strategy that is versatile and fully stereocontrolled.

Comparative normal mode analysis of LFA-1 integrin I-domains.

The conformational dynamics of the Inserted domain (I-domain) from the lymphocyte function-associated antigen-1 (LFA-1) was investigated by normal mode analysis of multiple structures of the low, intermediate, and high affinity states. LFA-1 is an integrin expressed on leukocytes and is of critical importance in adhesion reactions, like antigen-specific responses, homing, and diapedesis. The main ligand binding site of LFA-1 is the I-domain, which recognizes intercellular adhesion molecules (ICAMs), members of the immunoglobulin superfamily. From experimental crystal structures, a large-scale conformational change of, among others, the alpha7 helix of the I-domain has been observed leading to the proposal that these structural changes are linked to the conformational regulation of LFA-1. The results from the present calculations show that structural changes of the alpha7 helix consistent with those observed in the crystal structures are significantly sampled by the low frequency modes. This was found to be particularly true for the low affinity state of the I-domain, indicating that low frequency motions favor the conformational transition implicated in activation. However, beyond the simple downward shift of the helix implied by the crystal structures, the calculations further show that there is a noticeable swinging-out motion of the helix. The consequences of this motion are discussed in the context of integrin activation and inhibition. Moreover, significant changes in the atomic-level dynamics and in long-range correlated motions of the I-domain were found to occur upon binding of the natural ligand ICAM. These changes were more local upon binding of an allosteric inhibitor. The present study opens the question of how changes in dynamics may contribute to the long-range transmission of signal upon ICAM binding by the LFA-1 I-domain.

On the affinity regulation of the metal-ion-dependent adhesion sites in integrins.

Density functional theory and a polarizable continuum model are used to (i) understand the affinity modulating mechanisms of the interaction between the metal-ion-dependent adhesion site (MIDAS) of a selected integrin, lymphocyte function-associated antigen-1 (LFA-1) and a ligand mimetic acetate molecule and to (ii) propose a new, promising family of inhibitors to block the interaction of the integrin with intercellular adhesion molecule-1 (ICAM-1). We quantify the effect of isolated factors, such as the metal coordination, the nature of the ligand or the cation present on the MIDAS, and the effect of the permittivity of the media. We show that the affinity for ligand decreases when metal coordination changes from the open conformation to the closed conformation. In addition, Mn2+ and Zn2+ showed to be good competitors for the octahedrically coordinated Mg2+ and yielded excellent affinity values, whereas Ca2+ in an octahedric environment would decrease the affinity for the ligand. Our affinity studies of the open MIDAS showed that nitronate-derived or carboxylic acid-containing ligands may represent new promising scaffolds of future inhibitors. Finally, we show that affinities are always highly favored by low-dielectric environments, which explains the propensity of MIDAS motifs to be surrounded by hydrophobic residues in integrins and highlights the importance of including hydrophobic groups in the inhibitors.