Cross-Linked Crystals of Dirhodium Tetraacetate/RNase A Adduct Can Be Used as Heterogeneous Catalysts.

Due to their unique coordination structure, dirhodium paddlewheel complexes are of interest in several research fields, like medicinal chemistry, catalysis, etc. Previously, these complexes were conjugated to proteins and peptides for developing artificial metalloenzymes as homogeneous catalysts. Fixation of dirhodium complexes into protein crystals is interesting to develop heterogeneous catalysts. Porous solvent channels present in protein crystals can benefit the activity by increasing the probability of substrate collisions at the catalytic Rh binding sites. Toward this goal, the present work describes the use of bovine pancreatic ribonuclease (RNase A) crystals with a pore size of 4 nm (P3221 space group) for fixing [Rh2(OAc)4] and developing a heterogeneous catalyst to perform reactions in an aqueous medium. The structure of the [Rh2(OAc)4]/RNase A adduct was investigated by X-ray crystallography: the metal complex structure remains unperturbed upon protein binding. Using a number of crystal structures, metal complex accumulation over time, within the RNase A crystals, and structures at variable temperatures were evaluated. We also report the large-scale preparation of microcrystals (∼10-20 µm) of the [Rh2(OAc)4]/RNase A adduct and cross-linking reaction with glutaraldehyde. The catalytic olefin cyclopropanation reaction and self-coupling of diazo compounds by these cross-linked [Rh2(OAc)4]/RNase A crystals were demonstrated. The results of this work reveal that these systems can be used as heterogeneous catalysts to promote reactions in aqueous solution. Overall, our findings demonstrate that the dirhodium paddlewheel complexes can be fixed in porous biomolecule crystals, like those of RNase A, to prepare biohybrid materials for catalytic applications.

Unexpected Imidazole Coordination to the Dirhodium Center in a Protein Environment: Insights from X-ray Crystallography and Quantum Chemistry.

X-ray diffraction data demonstrate that the adduct formed upon the reaction of dirhodium(II,II) tetraacetate with RNase A reacts with imidazole, leading to the formation of an unexpected product with the imidazole that binds the dirhodium center at an equatorial site rather than an axial site. The origin of this result has been dissected using quantum-chemical calculations.

Digging into protein metalation differences triggered by fluorine containing-dirhodium tetracarboxylate analogues.

The catalytic and biological properties of dirhodium tetracarboxylates ([Rh2(µ-O2CR)4L2], L = axial ligand and R = CH3-, CH3CH2-, etc.) largely depend on the nature of bridging carboxylate equatorial µ-O2CR ligands, which can be easily exchanged by solvent molecules when R is CF3 (i.e. µ-O2CR is trifluoroacetate, tfa). Here, we prepared the [Rh2(OAc)(tfa)3] compound and investigated its interaction with bovine pancreatic ribonuclease and lysozyme under the same conditions used to study the reactivity of these proteins with [Rh2(OAc)4] and [cis-Rh2(OAc)2(tfa)2]. UV-vis absorption spectroscopy and 19F nuclear magnetic resonance studies indicate that [Rh2(OAc)(tfa)3] rapidly loses tfa ligands and interacts with the proteins. Crystallographic data demonstrate that the reaction of [Rh2(OAc)(tfa)3] with proteins can lead to products that are significantly different when compared to those obtained with [Rh2(OAc)4] and [cis-Rh2(OAc)2(tfa)2]: the dirhodium centre can bind the side chain of His residues at both axial and equatorial sites, at variance with what is found in the case of [Rh2(OAc)4] and [cis-Rh2(OAc)2(tfa)2]. These data indicate that the hydrolysis of dirhodium tetracarboxylates plays a significant role in defining their reaction with proteins allowing the formation of unexpected reaction products. These results suggest that [cis-Rh2(OAc)2(tfa)2] and [Rh2(OAc)(tfa)3] can be used to obtain different dirhodium/peptide and dirhodium/protein adducts with distinct catalytic properties and can explain the different cytotoxicity exhibited by tfa-containing dirhodium tetracarboxylates.

Protein-Based Delivery Systems for Anticancer Metallodrugs: Structure and Biological Activity of the Oxaliplatin/ß-Lactoglobulin Adduct.

ß-lactoglobulin is the major component of whey. Here, the adduct formed upon the reaction of the protein with oxaliplatin (OXA) has been prepared, structurally characterized by X-ray crystallography and electrospray ionization-mass spectrometry, and evaluated as a cytotoxic agent. The data demonstrate that OXA rapidly binds ß-lactoglobulin via coordination with a Met7 side chain upon release of the oxalate ligand. The adduct is significantly more cytotoxic than the free drug and induces apoptosis in cancer cells. Overall, our results suggest that metallodrug/ß-lactoglobulin adducts can be used as anticancer agents and that the protein can be used as a metallodrug delivery system.

