The redox-active defensive Selenoprotein T as a novel stress sensor protein playing a key role in the pathophysiology of heart failure.

Maladaptive cardiac hypertrophy contributes to the development of heart failure (HF). The oxidoreductase Selenoprotein T (SELENOT) emerged as a key regulator during rat cardiogenesis and acute cardiac protection. However, its action in chronic settings of cardiac dysfunction is not understood. Here, we investigated the role of SELENOT in the pathophysiology of HF: (i) by designing a small peptide (PSELT), recapitulating SELENOT activity via the redox site, and assessed its beneficial action in a preclinical model of HF [aged spontaneously hypertensive heart failure (SHHF) rats] and against isoproterenol (ISO)-induced hypertrophy in rat ventricular H9c2 and adult human AC16 cardiomyocytes; (ii) by evaluating the SELENOT intra-cardiomyocyte production and secretion under hypertrophied stimulation. Results showed that PSELT attenuated systemic inflammation, lipopolysaccharide (LPS)-induced macrophage M1 polarization, myocardial injury, and the severe ultrastructural alterations, while counteracting key mediators of cardiac fibrosis, aging, and DNA damage and restoring desmin downregulation and SELENOT upregulation in the failing hearts. In the hemodynamic assessment, PSELT improved the contractile impairment at baseline and following ischemia/reperfusion injury, and reduced infarct size in normal and failing hearts. At cellular level, PSELT counteracted ISO-mediated hypertrophy and ultrastructural alterations through its redox motif, while mitigating ISO-triggered SELENOT intracellular production and secretion, a phenomenon that presumably reflects the extent of cell damage. Altogether, these results indicate that SELENOT could represent a novel sensor of hypertrophied cardiomyocytes and a potential PSELT-based new therapeutic approach in myocardial hypertrophy and HF.

Mechanism of Action of Antitumor Au(I) N-Heterocyclic Carbene Complexes: A Computational Insight on the Targeting of TrxR Selenocysteine.

The targeting of human thioredoxin reductase is widely recognized to be crucially involved in the anticancer properties of several metallodrugs, including Au(I) complexes. In this study, the mechanism of reaction between a set of five N-heterocyclic carbene Au(I) complexes and models of the active Sec residue in human thioredoxin reductase was investigated by means of density functional theory approaches. The study was specifically addressed to the kinetics and thermodynamics of the tiled process by aiming at elucidating and explaining the differential inhibitory potency in this set of analogous Au(I) bis-carbene complexes. While the calculated free energy profile showed a substantially similar reactivity, we found that the binding of these Au(I) bis-carbene at the active CysSec dyad in the TrxR enzyme could be subjected to steric and orientational restraints, underlining both the approach of the bis-carbene scaffold and the attack of the selenol group at the metal center. A new and detailed mechanistic insight to the anticancer activity of these Au(I) organometallic complexes was thus provided by consolidating the TrxR targeting paradigm.

Inorganic Drugs as a Tool for Protein Structure Solving and Studies on Conformational Changes.

Inorganic drugs are capable of tight interactions with proteins through coordination towards aminoacidic residues, and this feature is recognized as a key aspect for their pharmacological action. However, the "protein metalation process" is exploitable for solving the phase problem and structural resolution. In fact, the use of inorganic drugs bearing specific metal centers and ligands capable to drive the binding towards the desired portions of the protein target could represent a very intriguing and fruitful strategy. In this context, a theoretical approach may further contribute to solve protein structures and their refinement. Here, we delineate the main features of a reliable experimental-theoretical integrated approach, based on the use of metallodrugs, for protein structure solving.

Mechanistic Evaluations of the Effects of Auranofin Triethylphosphine Replacement with a Trimethylphosphite Moiety.

