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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.

A straightforward modification route to obtain mono‐ and di‐substituted anthroyl ester bridge functionalized dinuclear Au(I) bis‐N‐heterocyclic carbene complexes is presented. The functionalization can be achieved starting from a hydroxyl‐functionalized ligand precursor followed by transmetallation of the corresponding Ag complex or via esterification of the hydroxyl‐functionalized gold complex. The compounds are characterized by NMR‐spectroscopy, ESI‐MS, elemental analysis and SC‐XRD. The mono‐ester Au complex shows quantum yields around 18%. In contrast, the corresponding syn‐di‐ester Au complex, exhibits significantly lower quantum yields of around 8%. Due to insufficient water solubility of the di‐ester, only the mono‐ester complex has been tested regarding its antiproliferative activity against HeLa‐ (cervix) and MCF‐7‐ (breast) cancer cell lines and a healthy fibroblast cell line (V79). IC50 values of 7.26 μM in the HeLa cell line and 7.92 μM in the MCF‐7 cell line along with selectivity indices of 8.8 (HeLa) and 8.0 (MCF‐7) are obtained. These selectivity indices are significantly higher than those obtained for the reference drugs cisplatin or auranofin., A simple modification route of hydroxyl functionalized bis‐NHC dinuclear Au(I) complexes with anthracene is presented, which leads to a significant improvement of the antiproliferative activity and quantum yields up to 18%. By controlling the reaction parameters, one hydroxyl group remains, allowing for further potential bioconjugation to obtain a selective theranostic metal complex.

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., Novel histidine‐derived Au(I) bis‐NHCs with different functional groups (ester, amide and carboxylic acid) show antiproliferative activity in MCF7, PC3 and A2780/A2780cisR cancer cell lines. A cellular uptake study revealed that the ester and the amide could have different intracellular distribution profiles but both complexes induce perturbations of the cellular physiological processes.

A novel N,N’‐allyl‐bridged bisimidazolium salt and a novel dinuclear Ag(I) and a Au(I) NHC complex are reported. Both metallacyclic complexes have a twisted structural shape due to the rigid allylic system and form two different isomers relating to the position of the double bonds. The allyl‐group shows photoisomerisation, but no reactivity towards bases for the additional coordination of Pd(II)., Building bridges: A novel N,N’‐allyl‐bridged bisimidazolium salt and a novel dinuclear Ag(I) and a Au(I) NHC complex are reported. Both metallacyclic complexes have a twisted structural shape due to the rigid allylic system and form two different isomers relating to the position of the double bonds. The allyl‐group shows photoisomerisation, but no reactivity towards bases for the additional coordination of Pd(II).

Paediatric Acquired ImmunoDeficiency Syndrome (AIDS) is a life-threatening and infectious disease in which the Human Immunodeficiency Virus (HIV) is mainly transmitted through Mother-To- Child Transmission (MTCT) during pregnancy, labour and delivery, or breastfeeding. This review provides an overview of the distinct therapeutic alternatives to abolish the systemic viral replication in paediatric HIV-1 infection. Numerous classes of antiretroviral agents have emerged as therapeutic tools for downregulation of different steps in the HIV replication process. These classes encompass Non- Nucleoside Analogue Reverse Transcriptase Inhibitors (NNRTIs), Nucleoside/Nucleotide Analogue Reverse Transcriptase Inhibitors (NRTIs/NtRTIs), INtegrase Inhibitors (INIs), Protease Inhibitors (PIs), and Entry Inhibitors (EIs). Co-administration of certain antiretroviral drugs with Pharmacokinetic Enhancers (PEs) may boost the effectiveness of the primary therapeutic agent. The combination of multiple antiretroviral drug regimens (Highly Active AntiRetroviral Therapy - HAART) is currently the standard therapeutic approach for HIV infection. So far, the use of HAART offers the best opportunity for prolonged and maximal viral suppression, and preservation of the immune system upon HIV infection. Still, the frequent administration of high doses of multiple drugs, their inefficient ability to reach the viral reservoirs in adequate doses, the development of drug resistance, and the lack of patient compliance compromise the complete HIV elimination. The development of nanotechnology-based drug delivery systems may enable targeted delivery of antiretroviral agents to inaccessible viral reservoir sites at therapeutic concentrations. In addition, the application of Computer-Aided Drug Design (CADD) approaches has provided valuable tools for the development of anti-HIV drug candidates with favourable pharmacodynamics and pharmacokinetic properties.

