DataSheet1_Evaluation of docking procedures reliability in affitins-partners interactions.pdf

Affitins constitute a class of small proteins belonging to Sul7d family, which, in microorganisms such as Sulfolobus acidocaldarius, bind DNA preventing its denaturation. Thanks to their stability and small size (60–66 residues in length) they have been considered as ideal candidates for engineering and have been used for more than 10 years now, for different applications. The individuation of a mutant able to recognize a specific target does not imply the knowledge of the binding geometry between the two proteins. However, its identification is of undoubted importance but not always experimentally accessible. For this reason, computational approaches such as protein-protein docking can be helpful for an initial structural characterization of the complex. This method, which produces tens of putative binding geometries ordered according to a binding score, needs to be followed by a further reranking procedure for finding the most plausible one. In the present paper, we use the server ClusPro for generating docking models of affitins with different protein partners whose experimental structures are available in the Protein Data Bank. Then, we apply two protocols for reranking the docking models. The first one investigates their stability by means of Molecular Dynamics simulations; the second one, instead, compares the docking models with the interacting residues predicted by the Matrix of Local Coupling Energies method. Results show that the more efficient way to deal with the reranking problem is to consider the information given by the two protocols together, i.e. employing a consensus approach.</p

Conformational Characterization of Lanthanide(III)−DOTA Complexes by ab Initio Investigation in Vacuo and in Aqueous Solution

The conformational behavior of four [Ln(DOTA)(H2O)]- systems (Ln = La, Gd, Ho, and Lu) has been characterized by means of ab initio calculations performed in vacuo and in aqueous solution, the latter by using the polarizable continuum model (PCM). Calculated molecular geometries and conformational energies of the [Ln(DOTA)(H2O)]- systems, and interconversion mechanisms, barriers, and 13C NMR spectra of the [Lu(DOTA)]- complex are compared with experimental values. For each system, geometry optimizations, performed in vacuo and in solution at the HF/3-21G level and using a 46+4fn core electron effective core potential (ECP) for lanthanides, provide two minima corresponding to a square antiprismatic (A) and an inverted antiprismatic (IA) coordination geometry. All the systems are nonacoordinated, with the exception of the IA isomer of the Lu complex that, from in solution calculations, is octacoordinated, in agreement with experimental data. On comparing the in vacuo relative free energies calculated at different theory levels it can be seen that the nonacoordinated species dominates at the beginning of the lanthanide series while the octacoordinated one does so at the end. Furthermore, on passing along the series the IA isomer becomes less and less favored with respect to A and for the Lu complex a stabilization of the IA isomer is observed in solution (but not in vacuo), in agreement with the experimental data. Investigation of the A↔IA isomerization process in the [Lu(DOTA)]- system provides two different interconversion mechanisms:  a single-step process, involving the simultaneous rotation of the acetate arms, and a multistep path, involving the inversion of the cyclen cycle configuration. While in vacuo the energy barrier for the acetate arm rotation is higher than that involved in the ring inversion (23.1 and 13.1 kcal mol-1 at the B3LYP/6-311G** level, respectively), in solution the two mechanisms present comparable barriers (14.7 and 13.5 kcal mol-1), in fairly good agreement with the experimental values. The NMR shielding constants for the two isomers of the [Lu(DOTA)]- complex have been calculated by means of the ab initio GIAO and CSGT methods, and using a 46-core-electron ECP for Lu. The calculated 13C NMR chemical shifts are in close agreement with the experimental values (rms 3.3 ppm, at the HF/6-311G** level) and confirm the structural assignment of the two isomers based on experimental NMR spectra in solution. The results demonstrate that our computational approach is able to predict several physicochemical properties of lanthanide complexes, allowing a better characterization of this class of compounds for their application as contrast agents in medical magnetic resonance imaging (MRI)

Ab Initio Investigation of Gadolinium Complexes with Polyamino Carboxylate Ligands and Force Fields Parametrization of Metal−Ligand Interactions

