Matteo Tegoni

Protein Design: Toward Functional Metalloenzymes

1. Overview A 2. Protein Redesign B 2.1. Making Use of Native Proteins: Protein Redesign B 2.2. Protein Redesign Based on Functions C 2.2.1. Redesign of Zinc Finger Structural Sites C 2.2.2. Redesign of Zinc Hydrolytic Centers E 2.2.3. Redesign of Heme Centers J 2.2.4. Redesign of Nonheme Redox Centers M 2.2.5. Artificial Metalloenzymes for Regioand Enantioselective Catalysis AA 2.2.6. Redesigned Protein Assemblies as Nanoreactors AK 2.3. Summary AN 3. De Novo Design AN 3.1. A Minimalist Approach: Designing Proteins from Scratch AN 3.2. Interactions between De Novo Designed Peptides and Metal Ions AN 3.2.1. Heavy Metal Toxicity AO 3.2.2. De Novo Designed Metal Centers Based on β-Structures AT 3.2.3. Metal-Induced Protein Folding AV 3.3. De Novo Designed Functional Metalloproteins: The Grail Quest of Protein Design AX 3.3.1. De Novo Designed Hydrolytic Centers AX 3.3.2. De Novo Designed Electron Transfer Centers BB 3.3.3. Other Catalytic Centers BG 3.4. Summary BN 4. Perspective BO Author Information BO Corresponding Author BO Notes BO Biographies BO Abbreviations BR References BR

Copper Binding Agents Acting as Copper Ionophores Lead to Caspase Inhibition and Paraptotic Cell Death in Human Cancer Cells

We report a quantitative structure-activity relationship study of a new class of pyrazole-pyridine copper complexes that establishes a clear correlation between the ability to promote copper accumulation and cytotoxicity. Intracellular metal accumulation is maximized when ligand lipophilicity allows the complex to rapidly cross the membrane. Copper and ligand follow different uptake kinetics and reach different intracellular equilibrium concentrations. These results support a model in which the ligand acts as an ionophore for the metal ion, cycling between intra- and extracellular compartments as dissociated or complexed entities. When treating cancer cells with structurally unrelated disulfiram and pyrazole-pyridine copper complexes, as well as with inorganic copper, the same morphological and molecular changes were reproduced, indicating that copper overload is responsible for the cytotoxic effects. Copper-based treatments drive sensitive cancer cells toward paraptotic cell death, a process hallmarked by endoplasmic reticulum stress and massive vacuolization in the absence of apoptotic features. A lack of caspase activation, as observed in copper-treated dying cells, is a consequence of metal-mediated inhibition of caspase-3. Thus, copper acts simultaneously as an endoplasmic reticulum (ER) stress inducer and a caspase-3 inhibitor, forcing the cell into caspase-independent paraptotic death. The establishment of a mechanism of action common to different copper binding agents provides a rationale for the exploitation of copper toxicity as an anticancer tool.

Copper-Dependent Cytotoxicity of 8-Hydroxyquinoline Derivatives Correlates with Their Hydrophobicity and Does Not Require Caspase Activation

This study reports the structure-activity relationship of a series of 8-hydroxoquinoline derivatives (8-HQs) and focuses on the cytotoxic activity of 5-Cl-7-I-8-HQ (clioquinol, CQ) copper complex (Cu(CQ)). 8-HQs alone cause a dose-dependent loss of viability of the human tumor HeLa and PC3 cells, but the coadministration of copper increases the ligands effects, with extensive cell death occurring in both cell lines. Cytotoxic doses of Cu(CQ) promote intracellular copper accumulation and massive endoplasmic reticulum vacuolization that precede a nonapoptotic (paraptotic) cell death. The cytotoxic effect of Cu(CQ) is reproduced in normal human endothelial cells (HUVEC) at concentrations double those effective in tumor cells, pointing to a potential therapeutic window for Cu(CQ). Finally, the results show that the paraptotic cell death induced by Cu(CQ) does not require nor involve caspases, giving an indication for the current clinical assessment of clioquinol as an antineoplastic agent.