© 2022 American Society for Microbiology. All Rights Reserved., 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., This study was supported in part by Associazione Italiana per la Ricerca sul Cancro (AIRC) grant 20108 (to A.C.) and by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement 828774 (to M.C.)

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.

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., 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., This research was funded by: the Portuguese Funding Agency, Fundacão para a Ciência e a Tecnologia, FCT IP: IF/01010/2013 and PTDC/BBB-BIO/0508/2014 to FAS, SFRH/BPD/100522/2014 and DL57/2016/CP1438/CT0002 to SIA, PD/BD/128281/2017, PTDC/BIA-BQM/5027/2020 and Dl 57/2016/CP1451/CT0023 to MC and VN, UIDB/04138/2020 and UIDP/04138/2020 to MMG, UID/Multi/04349/2020 and PTDC/QUI-NUC/30147/2017 to LG and JDGC, UID/NEU/04539/2019, UIDB/04539/2020 and ViraVector (CENTRO-01-0145-FEDER-022095) to RJN and LPA. Gilead Génese 2019 has provided support through project PGG/050/2019 to JNRD. CIISA has provided support through Project UIDB/00276/2020, funded by FCT. FAS and JG dedicate this study to the memory of Professor Carlos F. Barbas 3rd, an outstanding mentor and friend.

Infectious diseases like malaria, tuberculosis or HIV are among the leading causes of death worldwide according to WHO estimations. Nevertheless, the fight against infectious diseases is aggravated through growing development of resistance towards current drugs and due to their severe adverse effects. The introduction of the lipophilic organometallic moiety ferrocene, a compound with a sandwich-like structure, in an existing bioactive molecule is a promising tool for the development of new more efficient drugs with innovative mechanisms of action. Thus, this review summarizes recent developments in the field of ferrocene conjugation to bioactive molecules like natural products, synthetically derived drugs, peptides as well as heterobimetallic complexes. Hereby, we will provide the reader with a summary of the most potent ferrocene derivatives reported for a plethora of infectious diseases by tabulating and critically assessing the corresponding IC50 values and the minimal inhibitory concentrations (MIC). Owing to the diverse field of infectious diseases the reported ferrocene derivatives were classified according to their targets into four main groups: antiparasitic (with antimalarial agents as biggest group), antibacterial, antifungal and antiviral agents.

The overexpression of peptide receptors in certain tumors as compared to endogeneous expression levels represents the molecular basis for the design of peptide-based tools for targeted nuclear imaging and therapy. Receptor targeting with radiolabelled peptides became a very important imaging and/or therapeutic approach in nuclear medicine and oncology. A great variety of peptides has been radiolabelled with clinical relevant radionuclides, such as radiometals and radiohalogens. However, to the best of our knowledge concise and updated reviews providing information about the biomedical application of radioiodinated peptides are still missing. This review outlines the synthetic efforts in the preparation of radioiodinated peptides highlighting the importance of radioiodine in nuclear medicine, giving an overview of the most relevant radioiodination strategies that have been employed and describes relevant examples of their use in the biomedical field.

Novel bisphosphonate platinum complexes: [Pt(isopropylamine) 2 (BP)]NO 3 (BP = pamidronate and alendronate) have been synthesized and characterized. Their monomeric structure contains a bisphosphonate acting as chelate ligand through its oxygen atom donors, conferring the compound's cationic structure with a good solubility in water. The study of the compounds in solution showed high stability up to 24 h. The cytotoxicity in cancer cell lines has been assessed. We also present preliminary studies on the evaluation of the affinity towards biological targets such as DNA (both calf thymus DNA and supercoiled plasmid DNA) and hydroxyapatite where the complexes showed a low DNA interaction, but a clear affinity for hydroxyapatite comparing to their precursors.

