Cancer remains a leading cause of global mortality, with metastasis accounting for nearly 90% of related deaths. Matrix metalloproteinases (MMPs), and in particular MMP-1, play a pivotal role in tumor progression by degrading extracellular matrix components through a Zn(II)-dependent catalytic mechanism. Targeting the Zn(II) ion in the active site represents a potential approach for inhibitor design. In this study, we designed and investigated substrate-mimicking peptide inhibitors incorporating cysteine residues as zinc-binding groups (ZBGs) at distinct positions: CPQGLRG (Inh4, P4), PQGLCGR (Inh2', P2'), and PQGLRGC (Inh4', P4'). Using different techniques (potentiometry, mass spectrometry, NMR spectroscopy, and density functional theory calculations), we evaluated binary and ternary complexes formed between these peptides, Zn(II), and an MMP-1 active-site model. All inhibitors formed monomeric and bis(ligand) binary Zn(II)-complexes, with Inh4 demonstrating the highest thermodynamic stability. In ternary systems, the MMP-1 active site model served as the primary Zn(II) ligand coordinating through three histidine residues and reproducing the binding mode of the native enzyme. The inhibitors bound in the secondary step as the fourth coordination site, displacing the catalytic water. Ternary complexes of all inhibitors were predominant species formed above pH 6, coinciding with the optimal pH for MMP-1's activity. Among the peptides, Inh4, which stabilized ternary complexes most effectively, coordinates Zn(II) via its N-terminal amine. This binding mode is analogous to the strategy of tissue inhibitors of metalloproteinases. In contrast, Inh2' and Inh4' required structural rearrangements for Zn(II) coordination and formed less stable complexes due to steric constraints. The findings of this study identify N-terminal cysteine as the most effective ZBG placement for stabilizing Zn(II)-MMP-1 complexes, highlighting Inh4 as a promising lead for peptide-based MMP-1 inhibition. This work provides preliminary insights to guide the rational design of selective metalloproteinase inhibitors with therapeutic potential in cancer treatment.

Metallothioneins (MTs) are central to metal metabolism and contribute to organismal adaptation to variable metal bioavailability across ecosystems. Although well studied in chordates and mollusks, MTs remain poorly investigated in many arthropod lineages, particularly within the Myriapoda subphylum. Myriapods, comprising thousands of millipede (Diplopoda) and centipede (Chilopoda) species, are especially relevant for evolutionary studies because they are the sister group to Pancrustacea (crustaceans and insects), and they are some of the earliest arthropods to colonize land. Their MTs therefore provide critical insights into the origin and evolution of arthropod MTs and into the molecular adaptations underlying the colonization of new environments. In this work, we have identified 48 putative MTs from 30 myriapod species, all classified as type 1 (MT1) and occurring in two configurations: the bidomain MT1S (S for short) or the multidomain MT1L (L for long) variants. Evolutionary analyses suggest that MT1S represents the ancestral type not only in myriapods but across Arthropoda, whereas MT1L likely arose during chilopod diversification, probably restricted to the order Glomerida. Despite shared structural features, metal-binding characterization of three myriapod MTs-GminMT1Sa, GminMT1La from Glomeridella minima, and LforMT1S from Lithobius forficatus-revealed marked functional differences. The diplopod proteins GminMT1Sa and GminMT1La displayed a Cd-thionein character, while the chilopod LforMT1S was a multipurpose protein, binding cadmium, zinc, and copper without a clear metal preference. These differences likely reflect distinct metal uptake, retention, and excretion strategies in diplopods and chilopods, associated with their ecological adaptations as peaceful decomposers and voracious predators, respectively.