Pentacopper(II) 12-metallacrown-4 complexes with α- and β-aminohydroxamic acids in aqueous solution: a reinvestigation

A reinvestigation of the equilibria of (S)-α-alaninehydroxamic acid (α-Alaha) and (R)-aspartic-β-hydroxamic acid (Asp-β-ha) with copper(II) was performed in aqueous solution in order to clarify some contradictory literature reports regarding the stoichiometry of the polynuclear complexes formed. β-Alaninehydroxamic acid (β-Alaha, HL), for which the formation of a planar 12-metallacrown-4, [Cu5L4H−4]2+, was already reported, was also re-examined for comparison. Among the different techniques used (potentiometry, absorption spectrophotometry, spectropolarimetry and electrospray ionization mass spectrometry), ES data allowed to define unambiguously that all these three ligands form the same pentanuclear species. Therefore it can be concluded that in aqueous solution the hydroxamates of both α- and β-amino acids form 12-metallacrown-4 complexes, and that the formers are less stable.

Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)3+/2+, where Cu(I/II) is embeded in α-helical coiled coils, as a model for the CuT2 center of copper nitrite reductase. In Cu(I/II)(TRIL23H)3+/2+, Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)3 active site is characterized in both oxidation states, revealing similarities to the CuT2 site in the natural enzyme. The species Cu(II)(TRIL23H)32+ in aqueous solution can be reduced to Cu(I)(TRIL23H)3+ using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)32+ acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed.

Speciation and structure of copper(II) complexes with (S)-phenylalanine- and (S)-tryptophanhydroxamic acids in methanol/water solution: a combined potentiometric, spectrophotometric, CD and ESI-MS study

Abstract Complexation equilibria of ( S )-phenylalaninehydroxamic and ( S )-tryptophanhydroxamic acids (HL) with copper(II) were studied in methanol/water solution in order to avoid the precipitation of a polynuclear species observed in aqueous solution. Among the different techniques used (potentiometry, absorption spectrophotometry, spectropolarimetry and ESI-MS), mass spectrometry data allowed to detect a pentanuclear species [Cu 5 L 4 H −4 ] 2+ instead of the dinuclear one, [Cu 2 L 2 H −1 ] + , usually reported for the α-aminohydroxamates in aqueous solution. A cup structure of the pentanuclear cation is proposed, which is similar to that determined by X-ray analysis for the corresponding species of β-alaninehydroxamic acid.

Synthesis, molecular structure, solution equilibrium, and antiproliferative activity of thioxotriazoline and thioxotriazole complexes of copper(II) and palladium(II)

Preparations of copper(II) and palladium(II) complexes of 4-amino-5-methylthio-3-(2-pyridyl)-1,2,4-triazole (L(1)) and the copper(II) complex of 1,4-dihydro-4-amino-3-(2-pyridyl)-5-thioxo-1,2,4-triazole (HL) are described. These complexes have been characterized by means of spectroscopy and microanalysis. Molecular structures of HL (1), [CuCl(2)(H(2)L)]Cl.2H(2)O (2a), cis-[CuCl(2)(L(1))] (3), and cis-[PdCl(2)(L(1))] (4) have been determined by single-crystal X-ray diffraction. The HL ligand acts as a N,S bidentate through the thioxo moiety and the exo-amino group whilst the ligand L(1) forms N,N coordination complexes through the pyridine and triazole nitrogen atoms. Speciation in solution of the systems Cu/HL and Cu/L(1) have been determined by means of potentiometry and spectrophotometry as well as for the Cu/L(1)/A (HA=glycine) system in order to determine species present at physiological pH. Antiproliferative activity of these complexes and their ligands was evaluated, using the AlamarBlue Assay, on normal human fibroblasts (HF) and human fibrosarcoma tumor (HT1080) cells. The copper compounds cis-[CuCl(2)(H(2)L)]Cl and cis-[CuCl(2)(L(1))] exerted significant antiproliferative activity of both normal and neoplastic cells; although dose-response experiments revealed that the HT1080 cell line was more sensitive to the tested drugs than normal fibroblasts.