Four novel dinuclear Ag(I) and Au(I) NHC complexes bearing two 2,2-acetate-bridged bisimidazolylidene ligands (R = Me and iPr) of zwitterionic and metallacyclic forms are reported. The functionalized methylene bridge of the ligands leads to water soluble complexes, which have been characterized by NMR and IR spectroscopy, elemental analysis and single crystal X-ray diffraction in the case of La-H2-PF6, Ag2(La)2, Ag2(Lb)2 and Au2(La)2. Dimerization processes caused by hydrogen bonding or Ag(I)-carboxylate interactions in the solid state were observed for La-H2-PF6 and Ag2(La)2. DOSY NMR experiments confirmed that both bisimidazolium salts appear as dimers in aqueous solutions, in contrast to the corresponding monomeric Ag(I) and Au(I) complexes. Both gold(I) complexes form syn- and anti-isomers analogous to the reference coinage metal-based complexes. Protonation studies of the syn-isomer gold(I) complex Au2(La)2 were successful, whereas post-modification esterification or amidation reactions were not feasible. Additionally, decarboxylation reactions (thermally induced Krapcho- or oxidative Hunsdiecker-type) of the bisimidazolium salts were observed. Thus, the proximity of the carboxyl moiety to imidazolium/imidazolylidene rings seems to negatively affect stability and reactivity.

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 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.

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

Copyright © 2020 Cavaco, Pérez-Peinado, Valle, Silva, Correia, Andreu, Castanho and Neves. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms, 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., The authors thank the Portuguese Funding Agency, Fundação para a Ciência e a Tecnologia, FCT IP, for financial support (grants: PD/BD/128281/2017, PTDC/BBB-NAN/1578/2014); European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 828774; and “la Caixa” Banking Foundation under the project code HR17-00409.

In this work we explored the possibility of improving the selectivity of a cytotoxic Ru complex [RuCp(PPh3)(2,2'-bipy)][CF3SO3] (where Cp = η5-cyclopentadienyl) TM34 towards FGFR(+) breast cancer cells. Molecular dynamics (MD) simulations of TM34 in a phosphatidylcholine membrane model pinpointed the cyclopentadienyl group as a favorable derivatization position for the peptide conjugation approach. Three new Ru(ii) complexes presenting a functionalized η5-cyclopentadienyl were synthesized, namely [Ru(η5-C5H4COOH)(2,2'-bipy)(PPh3)][CF3SO3] (TM281) and its precursors, [Ru(η5-C5H4COOCH2CH3)(η2-2,2'-bipy)(PPh3)][CF3SO3] (3) and [Ru(η5-C5H4COOCH2CH3)(PPh3)2Cl] (2). Complex TM281 was prepared by the hydrolysis of the ethyl ester group appended to the η5-cyclopentadienyl ligand of complex 3 with K2CO3 in water/acetonitrile, followed by mild protonation using an ion exchange resin. The newly synthesized complexes were fully characterized by NMR, FTIR and UV-vis spectroscopic techniques. Also, electrochemical studies were carried out by means of cyclic voltammetry in order to evaluate the stability of the compounds. Single crystal X-ray diffraction studies were carried out for compounds 3 and TM281 which crystallized in the monoclinic system, space group P21/n. The unprecedented synthesis and characterization of three half-sandwich ruthenium(ii)-cyclopentadienyl peptide conjugates and their preliminary biological evaluation against human FGFR(+) and FGFR(-) breast cancer cells are also reported.