Metallothioneins (MTs) are a diverse family of proteins involved in metal homeostasis and detoxification, enabling organisms to adapt to varying metal availability across ecosystems. While extensively studied in vertebrates and gastropods, MTs remain poorly characterized in many arthropods, particularly chelicerates. Identifying and characterizing chelicerate MTs is therefore key to deciphering how this diverse group of animals -which includes spiders, scorpions, and ticks- copes with metal-related challenges in different environments. In this study, we have identified over 450 MTs from 221 chelicerate species and classified them into three structurally and phylogenetically distinct types: MT1, MT2, and MT3. Bidomain MTs are the most widespread across chelicerate lineages, while short monodomain forms and large variants occur in specific groups. Metal-binding characterization of eight representative MTs have revealed diverse metal selectivity -including Zn-, Cd-, and Cu-thioneins, as well as multipurpose forms- and biding capacity, ranging from 3 to 13 divalent ions per molecule. Evolutionary analyses suggest that MT1s are ancestral, MT2s appeared in Euchelicerates, and MT3s likely emerged in spiders. These findings establish a framework for understanding the MT diversity of chelicerates and reveal functional adaptations of potential evolutionary and ecological relevance, particularly those related to their ability to inhabit ecosystems with widely varying metal availability.

Metallodrugs are widely used in the treatment of several diseases, including cancer. Many bloodstream proteins exhibit high affinities for metals and metal-based compounds, and these interactions have been shown to impact metallodrug pharmacokinetics and pharmacodynamics, ultimately influencing their therapeutic performance. Several spectroscopic, spectrometric, and computational techniques have been used to further understand drug–protein binding modes at the molecular level over the last few years. However, there is still plenty of room to ascertain the full potential of integrated experimental–computational methodologies in the field of metallodrugs. These studies have mostly been limited to a few metal-containing systems, such as platinates and vanadium drugs. In this work, we aim to expand and validate the use of combined experimental–theoretical approaches in studying copper (Cu)-based drug interactions with proteins. To do so, a tetracoordinated Cu(II) complex has been employed as a practical case study, and its interactions with several relevant proteins using different experimental and computational techniques have been evaluated, including electron paramagnetic resonance spectroscopy, mass spectrometry, density functional theory, and protein–ligand docking calculations. Experimental data highlight interactions of the Cu(II) complex with albumin, myoglobin, and cytochrome C. Further insights into the preferential modes of binding were evaluated by density functional theory and docking calculations, which revealed three main outer surface binding sites for both albumin and myoglobin, as well as three preferential inner pockets in the case of albumin. The most relevant binding modes found for both proteins include noncoordinative interactions with the Cu(II) dimeric structure intact upon binding, as well as metal–ligand exchange and direct metal–amino acid coordination, mainly to glutamate/aspartate residues after cleavage of the dimer. Altogether, an example of the applicability of a mixed experimental–theoretical framework in the study of Cu-based drug interactions with relevant proteins is shown.

Protein modularity is acknowledged for promoting the emergence of new protein variants via domain rearrangements. Metallothioneins (MTs) offer an excellent model system for experimentally examining the consequences of domain rearrangements due to the possibility to assess the functional properties of native and artificially created variants using spectroscopic methods and metal tolerance assays. In this study, we have investigated the functional properties of AbiMT4 from the snail Alinda biplicata (Gastropoda, Mollusca), a large MT comprising 10 putative β domains (β3 9 β1), alongside four artificially designed variants differing in domain number, type, or order. Our findings reveal that AbiMT4 is a cadmium-selective protein with a high metal-binding capacity, characterized by structurally and functionally independent domains repeated in tandem along the protein. Our results indicate that due to its modular organization, AbiMT4 remains functional even when the number, type, and order of the domains are significantly altered. Furthermore, we demonstrate that the metal-binding properties of AbiMT4 are not dictated by the overall architecture of the protein but primarily arise from the properties of each individual domain. Using MTs as example, this work provides empirical evidence that domain rearrangements are an effective strategy for exploring new viable sequences and creating novel protein variants subject to adaptive selection. Thus, our study highlights the importance of the modular structure of proteins, as increasing their functional flexibility enhances their evolvability. Additionally, our work demonstrates a simple way to design and model new proteins for predefined functions.