Auranofin, a gold(I)-based complex, is under clinical trials for application as an anticancer agent for the treatment of nonsmall-cell lung cancer and ovarian cancer. In the past years, different derivatives have been developed, modifying gold linear ligands in the search for new gold complexes endowed with a better pharmacological profile. Recently, a panel of four gold(I) complexes, inspired by the clinically established compound auranofin, was reported by our research group. As described, all compounds possess an [Au{P(OMe)3}]+ cationic moiety, in which the triethylphosphine of the parent compound auranofin was replaced with an oxygen-rich trimethylphosphite ligand. The gold(I) linear coordination geometry was complemented by Cl-, Br-, I-, and the auranofin-like thioglucose tetraacetate ligand. As previously reported, despite their close similarity to auranofin, the panel compounds exhibited some peculiar and distinctive features, such as lower log P values which can induce relevant differences in the overall pharmacokinetic profiles. To get better insight into the P-Au strength and stability, an extensive study was carried out for relevant biological models, including three different vasopressin peptide analogues and cysteine, using 31P NMR and LC-ESI-MS. A DFT computational study was also carried out for a better understanding of the theoretical fundamentals of the disclosed differences with regard to triethylphosphine parent compounds.

Forensic Proteomics for the Discovery of New post mortem Interval Biomarkers: A Preliminary Study.

Estimating the time since death (post mortem interval, PMI) represents one of the most important tasks in daily forensic casework. For decades, forensic scientists have investigated changes in post mortem body composition, focusing on different physical, chemical, or biological aspects, to discover a reliable method for estimating PMI; nevertheless, all of these attempts remain unsuccessful considering the currently available methodical spectrum characterized by great inaccuracies and limitations. However, recent promising approaches focus on the post mortem decomposition of biomolecules. In particular, significant advances have been made in research on the post mortem degradation of proteins. In the present study, we investigated early post mortem changes (during the first 24 h) in the proteome profile of the pig skeletal muscle looking for new PMI specific biomarkers. By mass spectrometry (MS)-based proteomics, we were able to identify a total of nine potential PMI biomarkers, whose quantity changed constantly and progressively over time, directly or inversely proportional to the advancement of post mortem hours. Our preliminary study underlines the importance of the proteomic approach in the search for a reliable method for PMI determination and highlights the need to characterize a large number of reliable marker proteins useful in forensic practice for PMI estimation.

Kinetics of Reactions of Dirhodium and Diruthenium Paddlewheel Tetraacetate Complexes with Nucleophilic Protein Sites: Computational Insights.

Recently, dirhodium and diruthenium paddlewheel complexes have drawn attention as perspective anticancer drugs. In this study, the kinetics of reaction of typical paddlewheel scaffolds Rh2(µ-O2CCH3)4(H2O)2, Ru2(µ-O2CCH3)4(H2O)Cl, and [Ru2(µ-O2CCH3)4(HO)Cl]- with protein nucleophiles were investigated by means of the density functional theory. The substitution of axial ligands─water and chloride─by the models of protein residue side chains was analyzed, revealing the binding selectivity displayed by these paddlewheel metal scaffolds. The substitution of water is under a thermodynamic control, in which, although the Arg, Cys-, and Sec- residues are the most favorable, their binding is expected to be scarcely selective in a biological context. On the other hand, the replacement of the axial water with a more stable hydroxo ligand induces the chloride substitution in diRu complexes, which also targets Arg, Cys-, and Sec-, although with a moderately higher activation barrier for any examined protein residue. Additionally, the carried out characterization of the geometrical parameters of the transition states permitted determination of the impact of an increased steric hindrance of diRh and diRu complexes on their protein site selectivity. This study corroborates the idea of the substitution of the acetate ligands with biologically active, but more hindering, carboxylate ligands, in order to yield dual acting metallodrugs. This study allows us to assume that the delivery of diRu paddlewheel complexes in their monoanionic form [Ru2(µ-O2CR)4(OH)Cl]- decorated by the bulky substituents R may constitute an approach to augment the selectivity toward anticancer targets, such as TrxR in tumor cells, although under the condition that such a selectivity is operative only in high pH conditions.

