Molecular determinants for brain targeting by peptides: a meta-analysis approach with experimental validation.

Blood-brain barrier (BBB) peptide-shuttles (BBBpS) are able to translocate the BBB and reach the brain. Despite the importance of brain targeting in pharmacology, BBBpS are poorly characterized. Currently, their development relies on the empiric assumption that cell-penetrating peptides (CPPs), with proven ability to traverse lipid membranes, will likewise behave as a BBBpS. The relationship between CPPs/BBBpS remains elusive and, to the best of our knowledge, has not hitherto been subject to thorough experimental scrutiny. In this work, we have identified/quantified the main physicochemical properties of BBBpS and then searched for CPPs with these properties, hence potential BBBpS. The specific features found for BBBpS are: (i) small size, (ii) none or few aromatic residues, (iii) hydrophobic, and (iv) slight cationic nature. Then, we selected the 10 scoring best in an ordinary least squares analysis, and tested them in vitro and in vivo. Overall, we identified the molecular determinants for brain targeting by peptides, devised a methodology that can be used to assist in the design of peptides with potential brain penetration from amino acid residue sequences, and found four new BBBpS within the CPP library.

The use of a selective, nontoxic dual-acting peptide for breast cancer patients with brain metastasis.

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the absence of commonly targeted receptors. Unspecific chemotherapy is currently the main therapeutic option, with poor results. Another major challenge is the frequent appearance of brain metastasis (BM) associated with a significant decrease in patient overall survival. The treatment of BM is even more challenging due to the presence of the blood-brain barrier (BBB). Here, we present a dual-acting peptide (PepH3-vCPP2319) designed to tackle TNBC/BM, in which a TNBC-specific anticancer peptide (ACP) motif (vCPP2319) is joined to a BBB peptide shuttle (BBBpS) motif (PepH3). PepH3-vCPP2319 demonstrated selectivity and efficiency in eliminating TNBC both in monolayers (IC50≈5.0 µM) and in spheroids (IC50≈25.0 µM), with no stringent toxicity toward noncancerous cell lines and red blood cells (RBCs). PepH3-vCPP2319 was also able to cross the BBB in vitro and penetrate the brain in vivo, and was stable in serum with a half-life above 120 min. Tumor cell-peptide interaction is fast, with quick peptide internalization via clathrin-mediated endocytosis without membrane disruption. Upon internalization, the peptide is detected in the nucleus and the cytoplasm, indicating a multi-targeted mechanism of action that ultimately induces irreversible cell damage and apoptosis. In conclusion, we have designed a dual-acting peptide capable of brain penetration and TNBC cell elimination, thus expanding the drug arsenal to fight this BC subtype and its BM.

Dual FGFR-targeting and pH-activatable ruthenium-peptide conjugates for targeted therapy of breast cancer.

Dysregulation of Fibroblast Growth Factor Receptors (FGFRs) signaling has been associated with breast cancer, yet employing FGFR-targeted delivery systems to improve the efficacy of cytotoxic agents is still sparsely exploited. Herein, we report four new bi-functional ruthenium-peptide conjugates (RuPCs) with FGFR-targeting and pH-dependent releasing abilities, envisioning the selective delivery of cytotoxic Ru complexes to FGFR(+)-breast cancer cells, and controlled activation at the acidic tumoral microenvironment. The antiproliferative potential of the RuPCs and free Ru complexes was evaluated in four breast cancer cell lines with different FGFR expression levels (SKBR-3, MDA-MB-134-VI, MCF-7, and MDA-MB-231) and in human dermal fibroblasts (HDF), at pH 6.8 and pH 7.4 aimed at mimicking the tumor microenvironment and normal tissues/bloodstream pHs, respectively. The RuPCs showed higher cytotoxicity in cells with higher level of FGFR expression at acidic pH. Additionally, RuPCs showed up to 6-fold higher activity in the FGFR(+) breast cancer lines compared to the normal cell line. The release profile of Ru complexes from RuPCs corroborates the antiproliferative effects observed. Remarkably, the cytotoxicity and releasing ability of RuPCs were shown to be strongly dependent on the conjugation of the peptide position in the Ru complex. Complementary molecular dynamic simulations and computational calculations were performed to help interpret these findings at the molecular level. In summary, we identified a lead bi-functional RuPC that holds strong potential as a FGFR-targeted chemotherapeutic agent.

Exploiting click-chemistry: backbone post-functionalisation of homoleptic gold(I) 1,2,3-triazole-5-ylidene complexes.