Upconversion nanoparticles (UCNPs) are a class of interesting nanomaterials with unique multi-photon excitation photoluminescence properties, and they have been intensively explored as novel contrast agents for biomedical imaging and drug delivery. The development of photoinduced drug-release devices has been intensively developed in the last years, specially using UCNPs due to their properties to absorb single-band near infrared (NIR) light and subsequently emit high-energy UV-to-visible light which could photoactivate several prodrugs. Some examples of Pt(II) release have been described, all of them from Pt(IV) complexes taking advantage of the Pt(IV)/(II) redox couple. In this work, NIR light-responsive LiYF4:Yb/Tm UCNPs are presented as carrier systems to exert photoinduced Pt(II) drug release. For this, the surface of UCNPs were coated with an amphiphilic polymer to convert hydrophobic nanoparticles into hydrophilic and to load novel Pt(II) complexes. It is demonstrated that NIR radiation-induced Pt(II) drug release can be achieved without the need to use the Pt(IV)/(II) redox couple as a trigger. In this way, under NIR excitation, UCNPs can transform NIR irradiation into UV radiation which causes direct Pt(II) drug release in a spatial and temporal control manner. The release process has been monitored in real-time. Two platforms containing two different Pt(II) complexes have been studied, both showing similar results in terms of the enhancement of toxicity caused by the increase in Pt(II) concentration. Furthermore, a significant improvement of cytotoxicity against melanoma A375 cells was observed after irradiation of these platforms, confirming the feasibility of the proposed upconversion process to release Pt(II).

Metallothioneins (MTs) are a heterogenous family of proteins involved in metal homeostasis and detoxification, playing a crucial role in the adaptation of organisms to varying conditions of metal bioavailability across ecosystems. Despite their significance, MT characterization across animal groups remains uneven. While MTs have been extensively studied in vertebrates and gastropods, their presence and function in many arthropods, particularly within the Chelicerata subphylum, remain largely unexplored. Chelicerates, comprising thousands of species of spiders, scorpions and ticks, are a highly diverse group of animals inhabiting a wide range of environments. Identifying and characterizing their MTs is therefore essential for understanding how they manage metal availability of diverse habitats. In this study, we have identified over 400 chelicerate MTs, classifying them into three distinct types -types 1, 2, and 3- each exhibiting significant structural and phylogenetic differences. Our findings have revealed that most chelicerate MTs are bidomain proteins, with notable exceptions of monodomain forms and larger variants. Evolutionary analysis suggests that type 1 MTs represent the ancestral form, while types 2 and 3 likely emerged during the evolution of spiders. These results pave the way of a deeper understanding of the metal homeostasis and detoxification in chelicerates, laying the groundwork for future studies on the origin, evolution and functional diversification of MTs in arthropods and other metazoan lineages. ### Competing Interest Statement The authors have declared no competing interest.

Recently, we have studied the coordination chemistry of the Cu(II)-histidine-rich C-terminal tail (HRCT) complex of the mycobacterial GroEL1 protein. The structure of this domain differs significantly compared to the well-known methionine-glycine-rich GroEL chaperonin - it was predicted that mycobacterial GroEL1 could play a significant role in the metal homeostasis of Mycobacteria, especially copper. However, we found that this particular domain's pattern also repeats in a number of Ni(II)-binding proteins. Here, we present the studies concerning the properties of GroEL1 HRCT as a ligand for Ni(II) ions. For this purpose, we chose eight model peptides: L1 - Ac-DHDHHHGHAH, L2 - Ac-DKPAKAEDHDHHHGHAH, and 6 mutants of the latter in the pH range of 2-11. We examined the stoichiometry, stability, and spectroscopic features of copper complexes. We noticed that similar to the Cu(II)-complex, the presence of a Lys5 residue significantly increases the stability of the system. The impact of His mutations was also examined and carefully studied using NMR spectroscopy. His9 and His13 are the crucial residues for Ni(II) binding, whereas His12 has minimal relevance in complex formation.