Thermodynamics of Core Metal Replacement and Self-Assembly of Ca2+ 15-Metallacrown-5†

The equilibria for core Ca(2+) replacement by Ln(3+) in copper(II) 15-MC-5 complexes have been investigated using a series of visible spectrophotometric titrations of calcium(II) metallacrowns ({Ca(II)[15-MC(Cu(II)(N)(L))-5]}(2+)) with Ln(3+) ions (H(2)L = pheha, (S)-alpha-phenylalaninehydroxamic acid, or trpha, (S)-alpha-tryptophanhydroxamic acid). These studies allowed the determination of the equilibrium constants for the reaction {Ca(II)[15-MC(Cu(II)(N)(L))-5]}(2+) + Ln(3+) --> {Ln(III)[15-MC(Cu(II)(N)(L))-5]}(3+) + Ca(2+) in methanol/water 9:1 (Ln(3+) = La(3+), Gd(3+), Dy(3+), Er(3+)) or 99:1 (Ln(3+) = La(3+), Nd(3+), Gd(3+), Dy(3+), Er(3+), Yb(3+)), respectively. The log K for these reactions decreases with increasing atomic number of the lanthanide(III), ranging from 6.1 to 3.91 in methanol/water 9:1. The same behavior is observed in methanol/water 99:1, although the constants are uniformly lower (log K = 4.09-2.52). A significant thermodynamic selectivity was observed for the later lanthanides (Gd(3+)-Yb(3+)) while a smaller selectivity is present throughout the beginning of the series (La(3+)-Gd(3+)). This observation has been interpreted on the basis of the size correspondence between the metal ions and the metallacrown cavity. The overall stability of the {Ca(II)[15-MC(Cu(II)(N)(L))-5]}(2+) in methanol/water 9:1 has been determined by pH-spectrophotometric titrations with HCl. The resulting log K values are 63.46(12) and 65.05(13) for pheha and trpha, respectively (Ca(2+) + 5Cu(2+) + 5HL(-) = {Ca(II)[15-MC(Cu(II)(N)(L))-5]}(2+) +5H(+)). The stability of both the La(3+) and Ca(2+) 15-metallacrown-5 complexes in the presence of high Na(+) concentrations has also been demonstrated by spectophotometric studies. Based upon these observations, the preference of the 15-MC-5 for Ca(2+) complexation compared to crown ethers has been quantitatively demonstrated for the first time.

Thermodynamics of Self-Assembly of Copper(II) 15-Metallacrown-5 of Eu(III) or Gd(III) with (S)-α-Alaninehydroxamic Acid in Aqueous Solution