The conformational properties of five gadolinium(III) complexes with polyamino carboxylate (PAC) ligands used as magnetic resonance imaging contrast agents have been investigated by ab initio and molecular mechanics (MM) methods. Ab initio calculations were performed using an effective core potential (ECP) that includes 4f electrons in the core and an optimized valence basis set for the metal. To test the reliability of ECP calculations, full geometry optimizations of Gd complexes were performed at the RHF and DFT (B-LYP) levels using the 3-21G and the 6-31G* basis sets for the ligands. Comparison with experimental data shows that ab initio calculations provide quite accurate geometries and correct conformational energies at the RHF level. Within the framework of a valence force fields, parameters for Gd−ligand interactions were determined by fitting the empirical potential to the ab initio potential energy surface (PES) of the [Gd−DOTA(H2O)]-1 complex. Sampling of the PES was performed by moving the ion into the frozen coordination cage of the ab initio optimized geometry; for each generated structure the energy and first derivatives, with respect to the Cartesian coordinates of the metal and donor atoms, were calculated at the RHF level using both 3-21G and 6-31G* basis sets for the ligand. For each considered basis set, two sets of parameters, with the electrostatic contribution turned on or off in the force fields, were determined. All the implemented sets of parameters provide reliable molecular geometries for PAC complexes, but only sets derived including the electrostatic contribution correctly reproduce the observed trend of conformational energies

Cytotoxicity of <i>cis</i>-Platinum(II) Conjugate Models. The Effect of Chelating Arms and Leaving Groups on Cytotoxicity: A Quantitative Structure−Activity Relationship Approach

Thirteen newly synthesized or resynthesized diamine−platinum(II) complexes were characterized, and their cytotoxic activities (IC50) were tested on parental and resistant ovarian cancer cell lines. They represent models of conjugates between biologically active vectors and cytotoxic PtII moieties within the “drug targeting and delivery strategy”. Three drugs, routinely employed in the clinical treatment of cancer, namely, cisplatin, carboplatin, and oxaliplatin, were also included in the study as controls. The quantitative structure−activity relationship approach provides simple regression models able to predict log(1/IC50) of diamine−platinum(II) complexes on both parental and resistant ovarian cancer cell lines. The 16 complexes were characterized using 197 molecular descriptors, after which the best regression models relating a subset of these descriptors to the log(1/IC50) in the two cancer cell lines were calculated. Models with four variables proved to be endowed with very good predictive ability Q2LMO-50% ≥ 85.6%, making it possible to discard 50% of the molecules from the test set following for cross-validation procedure. A four-variable regression model also proved effective in predicting the resistance index RI, Q2LMO-50% = 84.4%

Water-Soluble [Tc(N)(PNP)] Moiety for Room-Temperature ^99mTc Labeling of Sensitive Target Vectors

The incorporation of bioactive molecules into a water-soluble [99mTc]­[Tc­(N)­(PNP)]-based mixed compound is described. The method, which exploits the chemical properties of the new [99mTc]­[Tc­(N)­(PNP3OH)]2+ synthon [PNP3OH = N,N-bis­(di-hydroxymethylenphosphinoethyl)­methoxyethylamine], was successfully applied to the labeling of small, medium (cysteine-functionalized biotin and c-RGDfK pentapeptide), and large molecules. Apomyoglobin was chosen as a model protein and derivatized via site-specific enzymatic reaction catalyzed by transglutaminase (TGase) with the H-Cys-Gly-Lys-Gly-OH tetrapeptide for the insertion in the protein sequence of a reactive N-terminal Cys for 99mTc chelation. Radiosyntheses were performed under physiological conditions at room temperature within 30 min. They were reproducible, highly specific, and quantitative. Heteroleptic complexes are hydrophilic and stable. Biodistributions of the selected compounds show favorable pharmacokinetics within 60 min post-injection and predominant elimination through the renal-urinary pathway. In a wider perspective, these data suggest a role of the [99mTc]­[Tc­(N)­(PNP)] technology in the labeling of temperature-sensitive biomolecules, especially targeting proteins for SPECT imaging

[Gd-AAZTA]^-: A New Structural Entry for an Improved Generation of MRI Contrast Agents

An innovative MRI contrast agent based on the unprecedented and easily obtained ligand AAZTA is described. The simple and straightforward synthesis of the ligand, together with the potentiometric and relaxometric behavior of the corresponding Gd(III) chelate, is reported. The complex [Gd(AAZTA)]- shows outstanding magnetic properties connected with high thermodynamic stability in aqueous solution and a nearly complete inertness toward the influence of bidentate endogenous anions, placing this compound as one of the most promising candidates for the development of high performance MRI contrast agents