Metallothioneins (MTs) constitute an important family of metal binding proteins. Mollusk MTs, in particular, have been used as model systems to better understand the evolution of their metal binding features and functional adaptation. In the present study two recombinantly produced MTs, LgiMT1 and LgiMT2, and their de novo evolved γ domain, of the marine limpet Lottia gigantea , were analyzed by electronic spectroscopy and mass spectrometry. Both MT proteins, as well as their γ domains, exhibit a strong binding specificity for Cd(II), but not for Zn(II) or Cu(I). The LgiMTs’ γ domain renders an M II 4 (SCys) 10 cluster with an increased Cd stoichiometry (binding 4 instead of 3 Cd 2+ ions), representing a novel structural element in the world of MTs, probably featuring an adamantane 3D structure. This cluster significantly improves the Cd(II)-binding performance of the full length proteins and thus contributes to the particularly high Cd coping capacity observed in free-living limpets.

The rapid spread of antibiotic-resistant bacteria continuously raises concerns about the future ineffectiveness of current antimicrobial treatments against infectious diseases. To address this problem, new therapeutic strategies and antimicrobial drugs with unique modes of action are urgently needed. Inhibition of metalloproteases, bacterial virulence factors, is a promising target for the development of antibacterial treatments. In this study, the interaction among Zn(II), Cu(II), and the metal-binding domains of two metalloproteases, AprA (Pseudomonas aureginosa) and CpaA (Acinetobacter baumanii), was investigated. The objective was to determine the coordination sphere of Zn(II) with a peptide model of two zinc-dependent metalloproteases. Additionally, the study explored the formation of Cu(II) complexes with the domains, as Cu(II) has been shown to inhibit metalloproteases. The third aim was to understand the role of nonbinding amino acids in stabilizing the metal complexes formed by these proteases. This work identified specific coordination patterns (HExxHxxxxxH) for both Zn(II) and Cu(II) complexes, with AprA and CpaA exhibiting a higher affinity for Cu(II) compared to Zn(II). The study also found that the CpaA domain has greater stability for both Zn(II) and Cu(II) complexes compared to AprA. The nonbinding amino acids of CpaA surrounding the metal ion contribute to the increased thermodynamic stability of the metal-peptide complex through various intramolecular interactions. These interactions can also influence the secondary structures of the peptides. The presence of certain amino acids, such as tyrosine, arginine, and glutamic acid, and their interactions contribute to the stability and, only in the case of Cu(II) complexes, the formation of a rare protein structure called a left-handed polyproline II helix (PPII), which is known to play a role in the stability and function of various proteins. These findings provide valuable insights into the coordination chemistry of bacterial metalloproteases and expand our understanding of potential mechanisms for inhibiting these enzymes.

The mycobacterial histidine-rich GroEL1 protein differs significantly compared to the well-known methionine/glycine-rich GroEL chaperonin. It was predicted that mycobacterial GroEL1 can play a significant role in the metal homeostasis of Mycobacteria but not, as its analogue, in protein folding. In this paper, we present the properties of the GroEL1 His-rich C-terminus as a ligand for Cu(II) ions. We studied the stoichiometry, stability, and spectroscopic features of copper complexes of the eight model peptides: L1─Ac-DHDHHHGHAH, L2─Ac-DKPAKAEDHDHHHGHAH, and six mutants of L2 in the pH range of 2-11. We revealed the impact of adjacent residues to the His-rich fragment on the complex stability: the presence of Lys and Asp residues significantly increases the stability of the system. The impact of His mutations was also examined: surprisingly, the exchange of each single His to the Gln residue did not disrupt the ability of the ligand to provide three binding sites for Cu(II) ions. Despite the most possible preference of the Cu(II) ion for the His9-His13 residues (Ac-DKPAKAEDHDHHH-) of the model peptide, especially the His11 residue, the study shows that there is not only one possible binding mode for Cu(II). The significance of this phenomenon is very important for the GroEL1 function─if the single mutation occurs naturally, the protein would be still able to interact with the metal ion.