Reactivity of N-Heterocyclic Carbene Half-Sandwich Ru-, Os-, Rh-, and Ir-Based Complexes with Cysteine and Selenocysteine: A Computational Study.

The structure and the reactivity of four half-sandwich metal complexes of RuII, OsII, RhIII, and IrIII were investigated by means of density functional theory approaches. These piano-stool complexes, grouped in cym-bound complexes, RuII(cym)(dmb)Cl2, 1, and OsII(cym)(dmb)Cl2, 2, and Cp*-bound complexes, RhIII(Cp*)(dmb)Cl2, 3, and IrIII(Cp*)(dmb)Cl2, 4, with cym = η6-p-cymene, Cp* = η5-pentamethylcyclopentadienyl, and dmb = 1,3-dimethylbenzimidazol-2-ylidene, were recently proposed as anticancer metallodrugs that preferably target Cys- or Sec-containing proteins. Thus, density functional theory calculations were performed here to characterize in detail the thermodynamics and the kinetics underlining the targeting of these metallodrugs at either neutral or anionic Cys and Sec side chains. Calculations evidenced that all these complexes preferably target at Cys or Sec via chloro exchange, although cym-bound and Cp*-bound complexes resulted to be more prone to bind at neutral or anionic forms, respectively, of these soft protein sites. Further decomposition analyses of the activation free energies for the reaction between 1-4 complexes and either Cys or Sec, paralleled with the comparison among the optimized transition-state structures, allowed us to spotlight the significant role played by solvation in determining the overall reactivity and selectivity expected for these prototypical metallodrugs.

Structural Reshaping of the Zinc-Finger Domain of the SARS-CoV-2 nsp13 Protein Using Bismuth(III) Ions: A Multilevel Computational Study.

The identification of novel therapeutics against the pandemic SARS-CoV-2 infection is an indispensable new address of current scientific research. In the search for anti-SARS-CoV-2 agents as alternatives to the vaccine or immune therapeutics whose efficacy naturally degrades with the occurrence of new variants, the salts of Bi3+ have been found to decrease the activity of the Zn2+-dependent non-structural protein 13 (nsp13) helicase, a key component of the SARS-CoV-2 molecular tool kit. Here, we present a multilevel computational investigation based on the articulation of DFT calculations, classical MD simulations, and MIF analyses, focused on the examination of the effects of Bi3+/Zn2+ exchange on the structure and molecular interaction features of the nsp13 protein. Our calculations confirmed that Bi3+ ions can replace Zn2+ in the zinc-finger metal centers and cause slight but appreciable structural modifications in the zinc-binding domain of nsp13. Nevertheless, by employing an in-house-developed ATOMIF tool, we evidenced that such a Bi3+/Zn2+ exchange may decrease the extension of a specific hydrophobic portion of nsp13, responsible for the interaction with the nsp12 protein. The present study provides for a detailed, atomistic insight into the potential anti-SARS-CoV-2 activity of Bi3+ and, more generally, evidences the hampering of the nsp13-nsp12 interaction as a plausible therapeutic strategy.

Reactions of Arsenoplatin-1 with Protein Targets: A Combined Experimental and Theoretical Study.