Reactivity of a fluorine-containing dirhodium tetracarboxylate compound with proteins.

Dirhodium complexes of general formula [Rh2(O2CR)4]L2 are a well-known class of bimetallic compounds that are used as efficient catalysts for a variety of reactions and have been shown to be potent antibacterial and anticancer agents. The catalytic and biological properties of these complexes largely depend on the nature of the bridging carboxylate ligands. Trifluoroacetate (tfa)-containing dirhodium compounds have been used to build artificial metalloenzymes upon reaction with peptides and have been shown to be more cytotoxic than dirhodium tetraacetate. However, there is no structural information on the interaction between these compounds and proteins. Here, cis-Rh2(µ-O2CCH3)2(µ-O2CCF3)2 ([cis-Rh2(OAc)2(tfa)2]) has been synthesized and its reaction with bovine pancreatic ribonuclease (RNase A) and hen egg white lysozyme (HEWL) was analyzed using a combination of different techniques, including Fluorine-19 nuclear magnetic resonance spectroscopy and macromolecular X-ray crystallography, with the aim to unveil the differences in the reactivity of tfa-containing dihrodium complexes with proteins when compared to [Rh2(OAc)4]. [cis-Rh2(OAc)2(tfa)2] and [Rh2(OAc)4] bind the N atoms of His side chains of RNase A at the axial position; however the fluorine-containing compound rapidly loses its tfa ligands, while [Rh2(OAc)4] can retain the acetate ligands upon protein binding. The reactivity of [cis-Rh2(OAc)2(tfa)2] with HEWL is slightly distinct when compared to that of [Rh2(OAc)4] under the same experimental conditions; however, both [cis-Rh2(OAc)2(tfa)2] and [Rh2(OAc)4] degrade when soaked within HEWL crystals. These results provide a structural-based guide for the design of new heterogenous chiral dirhodium/peptide and dirhodium/protein adducts with application in the fields of organic synthesis and asymmetric catalysis.

The crystal structure of the domain-swapped dimer of onconase highlights some catalytic and antitumor activity features of the enzyme.

Onconase (ONC) is a monomeric amphibian "pancreatic-type" RNase endowed with remarkable anticancer activity. ONC spontaneously forms traces of a dimer (ONC-D) in solution, while larger amounts can be formed when ONC is lyophilized from mildly acidic solutions. Here, we report the crystal structure of ONC-D and analyze its catalytic and antitumor activities in comparison to ONC. ONC-D forms via the three-dimensional swapping of the N-terminal α-helix between two monomers, but it displays a significantly different quaternary structure from that previously modeled [Fagagnini A et al., 2017, Biochem J 474, 3767-81], and based on the crystal structure of the RNase A N-terminal swapped dimer. ONC-D presents a variable quaternary assembly deriving from a variable open interface, while it retains a catalytic activity that is similar to that of ONC. Notably, ONC-D displays antitumor activity against two human melanoma cell lines, although it exerts a slightly lower cytostatic effect than the monomer. The inhibition of melanoma cell proliferation by ONC or ONC-D is associated with the reduction of the expression of the anti-apoptotic B cell lymphoma 2 (Bcl2), as well as of the total expression and phosphorylation of the Signal Transducer and Activator of Transcription (STAT)-3. Phosphorylation is inhibited in both STAT3 Tyr705 and Ser727 key-residues, as well as in its upstream tyrosine-kinase Src. Consequently, both ONC species should exert their anti-cancer action by inhibiting the pro-tumor pleiotropic STAT3 effects deriving either by its phospho-tyrosine activation or by its non-canonical signaling pathways. Both ONC species, indeed, increase the portion of A375 cells undergoing apoptotic cell death. This study expands the variety of RNase domain-swapped dimeric structures, underlining the unpredictability of the open interface arrangement upon domain swapping. Structural data also offer valuable insights to analyze the differences in the measured ONC or ONC-D biological activities.

Square-Planar vs. Trigonal Bipyramidal Geometry in Pt(II) Complexes Containing Triazole-Based Glucose Ligands as Potential Anticancer Agents.