The design of artificial helicoidal molecules derived from metal ions with biological properties is one of the objectives within metallosupramolecular chemistry. Herein, we report three zinc helicates derived from a family of bisthiosemicarbazone ligands with different terminal groups, Zn2(LMe)2∙2H2O 1, Zn2(LPh)2∙2H2O 2 and Zn2(LPhNO2)23, obtained by an electrochemical methodology. These helicates have been fully characterized by different techniques, including X-ray diffraction. Biological studies of the zinc(II) helicates such as toxicity assays with erythrocytes and interaction studies with proteins and oligonucleotides were performed, demonstrating in all cases low toxicity and an absence of covalent interaction with the proteins and oligonucleotides. The in vitro cytotoxicity of the helicates was tested against MCF-7 (human breast carcinoma), A2780 (human ovarian carcinoma cells), NCI-H460 (human lung carcinoma cells) and MRC-5 (normal human lung fibroblasts), comparing the IC50 values with cisplatin. We will try to demonstrate if the terminal substituent of the ligand precursor exerts any effect in toxicity or in the antitumor activity of the zinc helicates.

Metal-based chemotherapeutics like cisplatin are widely employed in cancer treatment. In the last years, the design of redox-active (transition) metal complexes, such as of copper (Cu), has attracted high interest as alternatives to overcome platinum-induced side-effects. However, several challenges are still faced, including optimal aqueous solubility and efficient intracellular delivery, and strategies like the use of cell-penetrating peptides have been encouraging. In this context, we previously designed a Cu(II) scaffold that exhibited significant reactive oxygen species (ROS)-mediated cytotoxicity. Herein, we build upon the promising Cu(II) redox-active metallic core and aim to potentiate its anticancer activity by rationally tailoring it with solubility- and uptake-enhancing functionalizations that do not alter the ROS-generating Cu(II) center. To this end, sulfonate, arginine and arginine-rich cell-penetrating peptide (CPP) derivatives have been prepared and characterized, and all the resulting complexes preserved the parent Cu(II) coordination core, thereby maintaining its reported redox capabilities. Comparative in vitro assays in several cancer cell lines reveal that while specific solubility-targeting derivatizations (i.e., sulfonate or arginine) did not translate into an improved cytotoxicity, increased intracellular copper delivery via CPP-conjugation promoted an enhanced anticancer activity, already detectable at short treatment times. Additionally, immunofluorescence assays show that the Cu(II) peptide-conjugate distributed throughout the cytosol without lysosomal colocalization, suggesting potential avoidance of endosomal entrapment. Overall, the systematic exploration of the tailored modifications enables us to provide further understanding on structure-activity relationships of redox-active metal-based (Cu(II)) cytotoxic complexes, which contributes to rationalize and improve the design of more efficient redox-mediated metal-based anticancer therapy.

Protein domains are independent structural and functional modules that can rearrange to create new proteins. While the evolution of multidomain proteins through the shuffling of different preexisting domains has been well documented, the evolution of domain repeat proteins and the origin of new domains are less understood. Metallothioneins (MTs) provide a good case study considering that they consist of metal-binding domain repeats, some of them with a likely de novo origin. In mollusks, for instance, most MTs are bidomain proteins that arose by lineage-specific rearrangements between six putative domains: α, β1, β2, β3, γ and δ. Some domains have been characterized in bivalves and gastropods, but nothing is known about the MTs and their domains of other Mollusca classes. To fill this gap, we investigated the metal-binding features of NpoMT1 of Nautilus pompilius (Cephalopoda class) and FcaMT1 of Falcidens caudatus (Caudofoveata class). Interestingly, whereas NpoMT1 consists of α and β1 domains and has a prototypical Cd2+ preference, FcaMT1 has a singular preference for Zn2+ ions and a distinct domain composition, including a new Caudofoveata-specific δ domain. Overall, our results suggest that the modular architecture of MTs has contributed to MT evolution during mollusk diversification, and exemplify how modularity increases MT evolvability.