Arsenoplatin-1 (AP-1) is a dual-action anticancer metallodrug with a promising pharmacological profile that features the simultaneous presence of a cisplatin-like center and an arsenite center. We investigated its interactions with proteins through a joint experimental and theoretical approach. The reactivity of AP-1 with a variety of proteins, including carbonic anhydrase (CA), superoxide dismutase (SOD), myoglobin (Mb), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and human serum albumin (HSA), was analyzed by means of electrospray ionization mass spectrometry (ESI MS) measurements. In accordance with previous observations, ESI MS experiments revealed that the obtained metallodrug-protein adducts originated from the binding of the [(AP-1)-Cl]+ fragment to accessible protein residues. Remarkably, in two cases, i.e., Mb and GAPDH, the formation of a bound metallic fragment that lacked the arsenic center was highlighted. The reactions of AP-1 with various nucleophiles side chains of neutral histidine, methionine, cysteine, and selenocysteine, in neutral form as well as cysteine and selenocysteine in anionic form, were subsequently analyzed through a computational approach. We found that the aquation of AP-1 is energetically disfavored, with a reaction free energy of +19.2 kcal/mol demonstrating that AP-1 presumably attacks its biological targets through the exchange of the chloride ligand. The theoretical analysis of thermodynamics and kinetics for the ligand-exchange processes of AP-1 with His, Met, Cys, Sec, Cys-, and Sec- side chain models unveils that only neutral histidine and deprotonated cysteine and selenocysteine are able to effectively replace the chloride ligand in AP-1.

The FMO2 analysis of the ligand-receptor binding energy: the Biscarbene-Gold(I)/DNA G-Quadruplex case study.

In this work, the ab initio fragment molecular orbital (FMO) method was applied to calculate and analyze the binding energy of two biscarbene-Au(I) derivatives, [Au(9-methylcaffein-8-ylidene)2]+ and [Au(1,3-dimethylbenzimidazol-2-ylidene)2]+, to the DNA G-Quadruplex structure. The FMO2 binding energy considers the ligand-receptor complex as well as the isolated forms of energy-minimum state of ligand and receptor, providing a better description of ligand-receptor affinity compared with simple pair interaction energies (PIE). Our results highlight important features of the binding process of biscarbene-Au(I) derivatives to DNA G-Quadruplex, indicating that the total deformation-polarization energy and desolvation penalty of the ligands are the main terms destabilizing the binding. The pair interaction energy decomposition analysis (PIEDA) between ligand and nucleobases suggest that the main interaction terms are electrostatic and charge-transfer energies supporting the hypothesis that Au(I) ion can be involved in π-cation interactions further stabilizing the ligand-receptor complex. Moreover, the presence of polar groups on the carbene ring, as C = O, can improve the charge-transfer interaction with K+ ion. These findings can be employed to design new powerful biscarbene-Au(I) DNA-G quadruplex binders as promising anticancer drugs. The procedure described in this work can be applied to investigate any ligand-receptor system and is particularly useful when the binding process is strongly characterized by polarization, charge-transfer and dispersion interactions, properly evaluated by ab initio methods.

Medicinal Hypervalent Tellurium Prodrugs Bearing Different Ligands: A Comparative Study of the Chemical Profiles of AS101 and Its Halido Replaced Analogues.

Ammonium trichloro (dioxoethylene-O,O') tellurate (AS101) is a potent immunomodulator prodrug that, in recent years, entered various clinical trials and was tested for a variety of potential therapeutic applications. It has been demonstrated that AS101 quickly activates in aqueous milieu, producing TeOCl3-, which likely represents the pharmacologically active species. Here we report on the study of the activation process of AS101 and of two its analogues. After the synthesis and characterization of AS101 and its derivatives, we have carried out a comparative study through a combined experimental and computational analysis. Based on the obtained results, we describe here, for the first time, the detailed reaction that AS101 and its bromido- and iodido-replaced analogues undergo in presence of water, allowing the conversion of the original molecule to the likely true pharmacophore. Interestingly, moving down in the halogens' group we observed a higher tendency to react, attributable to the ligands' effect. The chemical and mechanistic implications of these meaningful differences are discussed.

Anti-Staphylococcal Activity of the Auranofin Analogue Bearing Acetylcysteine in Place of the Thiosugar: An Experimental and Theoretical Investigation.