The synthesis of a homoleptic azide-functionalised Au(I) bis-1,2,3-triazole-5-ylidene complex is reported, starting from a backbone-modified 1,2,3-triazolium salt ligand precursor. The incorporated azide handle allows for a straightforward modification of the complex according to click-chemistry protocols without impacting the steric shielding around the metal center, demonstrating the superiority of the presented triazole ligand framework over imidazole based systems. Employing the SPAAC and the CuAAC reactions, post-modification of the complex is facilitated with two model substrates, while retaining very high antiproliferative activity (nanomolar range IC50 values) in A2780 and MCF-7 human cancer cells.

Panobinostat-loaded folate targeted liposomes as a promising drug delivery system for treatment of canine B-cell lymphoma.

Introduction: Cancer is a major public health problem with over 19 million cases reported in 2020. Similarly to humans, dogs are also largely affected by cancer, with non-Hodgkin's lymphoma (NHL) among the most common cancers in both species. Comparative medicine has the potential to accelerate the development of new therapeutic options in oncology by leveraging commonalities between diseases affecting both humans and animals. Within this context, in the present study, we investigated the potential of panobinostat (Pan)-loaded folate-targeted PEGylated liposomes (FA-PEG-Pan-Lip) for the treatment of canine B-cell lymphoma, while contributing to new perspectives in comparative oncology. Methods and results: Two formulations were developed, namely: PEG-Pan-Lip and FA-PEG-Pan-Lip. Firstly, folate receptor expression in the CLBL-1 canine B-cell lymphoma cell line was assessed. After confirming receptor expression, both Pan-loaded formulations (PEG-Pan-Lip, FA-PEG-Pan-Lip) demonstrated dose-dependent inhibitory effects on CLBL-1 cell proliferation. The FA-PEG-Pan-Lip formulation (IC50 = 10.9 ± 0.03 nM) showed higher cytotoxicity than the non-targeted PEG-Pan-Lip formulation (IC50 = 12.9 ± 0.03 nM) and the free panobinostat (Pan) compound (IC50 = 18.32±0.03 nM). Moreover, mechanistically, both Pan-containing formulations induced acetylation of H3 histone and apoptosis. Flow cytometry and immunofluorescence analysis of intracellular uptake of rhodamine-labeled liposome formulations in CLBL-1 cells confirmed cellular internalization of PEG-Lip and FA-PEG-Lip formulations and higher uptake profile for the latter. Biodistribution studies of both radiolabeled formulations in CD1 and SCID mice revealed a rapid clearance from the major organs and a 1.6-fold enhancement of tumor uptake at 24 h for 111In-FA-PEG-Pan-Lip (2.2 ± 0.1 %ID/g of tumor) compared to 111In-PEG-Pan-Lip formulation (1.2±0.2 %ID/g of tumor). Discussion: In summary, our results provide new data validating Pan-loaded folate liposomes as a promising targeted drug delivery system for the treatment of canine B-cell lymphoma and open innovative perspectives for comparative oncology.

Rabbit derived VL single-domains as promising scaffolds to generate antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.

Construction of HER2-Specific HIV-1-Based VLPs.

Virus-like particles (VLPs) are nanoplatforms comprised of one or more viral proteins with the capacity to self-assemble without viral genetic material. VLPs arise as promising nanoparticles (NPs) that can be exploited as vaccines, as drug delivery vehicles or as carriers of imaging agents. Engineered antibody constructs, namely single-chain variable fragments (scFv), have been explored as relevant molecules to direct NPs to their target. A vector containing the scFv of an antibody, aimed at the human epidermal growth factor receptor 2 (HER2) and fused to the human immunodeficiency virus (HIV) protein gp41, was previously constructed. The work herein describes the early results concerning the production and the characterization of HIV-1-based VLPs expressing this protein, which could function as potential non-toxic tools for transporting drugs and/or imaging agents.

How promising are HIV-1-based virus-like particles for medical applications.

New approaches aimed at identifying patient-specific drug targets and addressing unmet clinical needs in the framework of precision medicine are a strong motivation for researchers worldwide. As scientists learn more about proteins that drive known diseases, they are better able to design promising therapeutic approaches to target those proteins. The field of nanotechnology has been extensively explored in the past years, and nanoparticles (NPs) have emerged as promising systems for target-specific delivery of drugs. Virus-like particles (VLPs) arise as auspicious NPs due to their intrinsic properties. The lack of viral genetic material and the inability to replicate, together with tropism conservation and antigenicity characteristic of the native virus prompted extensive interest in their use as vaccines or as delivery systems for therapeutic and/or imaging agents. Owing to its simplicity and non-complex structure, one of the viruses currently under study for the construction of VLPs is the human immunodeficiency virus type 1 (HIV-1). Typically, HIV-1-based VLPs are used for antibody discovery, vaccines, diagnostic reagent development and protein-based assays. This review will be centered on the use of HIV-1-based VLPs and their potential biomedical applications.