The synthesis, full characterization, photochemical properties, and cytotoxic activity toward cisplatin-resistant cancer cell lines of new semisquaraine-type Pt(II) complexes are presented. The synthesis of eight semisquaraine-type ligands has been carried out by means of an innovative, straightforward methodology. A thorough structural NMR and X-ray diffraction analysis of the new ligands and complexes has been done. Density functional theory calculations have allowed to assign the trans configuration of the platinum center. Through the structural modification of the ligands, it has been possible to synthesize some complexes, which have turned out to be photoactive at wavelengths that allow their activation in cell cultures and, importantly, two of them show remarkable solubility in biological media. Photodegradation processes have been studied in depth, including the structural identification of photoproducts, thus justifying the changes observed after irradiation. From biological assessment, complexes C7 and C8 have been demonstrated to behave as promising photoactivatable compounds in the assayed cancer cell lines. Upon photoactivation, both complexes are capable of inducing a higher cytotoxic effect on the tested cells compared with nonphotoactivated compounds. Among the observed results, it is remarkable to note that C7 showed a PI > 50 in HeLa cells, and C8 showed a PI > 40 in A2780 cells, being also effective over cisplatin-resistant A2780cis cells (PI = 7 and PI = 4, respectively). The mechanism of action of these complexes has been studied, revealing that these photoactivated platinum complexes would actually present a combined mode of action, a therapeutically potential advantage.

We show for the first time glycosylation of recombinant metallothioneins (MTs) produced in E. coli. Interestingly, our results show that the glycosylation level of the recombinant MTs is inversely proportional to the degree of protein structuration, and reflects their different metal preferences.

Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxification, and therefore, MTs have been crucial for the adaptation of the living beings to variable situations of metal bioavailability. The evolution of MTs is, however, not yet fully understood, and to provide new insights into it, we have investigated the MTs in the diverse classes of Mollusks. We have shown that most molluskan MTs are bimodular proteins that combine six domains-α, β1, β2, β3, γ, and δ-in a lineage-specific manner. We have functionally characterized the Neritimorpha β3β1 and the Patellogastropoda γβ1 MTs, demonstrating the metal-binding capacity of the new γ domain. Our results have revealed a modular organization of mollusk MT, whose evolution has been impacted by duplication, loss, and de novo emergence of domains. MTs represent a paradigmatic example of modular evolution probably driven by the structural and functional requirements of metal binding.

The synthesis and characterization of four platinum(II) complexes using azobenzenes conveniently functionalized as ligands has been carried out. The characteristic photochemical behavior of the complexes due to the presence of azobenzene-type ligands and the role of the ligands in the activation of the complexes has been studied. Their promising cytotoxicity observed in HeLa cells prompted us to study the mechanism of action of these complexes as cytostatic agents. The interaction of the compounds with DNA, studied by circular dichroism, revealed a differential activity of the Pt(II) complexes upon irradiation. The intercalation abilities of the complexes as well as their reactivity with common proteins present in the blood stream allows to confirm some of the compounds obtained as good anticancer candidates.

Metallothioneins’ (MTs) biological function has been a matter of debate since their discovery. The importance to categorize these cysteine-rich proteins with high coordinating capacity into a specific group led to numerous classification proposals. We proposed a classification based on their metal-binding abilities, gradually sorting them from those with high selectivity towards Zn/Cd to those that are Cu-specific. However, the study of the NpeMT1 and NpeMT2isoforms of Nerita peloronta, has put a new perspective on this classification. N. peloronta has been chosen as a representative mollusk to elucidate the metal-binding abilities of Neritimorpha MTs, an order without any MTs characterized recently. Both isoforms have been recombinantly synthesized in cultures supplemented with ZnII, CdII, or CuII, and the purified metal–MT complexes have been thoroughly characterized by spectroscopic and spectrometric methods, leading to results that confirmed that Neritimorpha share Cd-selective MTs with Caenogastropoda and Heterobranchia, solving a so far unresolved question. NpeMTs show high coordinating preferences towards divalent metal ions, although one of them (NpeMT1) shares features with the so-called genuine Zn-thioneins, while the other (NpeMT2) exhibits a higher preference for Cd. The dissimilarities between the two isoforms let a window open to a new proposal of chemical MT classification.