Cystic fibrosis (CF), a life-shortening genetic disease, is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that codes for the CFTR protein, the major chloride channel expressed at the apical membrane of epithelial cells. The development of an imaging probe capable of non-invasively detect CFTR at the cell surface could be of great advantage for the management of CF. With that purpose, we synthesized the first extracellular loop of CFTR protein (ECL1) through fluorenylmethyloxycarbonyl (Fmoc)-based microwave-assisted solid-phase peptide synthesis (SPPS), according to a reported methodology. However, aspartimide formation, a well-characterized side reaction in Fmoc-SPPS, prompted us to adopt a different side-chain protection strategy for aspartic acid residues present in ECL1 sequence. The peptide was subsequently modified via PEGylation and biotinylation, and cyclized through disulfide bridge formation, mimicking the native loop conformation in CFTR protein. Herein, we report improvements in the synthesis of the first extracellular loop of CFTR, including peptide modifications that can be used to improve antigen presentation in phage display for selection of novel antibodies against plasma membrane CFTR.

Copyright © 2020 American Chemical Society, 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., This work was supported by Fundação para a Ciência e aTecnologia (FCT-MCTES, Portugal) project PTDC/QEQ-MED/4412/2014, and by the Institut National de la Santéetde la Recherche Médicale (Inserm). I.C.C., J.M.F., T.N.F., and I.B. acknowledge fellowships SFRH/BPD/65531/2009,SFRH/BD/70423/2010, SFRH/BD/5283/2013, and SFRH/BPD/76225/2011, respectively, funded by FCT-MCTES. A.T. is the recipient of a fellowship from Agence Nationale de Recherche sur le Sida et les Hépatites Virales (ANRS). A.S.V.acknowledges funding under the Investigator Programme (IF/00803/2012) from FCT-MCTES. Work at Pompeu Fabra University was supported by grants AGL2014-52395-C2 and AGL2017-84097-C2-2-R and by the“María de Maeztu”Program for Units of Excellence in R&D from the Spanish Ministry of Innovation and Competitiveness (MINECO).

Phosphines are the most studied ligands on gold-based metallodrugs, whereas N-heterocyclic carbenes (NHCs) have been emerging in the past years as potential alternative ligands for medicinal applications. Indeed, in both cases, phosphine and NHC allow the preparation of a plethora of transition metal-based complexes with attractive properties for medicinal applications. This review offers an update on phosphine and NHC ligands used in gold complexes with potential anti-cancer, anti-bacterial, anti-parasitic (malaria, leishmaniasis and trypanosomasis) and anti-viral (HIV-1) activity. Structural information about diverse ligands is discussed and the biological activity of the corresponding homo-metallic Au(I), Au(III) and hetero-metallic complexes is summarized in tables and discussed as well. Additionally, these complexes are compared with reference compounds and facts about mechanistic studies and luminescence properties are highlighted. The main target of this review is to find trends in ligand design for medicinal purposes. Particularly, questions about the balance between lipophilicity and hydrophilicity, the presence of functional groups, the utilization of multinuclear complexes and the comparison of neutral versus cationic complexes will be addressed.

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to almost total absence of CFTR at the plasma membrane, a defect potentially corrected via drug-based therapies. Herein, we report the first proof-of-principle study of a noninvasive imaging probe able to detect CFTR at the plasma membrane. We radiolabeled the CFTR inhibitor, CFTRinh -172a, with technetium-99m via a pyrazolyl-diamine chelating unit, yielding a novel 99m Tc(CO)3 complex. A non-radioactive surrogate showed that the structural modifications introduced in the inhibitor did not affect its activity. The radioactive complex was able to detect plasma membrane CFTR, shown by its significantly higher uptake in wild-type versus mutated cells. Furthermore, assessment of F508del CFTR pharmacological correction in human cells using the radioactive complex revealed differences in corrector versus control uptake, recapitulating the biochemical correction observed for the protein.