The HIV-1 Matrix Protein p17 Does Cross the Blood-Brain Barrier.

Human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND) remains an important neurological manifestation in HIV-1-infected (HIV+) patients. Furthermore, detection of the HIV-1 matrix protein p17 (p17) in the central nervous system (CNS) and its ability to form toxic assemblies in the brain have been recently confirmed. Here, we show for the first time, using both an in vitro blood-brain barrier (BBB) model and in vivo biodistribution studies in healthy mice, that p17 can cross the BBB. There is rapid brain uptake with 0.35% ± 0.19% of injected activity per gram of tissue (IA/g) 2 min after administration, followed by brain accumulation with 0.28% ± 0.09% IA/g after 1 h. The interaction of p17 with chemokine receptor 2 (CXCR2) at the surface of brain endothelial cells triggers transcytosis. The present study supports the hypothesis of a direct role of free p17 in neuronal dysfunction in HAND by demonstrating its intrinsic ability to reach the CNS. IMPORTANCE The percentage of patients affected by HIV-1-associated neurocognitive disorder (HAND) ranges from 30% to 50% of HIV-infected (HIV+) patients. The mechanisms leading to HAND development need to be elucidated, but the roles of secreted viral proteins, chemokines, and proinflammatory molecules appear to be clear. In particular, the blood-brain barrier (BBB) represents a route for entry into the central nervous system (CNS) and thus plays an important role in HAND. Several findings suggest a key role for the HIV-1 matrix protein p17 (p17) as a microenvironmental factor capable of inducing neurocognitive disorders. Here, we show the ability of the p17 to cross the BBB and to reach the CNS, thus playing a crucial role in neuronal dysfunction in HAND.

Ultrasonication Improves Solid Phase Synthesis of Peptides Specific for Fibroblast Growth Factor Receptor and for the Protein-Protein Interface RANK-TRAF6.

Considering our interest in the use of peptides as potential target-specific drugs or as delivery vectors of metallodrugs for various biomedical applications, it is crucial to explore improved synthetic methodologies to accomplish the highest peptide crude purity in the shortest time possible. Therefore, we compared "classical" fluorenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) with ultrasound(US)-assisted SPPS based on the preparation of three peptides, namely the fibroblast growth factor receptor 3(FGFR3)-specific peptide Pep1 (VSPPLTLGQLLS-NH2) and the novel peptides Pep2 (RQMATADEA-NH2) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH2), which are being developed aimed at interfering with the intracellular protein-protein interaction(PPI) RANK-TRAF6. Our results demonstrated that US-assisted SPPS led to a 14-fold (Pep1) and 4-fold time reduction (Pep2) in peptide assembly compared to the "classical" method. Interestingly, US-assisted SPPS yielded Pep1 in higher purity (82%) than the "classical" SPPS (73%). The significant time reduction combined with high crude peptide purity attained prompted use to apply US-assisted SPPS to the large peptide Pep3, which displays a high number of hydrophobic amino acids and homooligo-sequences. Remarkably, the synthesis of this 25-mer peptide was attained during a "working day" (347 min) in moderate purity (approx. 49%). In conclusion, we have reinforced the importance of using US-SPPS towards facilitating the production of peptides in shorter time with increased efficacy in moderate to high crude purity. This is of special importance for long peptides such as the case of Pep3.

Highly Specific Blood-Brain Barrier Transmigrating Single-Domain Antibodies Selected by an In Vivo Phage Display Screening.

A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.

Gold(I) Bis(1,2,3-triazol-5-ylidene) Complexes as Promising Selective Anticancer Compounds.

The synthesis and antiproliferative activity of Mes- and iPr-substituted gold(I) bis(1,2,3-triazol-5-ylidene) complexes in various cancer cell lines are reported, showing nanomolar IC50 values of 50 nM (lymphoma cells) and 500 nM (leukemia cells), respectively (Mes < iPr). The compounds exclusively induce apoptosis (50 nM to 5 µM) instead of necrosis in common malignant blood cells (leukemia cells) and do not affect non-malignant leucocytes. Remarkably, the complexes not only overcome resistances against the well-established cytostatic etoposide, cytarabine, daunorubicin, and cisplatin but also promote a synergistic effect of up to 182% when used with daunorubicin. The present results demonstrate that gold(I) bis(1,2,3-triazol-5-ylidene) complexes are highly promising and easily modifiable anticancer metallodrugs.