Auranofin (AF, hereafter) is an orally administered chrysotherapeutic agent approved for the treatment of rheumatoid arthritis that is being repurposed for various indications including bacterial infections. Its likely mode of action involves the impairment of the TrxR system through the binding of the pharmacophoric cation [AuPEt3]+. Accordingly, a reliable strategy to expand the medicinal profile of AF is the replacement of the thiosugar moiety with different ligands. Herein, we aimed to prepare the AF analogue bearing the acetylcysteine ligand (AF-AcCys, hereafter) and characterize its anti-staphylococcal activity. Biological studies revealed that AF-AcCys retains an antibacterial effect superimposable with that of AF against Staphylococcus aureus, whereas it is about 20 times less effective against Staphylococcus epidermidis. Bioinorganic studies confirmed that upon incubation with human serum albumin, AF-AcCys, similarly to AF, induced protein metalation through the [AuPEt3]+ fragment. Additionally, AF-AcCys appeared capable of binding the dodecapeptide Ac-SGGDILQSGCUG-NH2, corresponding to the tryptic C-terminal fragment (488-499) of hTrxR. To shed light on the pharmacological differences between AF and AF-AcCys, we carried out a comparative experimental stability study and a theoretical estimation of bond dissociation energies, unveiling the higher strength of the Au-S bond in AF-AcCys. From the results, it emerged that the lower lipophilicity of AF-AcCys with respect to AF could be a key feature for its different antibacterial activity. The differences and similarities between AF and AF-AcCys are discussed, alongside the opportunities and consequences that chemical structure modifications imply.

Hampering the early aggregation of PrP-E200K protein by charge-based inhibitors: a computational study.

A multilayered computational workflow was designed to identify a druggable binding site on the surface of the E200K pathogenic mutant of the human prion protein, and to investigate the effect of the binding of small molecules in the inhibition of the early aggregation of this protein. At this purpose, we developed an efficient computational tool to scan the molecular interaction properties of a whole MD trajectory, thus leading to the characterization of plausible binding regions on the surface of PrP-E200K. These structural data were then employed to drive structure-based virtual screening and fragment-based approaches to the seeking of small molecular binders of the PrP-E200K. Six promising compounds were identified, and their binding stabilities were assessed by MD simulations. Therefore, analyses of the molecular electrostatic potential similarity between the bound complexes and unbound protein evidenced their potential activity as charged-based inhibitors of the PrP-E200K early aggregation.

Computational Studies of Au(I) and Au(III) Anticancer MetalLodrugs: A Survey.

Owing to the growing hardware capabilities and the enhancing efficacy of computational methodologies, computational chemistry approaches have constantly become more important in the development of novel anticancer metallodrugs. Besides traditional Pt-based drugs, inorganic and organometallic complexes of other transition metals are showing increasing potential in the treatment of cancer. Among them, Au(I)- and Au(III)-based compounds are promising candidates due to the strong affinity of Au(I) cations to cysteine and selenocysteine side chains of the protein residues and to Au(III) complexes being more labile and prone to the reduction to either Au(I) or Au(0) in the physiological milieu. A correct prediction of metal complexes' properties and of their bonding interactions with potential ligands requires QM computations, usually at the ab initio or DFT level. However, MM, MD, and docking approaches can also give useful information on their binding site on large biomolecular targets, such as proteins or DNA, provided a careful parametrization of the metal force field is employed. In this review, we provide an overview of the recent computational studies of Au(I) and Au(III) antitumor compounds and of their interactions with biomolecular targets, such as sulfur- and selenium-containing enzymes, like glutathione reductases, glutathione peroxidase, glutathione-S-transferase, cysteine protease, thioredoxin reductase and poly (ADP-ribose) polymerase 1.

Short and Long Time Bloodstains Age Determination by Colorimetric Analysis: A Pilot Study.

Bloodstains found at crime scenes represent a crucial source of information for investigative purposes. However, in forensic practice, no technique is currently used to estimate the time from deposition of bloodstains. This preliminary study focuses on the age estimation of bloodstains by exploiting the color variations over time due to the oxidation of the blood. For this purpose, we used a colorimetric methodology in order to easily obtain objective, univocal and reproducible results. We developed two bloodstain age prediction algorithms: a short-term and a long-term useful model for the first 24h and 60 days, respectively. Both models showed high levels of classification accuracy, particularly for the long-term model. Although a small-scale study, these results improve the potential application of colorimetric analysis in the time-line reconstruction of violent criminal events.