Although relevant from the clinical point of view, radiotracers targeting cationic amino acid transporters are relatively unexplored and, in particular, no metal-based radiotracers are known. The rare examples of complexes recognized by amino acid transporters, namely by the Na+-independent neutral l-type amino acid transporter 1 (LAT1), are 99mTc(i)/Re(i) compounds. Herein, we describe conjugates comprising a pyrazolyl-diamine chelating unit and the cationic amino acid l-arginine (l-Arg) linked by a propyl (L1) or hexyl linker (L2), which allowed the preparation of stable complexes of the type fac-[99mTc(CO)3(k3-L)]+ (Tc1, L = L1; Tc2, L = L2) and of the respective surrogates Re1 and Re2. Interestingly, complex Tc2 exhibited moderate levels of time-dependent internalization in three human tumoural cell lines, with approximately 3% of total applied activity internalized, corresponding to 21% of the cell-associated activity. A putative mechanism of retention in the cytoplasm of cells could be the interaction of the complex with inducible nitric oxide synthase (iNOS), which is the enzyme responsible for the catalytic oxidation of l-Arg to citrulline and nitric oxide. However, the surrogate complex Re2 does not recognize iNOS, as demonstrated by the in vitro assays with purified iNOS and in studies with lipopolysaccharide(LPS)-activated macrophages. Preliminary mechanistic studies suggest that the internalization of Tc2 is linked to the cationic amino acid transporters, namely system y+. This finding might open the way towards the development of novel families of metal-based radiotracers for probing metabolically active cancer cells.

Human epidermal growth factor 2 (HER2) is a ligand-free tyrosine kinase receptor of the HER family that is overexpressed in some of the most aggressive tumours. Although it is known that HER2 dimerization involves a specific region of its extracellular domain, the so-called &ldquo, dimerization arm&rdquo, the mechanism of dimerization inhibition remains uncertain. However, uncovering how antibody interactions lead to inhibition of HER2 dimerization is of key importance in understanding its role in tumour progression and therapy. Herein, we employed several computational modelling techniques for a molecular-level understanding of the interactions between HER and specific anti-HER2 antibodies, namely an antigen-binding (Fab) fragment (F0178) and a single-chain variable fragment from Trastuzumab (scFv). Specifically, we investigated the effects of antibody-HER2 interactions on the key residues of &ldquo, from molecular dynamics (MD) simulations of unbound HER (in a total of 1 &micro, s), as well as ScFv:HER2 and F0178:HER2 complexes (for a total of 2.5 &micro, s). A deep surface analysis of HER receptor revealed that the binding of specific anti-HER2 antibodies induced conformational changes both in the interfacial residues, which was expected, and in the ECDII (extracellular domain), in particular at the &ldquo, which is critical in establishing protein&ndash, protein interface (PPI) interactions. Our results support and advance the knowledge on the already described trastuzumab effect on blocking HER2 dimerization through synergistic inhibition and/or steric hindrance. Furthermore, our approach offers a new strategy for fine-tuning target activity through allosteric ligands.

Synthesis and characterisation of the first macrocyclic Cu(I), Ag(I) and Au(I) tricarbene/urea N-heterocyclic carbene (NHC) complexes is described and their fluorescence properties are compared to related tetracarbene complexes. The newly described silver and gold complexes show luminescence with emission maxima at λem,max = 481 nm and λem,max = 334 nm, respectively. Moreover, their ability to inhibit the growth of bacteria (S. aureus and E. coli) and cancer cells (HeLa and MCF7) is investigated. A strong influence of the metal is observed. While the copper and gold complexes are inactive against bacteria, the silver complex depicts a moderate activity (MIC ≈ 30 µM). The copper and silver complexes exhibit antiproliferative activity in both cell lines, ranging from 25.1 µM to 3.03 µM.

The lack of selectivity for cancer cells and the resulting\ud negative impact on healthy tissue is a severe drawback of actual\ud cancer chemotherapy. Tethering of cytotoxic drugs to targeting\ud vectors such as peptides, which recognize receptors overexpressed\ud on the surface of tumor cells, is one possible strategy to overcome\ud such a problem. The pentapeptide cyc(RGDfK) targets the integrin\ud receptor αvβ3, important for tumor growth and metastasis formation.\ud In this work, two terpyridine based Ru(II) complexes were prepared\ud and for the first time conjugated to cyc(RGDfK) via amide bond\ud formation resulting in a monomeric and a dimeric bioconjugate. Both\ud Ru(II) complexes bind strongly and selectively to integrin αvβ3, with\ud the dimeric molecule displaying a 20-fold higher affinity to the\ud receptor than the monomeric one. However, the cytotoxicity of the\ud complexes and related bioconjugates against human A549 and\ud SKOV-3 cell lines is still not sufficient for application as anticancer\ud agents. Nevertheless, considering the high selectivity for integrin\ud receptor αvβ3, the synthesis of Ru-based bioconjugates with\ud cyc(RGDfK) paves a promising way towards the design of effective\ud targeted anticancer agents.