Data_Sheet_1_Equilibrium Thermodynamics, Formation, and Dissociation Kinetics of Trivalent Iron and Gallium Complexes of Triazacyclononane-Triphosphinate (TRAP) Chelators: Unraveling the Foundations of Highly Selective Ga-68 Labeling.doc

<p>In order to rationalize the influence of Fe^III contamination on labeling with the ⁶⁸Ga eluted from ⁶⁸Ge/⁶⁸Ga-generator, a detailed investigation was carried out on the equilibrium properties, formation and dissociation kinetics of Ga^III- and Fe^III-complexes of 1,4,7-triazacyclononane-1,4,7-tris(methylene[2-carboxyethylphosphinic acid]) (H₆TRAP). The stability and protonation constants of the [Fe(TRAP)]³⁻ complex were determined by pH-potentiometry and spectrophotometry by following the competition reaction between the TRAP ligand and benzhydroxamic acid (0.15 M NaNO₃, 25°C). The formation rates of [Fe(TRAP)] and [Ga(TRAP)] complexes were determined by spectrophotometry and ³¹P-NMR spectroscopy in the pH range 4.5–6.5 in the presence of 5–40 fold H_xTRAP^(x−6) excess (x = 1 and 2, 0.15 M NaNO₃, 25°C). The kinetic inertness of [Fe(TRAP)]³⁻ and [Ga(TRAP)]³⁻ was examined by the trans-chelation reactions with 10 to 20-fold excess of H_xHBED^(x−4) ligand by spectrophotometry at 25°C in 0.15 M NaCl (x = 0,1 and 2). The stability constant of [Fe(TRAP)]³⁻ (logK_FeL = 26.7) is very similar to that of [Ga(TRAP)]³⁻ (logK_GaL = 26.2). The rates of ligand exchange reaction of [Fe(TRAP)]³⁻ and [Ga(TRAP)]³⁻ with H_xHBED^(x−4) are similar. The reactions take place quite slowly via spontaneous dissociation of [M(TRAP)]³⁻, [M(TRAP)OH]⁴⁻ and [M(TRAP)(OH)₂]⁵⁻ species. Dissociation half-lives (t_1/2) of [Fe(TRAP)]³⁻ and [Ga(TRAP)]³⁻ complexes are 1.1 × 10⁵ and 1.4 × 10⁵ h at pH = 7.4 and 25°C. The formation reactions of [Fe(TRAP)]³⁻ and [Ga(TRAP)]³⁻ are also slow due to the formation of the unusually stable monoprotonated [^*M(HTRAP)]²⁻ intermediates [^*logK_Ga(HL) = 10.4 and ^*logK_Fe(HL) = 9.9], which are much more stable than the [^*Ga(HNOTA)]⁺ intermediate [^*logK_Ga(HL) = 4.2]. Deprotonation and transformation of the monoprotonated [^*M(HTRAP)]²⁻ intermediates into the final complex occur via OH⁻-assisted reactions. Rate constants (k_OH) characterizing the OH⁻-driven deprotonation and transformation of [^* Ga(HTRAP)]²⁻ and [^*Fe(HTRAP)]²⁻ intermediates are 1.4 × 10⁵ M⁻¹s⁻¹ and 3.4 × 10⁴ M⁻¹s⁻¹, respectively. In conclusion, the equilibrium and kinetic properties of [Fe(TRAP)] and [Ga(TRAP)] complexes are remarkably similar due to the close physico-chemical properties of Fe^III and Ga^III-ions. However, a slightly faster formation of [Ga(TRAP)] over [Fe(TRAP)] provides a rationale for a previously observed, selective complexation of ⁶⁸Ga^III in presence of excess Fe^III.</p

Improved Efficacy of Synthesizing *M^III-Labeled DOTA Complexes in Binary Mixtures of Water and Organic Solvents. A Combined Radio- and Physicochemical Study