Emerging Molecular Receptors for the Specific-Target Delivery of Ruthenium and Gold Complexes into Cancer Cells.

Cisplatin and derivatives are highly effective in the treatment of a wide range of cancer types; however, these metallodrugs display low selectivity, leading to severe side effects. Additionally, their administration often results in the development of chemoresistance, which ultimately results in therapeutic failure. This scenario triggered the study of other transition metals with innovative pharmacological profiles as alternatives to platinum, ruthenium- (e.g., KP1339 and NAMI-A) and gold-based (e.g., Auranofin) complexes being among the most advanced in terms of clinical evaluation. Concerning the importance of improving the in vivo selectivity of metal complexes and the current relevance of ruthenium and gold metals, this review article aims to survey the main research efforts made in the past few years toward the design and biological evaluation of target-specific ruthenium and gold complexes. Herein, we give an overview of the inorganic and organometallic molecules conjugated to different biomolecules for targeting membrane proteins, namely cell adhesion molecules, G-protein coupled receptors, and growth factor receptors. Complexes that recognize the progesterone receptors or other targets involved in metabolic pathways such as glucose transporters are discussed as well. Finally, we describe some complexes aimed at recognizing cell organelles or compartments, mitochondria being the most explored. The few complexes addressing targeted gene therapy are also presented and discussed.

Integrated in Silico and Experimental Approach towards the Design of a Novel Recombinant Protein Containing an Anti-HER2 scFv.

Biological therapies, such as recombinant proteins, are nowadays amongst the most promising approaches towards precision medicine. One of the most innovative methodologies currently available aimed at improving the production yield of recombinant proteins with minimization of costs relies on the combination of in silico studies to predict and deepen the understanding of the modified proteins with an experimental approach. The work described herein aims at the design and production of a biomimetic vector containing the single-chain variable domain fragment (scFv) of an anti-HER2 antibody fragment as a targeting motif fused with HIV gp41. Molecular modeling and docking studies were performed to develop the recombinant protein sequence. Subsequently, the DNA plasmid was produced and HEK-293T cells were transfected to evaluate the designed vector. The obtained results demonstrated that the plasmid construction is robust and can be expressed in the selected cell line. The multidisciplinary integrated in silico and experimental strategy adopted for the construction of a recombinant protein which can be used in HER2+-targeted therapy paves the way towards the production of other therapeutic proteins in a more cost-effective way.

Fluorescent palladium(II) and platinum(II) NHC/1,2,3-triazole complexes: antiproliferative activity and selectivity against cancer cells.

Fluorescent Pd(ii) and Pt(ii) complexes bearing 4-methylene-7-methoxycoumarin (MMC) and 2,6-diispropylphenyl (Dipp) substituted NHC/1,2,3-triazole hybrid ligands are described. Depending on the reaction conditions two different ligand coordination modes are observed, i.e., bidentate solely coordinating via NHCs or tetradentate coordinating via NHCs and 1,2,3-triazoles. All Dipp substituted complexes show antiproliferative activity against cervix (HeLa) and breast (MCF-7) human carcinoma cells. The activity significantly depends on the coordination mode, with the tetradentate motif being notably more effective (HeLa: IC50 = 3.9 µM to 4.7 µM; MCF-7: IC50 = 2.07 µM to 2.35 µM). Amongst the MMC series, only the Pd(ii) complex featuring the bidentate coordination mode is active against HeLa (IC50 = 6.1 µM). In contrast to its structurally related Dipp derivative (SI = 0.6), it shows a high selectivity for HeLa (SI > 16) compared to healthy skin cells (HaCaT). According to fluorescence microscopy, this compound is presumably located in late endosomes or lysosomes.

Bioconjugate Supramolecular Pd2+ Metallacages Penetrate the Blood Brain Barrier In Vitro and In Vivo.

The biomedical application of discrete supramolecular metal-based structures, specifically self-assembled metallacages, is still an emergent field of study. Capitalizing on the knowledge gained in recent years on the development of 3-dimensional (3D) metallacages as novel drug delivery systems and theranostic agents, we explore here the possibility to target [Pd2L4]4+ cages (L = 3,5-bis(3-ethynylpyridine)phenyl ligand) to the brain. In detail, a new water-soluble homoleptic cage (CPepH3) tethered to a blood brain barrier (BBB)-translocating peptide was synthesized by a combination of solid-phase peptide synthesis (SPPS) and self-assembly procedures. The cage translocation efficacy was assessed by inductively coupled mass spectrometry (ICP-MS) in a BBB cellular model in vitro. Biodistribution studies of the radiolabeled cage [[99mTcO4]- ⊂ CPepH3] in the CD1 mice model demonstrate its brain penetration properties in vivo. Further DFT studies were conducted to model the structure of the [[99mTcO4]- ⊂ cage] complex. Moreover, the encapsulation capabilities and stability of the cage were investigated using the [ReO4]- anion, the "cold" analogue of [99mTcO4]-, by 1H NMR spectroscopy. Overall, our study constitutes another proof-of-concept of the unique potential of supramolecular coordination complexes for modifying the physiochemical and biodistribution properties of diagnostic species.