Insight into the Substitution Mechanism of Antitumor Au(I) N-Heterocyclic Carbene Complexes by Cysteine and Selenocysteine.

We carried out a detailed theoretical study on the mechanism of the carbene ligand substitution by cysteine and selenocysteine residues in an Au(I) bis-N-heterocyclic carbene complex in order to model the initial stages of the mechanism of action of this promising class of antitumor metallodrug. Both neutral and deprotonated capped Cys and Sec species were considered as possible nucleophiles in the ligand exchange reaction on the metal center to model the corresponding protein side chains. Energies and geometric structures of the possible transition states and reactant- and product-adducts involved in the substitution process have been calculated using density functional theory and local MP2. Reaction and activation enthalpies and free energies have been evaluated and indicate a slightly exothermic and exergonic process with reasonably low barriers, 21.3 and 19.6 kcal mol-1, respectively, for capped Cys and Sec, in good agreement with the experimental data available for the reaction with free amino acids. The results suggest a mechanism for the ligand exchange reaction involving an anionic thiolate or selenothiolate species coupled to an explicit proton transfer to the leaving carbene from the acidic component of the buffer. The presence of a buffer is necessary both in in vitro experiments and under physiological conditions, and its proton reservoir behavior reveals the importance of the environmental effects in carbene substitution by biological nucleophiles.

Determinants of the Lead(II) Affinity in pbrR Protein: A Computational Study.

Investigation of the diverse evolutionary developed mechanisms enabling bacteria to maintain homeostasis and to be resistant to lead is crucial for the discovery of novel strategies for isolation of this highly toxic metal and its subsequent elimination from contaminated environments. The metalloregulatory protein pbrR and its homologues that were identified in the Cupriavidus metallidurans CH34 chromosome are the only characterized natural metalloproteins that have a special affinity toward Pb(II) and that bind it with at least a 1000-fold selectivity over other heavy metals. The X-ray structures of apo and Pb(II)-bound pbrR have been recently reported. In the present study, the binding of Pb(II) at pbrR was investigated by means of multiscale computational modeling. Molecular dynamics simulations substantiated how conformations amenable for the Pb(II) complexation through the tris-cysteine motif are formed from the antiparallel coiled-coil packing interaction of two dimerization helices of two pbrR monomers, and the phase space of apo-pbrR has been extensively sampled. Hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on metal-bound structures of pbrR also allowed us to determine the most probable protonation state for the lead binding motif and evaluate the structural features mostly affecting the Pb(II) coordination in this protein. In agreement with available experimental data, we found that pbrR may control its Pb(II) affinity, probably, by conformational changes that affect the distance between Cys78' and Cys122 and their protonation states, thus being able to switch on the Pb(II) sequestration/release-prone states in response to external stimuli. The protein structure enveloping the metal binding motif favors the thiol-thiolate-thiolate protonation state of Pb(II)-pbrR, thus probably enhancing the binding selectivity for Pb(II), compared to other metal ions.

An insight of early PrP-E200K aggregation by combined molecular dynamics/fragment molecular orbital approaches.

Unveiling the events leading to the formation of prion particles is a nowadays challenge in the field of neurochemistry. Pathogenic mutants of prion protein (PrP) are characterized by both an intrinsic tendency to aggregation and scrapie conversion propensity. However, the question about a possible correlation between these two events lasts still unanswered. Here, a multilayered computational workflow was employed to investigate structure, stability, and molecular interaction properties of a dimer of PrPC -E200K, a well-known mutant of the PrP that represents a reduced model of early aggregates of this protein. Based on the combination of molecular dynamics and quantum mechanical approaches, this study provided for an in depth insight of PrPC -E200K dimer in terms of residue-residue interactions. Assembly hypotheses for the early aggregation of PrPC -E200K are paved and compared with PrPSc models reported in the literature to find a structural link between early and late (scrapie) aggregates of this protein.