Typically, the synthesis of radiometal-based radiopharmaceuticals is performed in buffered aqueous solutions. We found that the presence of organic solvents like ethanol increased the radiolabeling yields of [68Ga]­Ga-DOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacatic acid). In the present study, the effect of organic cosolvents [ethanol (EtOH), isopropyl alcohol, and acetonitrile] on the radiolabeling yields of the macrocyclic chelator DOTA with several trivalent radiometals (gallium-68, scandium-44, and lutetium-177) was systematically investigated. Various binary water (H2O)/organic solvent mixtures allowed the radiolabeling of DOTA at a significantly lower temperature than 95 °C, which is relevant for the labeling of sensitive biological molecules. Simultaneously, much lower amounts of the chelators were required. This strategy may have a fundamental impact on the formulation of trivalent radiometal-based radiopharmaceuticals. The equilibrium properties and formation kinetics of [M­(DOTA)]− (MIII= GaIII, CeIII, EuIII, YIII, and LuIII) complexes were investigated in H2O/EtOH mixtures (up to 70 vol % EtOH). The protonation constants of DOTA were determined by pH potentiometry in H2O/EtOH mixtures (0–70 vol % EtOH, 0.15 M NaCl, 25 °C). The log K1H and log K2H values associated with protonation of the ring N atoms decreased with an increase of the EtOH content. The formation rates of [M­(DOTA)]− complexes increase with an increase of the pH and [EtOH]. Complexation occurs through rapid formation of the diprotonated [M­(H2DOTA)]+ intermediates, which are in equilibrium with the kinetically active monoprotonated [M­(HDOTA)] intermediates. The rate-controlling step is deprotonation (and rearrangement) of the monoprotonated intermediate, which occurs through H2O (*M(HL)kH2O) and OH– (*M(HL)kOH) assisted reaction pathways. The rate constants are essentially independent of the EtOH concentration, but the M(HL)kH2O values increase from CeIII to LuIII. However, the logKM(HL)H protonation constants, analogous to the log KH2 value, decrease with increasing [EtOH], which increases the concentration of the monoprotonated M­(HDOTA) intermediate and accelerates formation of the final complexes. The overall rates of complex formation calculated by the obtained rate constants at different EtOH concentrations show a trend similar to that of the complexation rates determined with the use of radioactive isotopes

Design, Synthesis, Biological Evaluation, and NMR Studies of a New Series of Arylsulfones As Selective and Potent Matrix Metalloproteinase-12 Inhibitors

Overexpression of macrophage elastase (MMP-12), a member of the matrix metalloproteinases family, can be linked to tissue remodeling and degradation in some inflammatory processes, such as chronic obstructive pulmonary disease (COPD), emphysema, rheumatoid arthritis (RA), and atherosclerosis. On this basis, MMP-12 can be considered an attractive target for studying selective inhibitors that are useful in the development of new therapies for COPD and other inflammatory diseases. We report herein the design, synthesis, and in vitro evaluation of a new series of compounds, possessing an arylsulfonyl scaffold, for their potential as selective inhibitors of MMP-12. The best compound in the series showed an IC50 value of 0.2 nM, with good selectivity over MMP-1 and MMP-14. A docking study was carried out on this compound in order to investigate its binding interactions with MMP-12, and NMR studies on the complex with the MMP-12 catalytic domain were able to validate the proposed binding mode

Synthesis and Preliminary Evaluation in Tumor Bearing Mice of New ¹⁸F‑Labeled Arylsulfone Matrix Metalloproteinase Inhibitors as Tracers for Positron Emission Tomography

New fluorinated, arylsulfone-based matrix metalloproteinase (MMP) inhibitors containing carboxylate as the zinc binding group were synthesized as radiotracers for positron emission tomography. Inhibitors were characterized by <i>K</i>_i for MMP-2 in the nanomolar range and by a fair selectivity for MMP-2/9/12/13 over MMP-1/3/14. Two of these compounds were obtained in the ¹⁸F-radiolabeled form, with radiochemical purity and yield suitable for preliminary studies in mice xenografted with a human U-87 MG glioblastoma. Target density in xenografts was assessed by Western blot, yielding <i>B</i>_max/<i>K</i>_d = 14. The biodistribution of the tracer was dominated by liver uptake and hepatobiliary clearance. Tumor uptake of ¹⁸F-labeled MMP inhibitors was about 30% that of [¹⁸F]­fluorodeoxyglucose. Accumulation of radioactivity within the tumor periphery colocalized with MMP-2 activity (evaluated by in situ zimography). However, specific tumor uptake accounted for only 18% of total uptake. The aspecific uptake was ascribed to the high binding affinity between the radiotracer and serum albumin