Amidated kyotorphin (L-Tyr-L-Arg-NH2; KTP-NH2) shows analgesic properties following systemic administration. Although KTP-NH2 does not have toxic effects in the liver, its biodistribution is unknown. KTP-NH2 was radioiodinated to evaluate its biological fate in vivo. Mono-radioiodinated KTP-NH2 ([125I]MIK) was radiochemically stable in vitro, namely in saline at 4 °C up to 1 week, and moderately stable when incubated with human serum at 37 °C. Although the radioiodinated peptide could translocate a cellular model of the blood–brain barrier (BBB), the levels of radioactivity in the brain were minimal, 5 and 10 min after intraperitoneal injection (i.p.). Significant accumulation of 125I was found in the thyroid probably reflecting the hydrolysis of the iodine–tyrosine bond by liver deiodinases. HPLC analysis of plasma samples collected 5 min post-injection showed that intact [125I]MIK accounted for 72.5% of the total radioactivity, whereas the remaining radioactivity was associated with free radioiodide (I−). These findings were subsequently confirmed by the low radioactivity found in the liver and kidney homogenates from rats sacrificed 10 min after i.p. administration. The analgesic activity of mono- and di-iodinated KTP-NH2 was also evaluated by a hot plate assay showing a delayed peak of maximal efficacy compared to KTP-NH2 (30 vs. 15 minutes). Overall, the peripheral effects of the peptides cannot be excluded.

Block copolymer micelles (BCMs), nano-self-assemblies of amphiphilic copolymers with a hydrophobic core surrounded by a hydrophilic corona, were explored for the simultaneous delivery of a lipophilic chemotherapeutic drug and radiation to tumors. Docetaxel (DTX)-functionalized BCMs were radiolabeled with the β− emitter 188Re by two different approaches (direct and indirect labelling) affording in both cases 188Re-Pz-DTX-BCMs with high purity (>98%). The 188Re-labeled BCMs are stable in PBS pH 7.4 and in cell culture medium, at 37 °C. Cell uptake of 188Re-Pz-DTX-BCMs in MDA-MB-231 metastatic breast cancer cells and MNNG/HOS osteosarcoma cells presented a maximal uptake of ca. 12% at 24 h incubation time. Biodistribution studies in tumor-bearing nude mice (MDA-MB-231 human xenografts) has shown prolonged circulation lifetime in the bloodstream and moderate uptake in the tumor (1.8 ± 0.8% I.A./g at 24 h p.i.). The prolonged blood circulation lifetime together with the in vivo stability, suggests that the micelles are potentially useful as drug delivery system to tumors by the EPR effect. As far as we are aware, we describe herein the first example of BCMs, 188Re-Pz-DTX-BCMs, as versatile nanoplatforms for the simultaneous delivery of beta radiation (188Re) and the chemotherapeutic drug (DTX) potentially useful for cancer therapy. The expected synergistic effect of such combination in pain therapy and/or cancer progression holds promise when compared to the conventional sequential treatment.

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

Precision medicine relies on individually tailored therapeutic intervention taking into account individual variability. It is strongly dependent on the availability of target-specific drugs and/or imaging agents that recognize molecular targets and patient-specific disease mechanisms. The most sensitive molecular imaging modalities, Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET), rely on the interaction between an imaging radioprobe and a target. Moreover, the use of target-specific molecular tools for both diagnostics and therapy, theranostic agents, represent an established methodology in nuclear medicine that is assuming an increasingly important role in precision medicine. The design of innovative imaging and/or theranostic agents is key for further accomplishments in the field. G-protein-coupled receptors (GPCRs), apart from being highly relevant drug targets, have also been largely exploited as molecular targets for non-invasive imaging and/or systemic radiotherapy of various diseases. Herein, we will discuss recent efforts towards the development of innovative imaging and/or theranostic agents targeting selected emergent GPCRs, namely the Frizzled receptor (FZD), Ghrelin receptor (GHSR-1a), G protein-coupled estrogen receptor (GPER), and Sphingosine-1-phosphate receptor (S1PR). The pharmacological and clinical relevance will be highlighted, giving particular attention to the studies on the synthesis and characterization of targeted molecular imaging agents, biological evaluation, and potential clinical applications in oncology and non-oncology diseases. Whenever relevant, supporting computational studies will be also discussed.