An Insight on the Gold(I) Affinity of golB Protein via Multilevel Computational Approaches.

Several bacterial species have evolutionary developed protein systems specialized in the control of intracellular gold ion concentration. In order to prevent the detrimental consequences that may be induced even at very low concentrations, bacteria such as Salmonella enterica and Cupriavidus metallidurans utilize Au-specific merR-type transcriptional regulators that detect these toxic ions and control the expression of specific resistance factors. Among these highly specialized proteins, golB has been investigated in depth, and X-ray structures of both apo and Au(I)-bound golB have been recently reported. Here, the binding of Au(I) at golB was investigated by means of multilevel computational approaches. Molecular dynamics simulations evidenced how conformations amenable for the Au(I) chelation through the Cys-XX-Cys motif on helix 1 are extensively sampled in the phase space of apo-golB. Hybrid QM/MM calculations on metal-bound structures of golB also allowed to characterize the most probable protonation state for gold binding motif and to assess the structural features mostly influencing the Au(I) coordination in this protein. Consistently with experimental evidence, we found that golB may control its Au(I) affinity by conformational changes that affect the distance between Cys10 and Cys13, thus being able to switch between the Au(I) sequestration/release-prone states in response to external stimuli. The protein structure enveloping the metal binding motif favors the thiol-thiolate protonation state of Au(I)-golB, thus probably enhancing the binding selectivity for Au(I) compared to other cations.

Insights into the Mechanisms of Aquaporin-3 Inhibition by Gold(III) Complexes: the Importance of Non-Coordinative Adduct Formation.

Following our recent reports on the inhibition of the water and glycerol channel aquaglyceroporin-3 (AQP3) by the coordination complex [AuIII(1,10-phenanthroline)Cl2] (Auphen), a series of six new Au(III) complexes featuring substituted 1,10-phenanthroline ligands (1-6) have been synthesized and characterized. The speciation of the compounds studied in buffered solution by UV-visible spectrophotometry showed that most of the complexes remain stable for several hours. Quantum mechanics (QM) studies of the hydrolysis processes of the compounds suggest that they are thermodynamically less prone to exchange the chlorido ligands with H2O or OH- in comparison to Au(III) bipyridyl complexes. Preliminary data on the antiproliferative activity against A549 human lung cancer cells indicate that the compounds are able to inhibit cell proliferation in vitro. Stopped-flow spectroscopy showed that these complexes potently inhibit glycerol permeation in human red blood cells (hRBC) through AQP3 blockage. The QM investigation of the ligand exchange with methanethiol, used as a model of Cys40 of AQP3, was carried out for some derivatives and showed that the affinity of the compounds' binding for thiols is higher in comparison to the Aubipy complex ([AuIII(bipy)Cl2]PF6, bipy = 2,2'-bipyridine). In addition, both noncovalent and coordinative binding of complex 3 ( [AuIII(5-chloro-1,10-phenanthroline)Cl2]PF6) to the protein channel has been investigated in comparison to the benchmark Auphen and Aubipy using a computational workflow, including QM, molecular dynamics (MD), and quantum mechanics/molecular mechanics (QM/MM) approaches. Finally, atoms in molecules (AIM) and natural bond orbital (NBO) analyses corroborate the MD predictions, providing quantification of the noncoordinative interactions between the compounds and AQP3. AQP3 inhibition is the result of protein conformational changes, upon coordinative gold binding, which induce pore closure. The importance of noncoordinative adducts in modulating the AQP3 inhibition properties of the investigated Au(III) compounds has been elucidated, and these interactions should be further considered in the future design of isoform-selective AQP inhibitors.