The equilibria of self-assembly of 15-metallacrown-5 (15-MC-5) complexes of Cu(2+) and (S)-alpha-alaninehydroxamic acid (alpha-Alaha, HL) with the lanthanide (Ln) ions Eu(3+) or Gd(3+) in aqueous solution are described. The binary Ln(3+)/alpha-Alaha systems were first studied by potentiometric and calorimetric in-cell titrations; the latter technique allowed us to define the most suitable speciation model. On the contrary, because the kinetics of formation of the Ln(3+) 15-MC-5 complexes is slow, their stability constants were determined by out-of-cell (batch) potentiometric titrations. Two 15-MC-5 complexes are formed with both Eu(3+) and Gd(3+), namely, {Ln[Cu(5)L(5)H(-5)]}(3+) and {Ln[Cu(5)L(5)H(-5)](OH)}(2+), with the latter being the hydroxo species of the former. The acidity of the former to give the hydroxo species is remarkably high (log K = 4.40-4.69). Moreover, our potentiometric and spectrophotometric investigations clearly indicate that the hydroxide ion is coordinated to the central Ln ion, as was reported for several 15-MC-5 in the solid state. The formation of {Ln[Cu(5)L(5)H(-5)]}(3+) starts at ca. pH 3.5, which converts at ca. pH 4.5 into the {Ln[Cu(5)L(5)H(-5)](OH)}(2+) species, which predominates up to pH 7, where a purple precipitate occurs. The coexistence of both 15-MC-5 species and the copper(II) 12-MC-4 species of alpha-Alaha ([Cu(5)L(4)H(-4)](2+)) was observed under appropriate experimental conditions (pH and ligand and metal concentrations). A complete ESI-MS investigation of the Ln(3+)/Cu(2+)/alpha-Alaha system at different pHs confirmed the formation of the two 15-MC-5 species. The 15-MC-5 stability constants were employed to quantitatively evaluate the solution behavior of Ln(III) MCs regarding their integrity, ligand substitution, and transmetalation processes. In particular, EDTA or DOTA, added in equimolar amounts, should not appreciably interfere with the MC integrity, as found in previous experimental investigations, although it is expected that at higher amounts of EDTA, the MC should be disrupted. Our results also demonstrate that an excess of alpha-aminohydroxamate does not interfere with the integrity of the MC, and the disappearance of the CD spectra upon addition of the R enantiomer to 15-MC-5 containing the S enantiomer is due to a very rapid ligand exchange with formation of all possible isomers with no selectivity. The stability of the 15-MC-5 complexes in the presence of transferrin, serum albumin, or an excess of Zn(2+) is also discussed. With regards to the latter metal ion, we found that the MCs are stable toward Gd(3+)/Zn(2+) transmetalation. although the presence of a phosphate buffer promotes the disruption of the MC scaffold by formation of stable Gd(3+)/phosphate species.

Copper(II) 12-metallacrown-4 complexes of alpha-, beta- and gamma-aminohydroxamic acids: a comparative thermodynamic study in aqueous solution.

A complete thermodynamic study of the protonation and Cu(II) complex formation equilibria of a series of alpha- and beta-aminohydroxamic acids in aqueous solution was performed. The thermodynamic parameters obtained for the protonation of glycine-, (S)-alpha-alanine-, (R,S)-valine-, (S)-leucine-, beta-alanine- and (R)-aspartic-beta-hydroxamic acids were compared with those previously reported for gamma-amino- and (S)-glutamic-gamma-hydroxamic acids. The enthalpy/entropy parameters calculated for the protonation microequilibria of these three types of ligands are in very good agreement with the literature values for simple amines and hydroxamic acids. The pentanuclear complexes [Cu5L4H(-4)]2+ contain the ligands acting as (NH2,N-)-(O,O-) bridging bis-chelating and correspond to 12-metallacrown-4 (12-MC-4) which are formed by self-assembly between pH 4 and 6 with alpha-aminohydroxamates (HL), while those with beta- and gamma-derivatives exist in a wider pH range (4-11). The stability order of these metallomacrocycles is beta- >> alpha- > gamma-aminohydroxamates. The formation of 12-MC-4 with alpha-aminohydroxamates is entropy-driven, and that with beta-derivatives is enthalpy-driven, while with gamma-GABAhydroxamate both effects occur. These results are interpreted on the basis of specific enthalpies or entropy contributions related to chelate ring dimensions, charge neutralization and solvation-desolvation effects. The enthalpy/entropy parameters of 12-MC-4 with alpha-aminohydroxamic acids considered are also dependent on the optical purity of the ligands. Actually, that with (R,S)-valinehydroxamic acid presents an higher entropy and a lower enthalpy value than those of enantiopure ligands, although the corresponding stabilities are almost equivalent. Moreover, DFT calculations are in agreement with a more exothermic enthalpy found for metallacrowns with enantiomerically pure ligands.