Anti-HIV-1 Activity of pepRF1, a Proteolysis-Resistant CXCR4 Antagonist Derived from Dengue Virus Capsid Protein.

There is an urgent need for the development of new anti-HIV drugs that can complement existing medicines to be used against resistant strains. Here, we report the anti-HIV-1 peptide pepRF1, a human serum-resistant peptide derived from the Dengue virus capsid protein. In vitro, pepRF1 shows a 50% inhibitory concentration of 1.5 nM with a potential therapeutic window higher than 53 000. This peptide is specific for CXCR4-tropic strains, preventing viral entry into target cells by binding to the viral coreceptor CXCR4, acting as an antagonist of this receptor. pepRF1 is more effective than T20, the only peptide-based HIV-1 entry inhibitor approved, and excels in inhibiting a HIV-1 strain resistant to T20. Potentially, pepRF1 can be used alone or in combination with other anti-HIV drugs. Furthermore, one can also envisage its use as a novel therapeutic strategy for other CXCR4-related diseases.

To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models.

The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood-brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides.

Mechanisms underlying the cytotoxic activity of syn/anti-isomers of dinuclear Au(I) NHC complexes.

The syn- and anti-isomers of dinuclear Au(I) complexes of the type Au2(RLOH)(PF6)2 (R = isopropyl or mesityl) bearing 2-hydroxyethane-1,1-diyl-bridged bisimidazolylidene ligands were separated by reversed phase high performance liquid chromatography (HPLC) and characterized by NMR spectroscopy, elemental analysis, ESI mass spectrometry as well as single crystal X-ray diffraction analysis. Evaluation of the antiproliferative activity of the isolated isomers has shown very small difference in their cytotoxic behavior in various cancer cell lines. Additional counter-anion exchange (hexafluorophosphate to chloride) allows to increase the water solubility of synAu2(MesLOH)(PF6)2 and leads to higher antiproliferative activity when compared to the hexafluorophosphate-complex. Both isomers were treated with l-cysteine as nucleophilic thiol source and only the anti-isomer shows dissociation of one bisimidazolylidene ligand after 24 h. In the case of the syn-isomer, density functional theory calculations indicate a lower reactivity due to the higher steric hindrance of the N-substituents and additional hydrogen bond interaction, which prevents a nucleophilic attack. When the N-substituent is replaced by the bulkier mesityl group, both conformations remain unreactive and result to be the most cytotoxic complexes in the above-mentioned cancer cell lines. Interestingly, synAu2(MesLOH)(PF6)2 exhibits a high selectivity in the MCF-7 cell line with a selectivity index (SI) of 19, which is superior to auranofin (SI < 1), making this compound an ideal candidate for further studies. Preliminary mechanistic studies reveal that the cytotoxic complexes possess mitochondrial-TrxR inhibition properties in the nanomolar range. Additionally, the cellular distribution studies by ICP-MS and nuclear microscopy have shown that the compound accumulates in the membranes. These results suggest that the mitochondrial membrane is the main target for this type of dinuclear complexes, causing oxidative stress by inhibiting mitochondrial thioredoxin reductase.

Antiproliferative Activity of Functionalized Histidine-derived Au(I) bis-NHC Complexes for Bioconjugation.

A series of histidine derived Au(I) bis-NHC complexes bearing different ester, amide and carboxylic acid functionalities as well as wingtip substituents is synthesized and characterized. The stability in aqueous media, in vitro cytotoxicity in a set of cancer cell lines (MCF7, PC3 and A2780/A2780cisR) along with the cellular uptake are evaluated. Stability tests suggest hydrolysis of the ester within 8 h, which might lead to deactivation. Furthermore, the bis-NHC system shows a sufficient stability against cysteine and the thiol containing peptide GSH. The benzyl ester and amide show the highest activity comparable to the benchmark compound cisplatin, with the ester only displaying a slightly lower cytotoxicity than the amide. A cellular uptake study revealed that the benzyl ester and the amide could have different intracellular distribution profiles but both complexes induce perturbations of the cellular physiological processes. The simple modifiability and high stability of the complexes provides a promising system for upcoming post modifications to enable targeted cancer therapy.