This article describes the synthesis, characterization, and biological activity of novel square-planar cationic platinum(II) complexes containing glucoconjugated triazole ligands and a comparison with the results obtained from the corresponding five-coordinate complexes bearing the same triazole ligands. Stability in solution, reactivity with DNA and small molecules of the new compounds were evaluated by NMR, fluorescence, and UV-vis absorption spectroscopy, together with their cytotoxic action against pairs of immortalized and tumorigenic cell lines. The results show that the square-planar species exhibit greater stability than the corresponding five-coordinate ones. Furthermore, although the square-planar complexes are less cytotoxic than the latter ones, they exhibit a certain selectivity. These results simultaneously demonstrate that overall stability is a fundamental prerequisite for preserving the performance of the agents and that coordinative saturation constitutes a point in favor of their biological action.

Unusual Structural Features in the Adduct of Dirhodium Tetraacetate with Lysozyme.

The structures of the adducts formed upon reaction of the cytotoxic paddlewheel dirhodium complex [Rh2(µ-O2CCH3)4] with the model protein hen egg white lysozyme (HEWL) under different experimental conditions are reported. Results indicate that [Rh2(µ-O2CCH3)4] extensively reacts with HEWL:it in part breaks down, at variance with what happens in reactions with other proteins. A Rh center coordinates the side chains of Arg14 and His15. Dimeric Rh-Rh units with Rh-Rh distances between 2.3 and 2.5 Å are bound to the side chains of Asp18, Asp101, Asn93, and Lys96, while a dirhodium unit with a Rh-Rh distance of 3.2-3.4 Å binds the C-terminal carboxylate and the side chain of Lys13 at the interface between two symmetry-related molecules. An additional monometallic fragment binds the side chain of Lys33. These data, which are supported by replicated structural determinations, shed light on the reactivity of dirhodium tetracarboxylates with proteins, providing useful information for the design of new Rh-containing biomaterials with an array of potential applications in the field of catalysis or of medicinal chemistry and valuable insight into the mechanism of action of these potential anticancer agents.

Interaction of Platinum-based Drugs with Proteins: An Overview of Representative Crystallographic Studies.

Pt-based drugs are widely used in clinics for the treatment of cancer. The mechanism of action of these molecules relies on their interaction with DNA. However, the recognition of these metal compounds by proteins plays an important role in defining pharmacokinetics, side effects and their overall pharmacological profiles. Single crystal X-ray diffraction studies provided important information on the molecular mechanisms at the basis of this process. Here, the molecular structures of representative adducts obtained upon reaction with proteins of selected Pt-based drugs, including cisplatin, carboplatin and oxaliplatin, are briefly described and comparatively examined. Data indicate that metal ligands play a significant role in driving the reaction of Pt compounds with proteins; non-covalent interactions that occur in the early steps of Pt compound/protein recognition process play a crucial role in defining the structure of the final Pt-protein adduct. In the metallated protein structures, Pt centers coordinate few protein side chains, such as His, Met, Cys, Asp, Glu and Lys residues upon releasing labile ligands.

Cisplatin binding to ß-lactoglobulin: a structural study.

ß-Lactoglobulin is a major globular milk whey carrier with potential applications as an oral drug delivery system. Herein, the interactions between ß-lactoglobulin and cisplatin are investigated by UV-Vis absorption spectroscopy, circular dichroism, X-ray crystallography and electrospray ionization mass spectrometry. Structural data indicate that the protein retains its conformation upon cisplatin binding. Pt-containing fragments bind the side chains of Met7, His146 and Lys8, with the number of binding sites increasing over time. Mass spectrometry data indicate that [Pt(NH3)2Cl+], [Pt(NH3)2OH22+] and [Pt(NH3)22+] fragments interact with ß-lactoglobulin; up to 3 cisplatin fragments can bind the protein and the number of cisplatin binding sites increases over time. This work opens a new pathway in pharmaceutical studies based on a rational design of metal-based drug/ß-lactoglobulin adducts as delivering vehicles of metallodrugs.

Protein-metallodrugs interactions: Effects on the overall protein structure and characterization of Au, Ru and Pt binding sites.

The effects of metalation process by inorganic compounds containing Au, Pt and Ru on protein structure and on conformation and flexibility of the residues involved in the metal compound binding have been here investigated by analysing 204 structures of protein/metallodrug adducts and the corresponding metal-free forms. The overall structure of the proteins is not significantly affected by the metal label. 162 non-redundant protein residues involved in Au, Pt and Ru coordination have been identified. In the metal-free protein structures these residues often belong to α-helical regions and show low flexibility. They do not necessarily belong to outer layers of the protein structure. In the majority of the adducts, the side chains of these residues adopt a conformation that is similar to that observed in the metal-free protein. The metal coordination reduces their solvent accessible surface area without altering their overall flexibility. These results could be useful for the prediction of residues able to bind Au, Pt and Ru compounds.