Cancer has become one of the main leading causes of morbidity and mortality worldwide. One of the critical drawbacks of current cancer therapeutics has been the lack of the target-selectivity, as these drugs should have an effect exclusively on cancer cells while not perturbing healthy ones. In addition, their mechanism of action should be sufficiently fast to avoid the invasion of neighbouring healthy tissues by cancer cells. The use of conventional chemotherapeutic agents and other traditional therapies, such as surgery and radiotherapy, leads to off-target interactions with serious side effects. In this respect, recently developed target-selective Antibody-Drug Conjugates (ADCs) are more effective than traditional therapies, presumably due to their modular structures that combine many chemical properties simultaneously. In particular, ADCs are made up of three different units: a highly selective Monoclonal antibody (Mab) which is developed against a tumour-associated antigen, the payload (cytotoxic agent), and the linker. The latter should be stable in circulation while allowing the release of the cytotoxic agent in target cells. The modular nature of these drugs provides a platform to manipulate and improve selectivity and the toxicity of these molecules independently from each other. This in turn leads to generation of second- and third-generation ADCs, which have been more effective than the previous ones in terms of either selectivity or toxicity or both. Development of ADCs with improved efficacy requires knowledge at the atomic level regarding the structure and dynamics of the molecule. As such, we reviewed all the most recent computational methods used to attain all-atom description of the structure, energetics and dynamics of these systems. In particular, this includes homology modelling, molecular docking and refinement, atomistic and coarse-grained molecular dynamics simulations, principal component and cross-correlation analysis. The full characterization of the structure-activity relationship devoted to ADCs is critical for antibody-drug conjugate research and development.

Non-Hodgkin lymphoma (NHL) is one of the most common causes of cancer-related death in the United States and Europe. Although the outcome of NHL patients has improved over the last years with current therapies, the rate of mortality is still high. A plethora of new drugs is entering clinical development for NHL treatment; however, the approval of new treatments remains low due in part to the paucity of clinically relevant models for validation. Canine lymphoma shares remarkable similarities with its human counterpart, making the dog an excellent animal model to explore novel therapeutic molecules and approaches. Histone deacetylase inhibitors (HDACis) have emerged as a powerful new class of anti-cancer drugs for human therapy. To investigate HDACi antitumor properties on canine diffuse large B-cell lymphoma, a panel of seven HDACi compounds (CI-994, panobinostat, SBHA, SAHA, scriptaid, trichostatin A and tubacin) was screened on CLBL-1 canine B-cell lymphoma cell line. Our results demonstrated that all HDACis tested exhibited dose-dependent inhibitory effects on proliferation of CLBL-1 cells, while promoting increased H3 histone acetylation. Amongst all HDACis studied, panobinostat proved to be the most promising compound and was selected for further in vitro and in vivo evaluation. Panobinostat cytotoxicity was linked to H3 histone and α-tubulin acetylation, and to apoptosis induction. Importantly, panobinostat efficiently inhibited CLBL-1 xenograft tumor growth, and strongly induced acetylation of H3 histone and apoptosis in vivo. In conclusion, these results provide new data validating HDACis and, especially, panobinostat as a novel anti-cancer therapy for veterinary applications, while contributing to comparative oncology.

Canine diffuse large B-cell lymphoma (DLBCL) is one of the most common cancers in dogs which shares remarkable similarities with its human counterpart, making the dog an excellent model for the investigation of novel therapeutic agents. However, the integration of canine lymphoma in comparative studies has been limited due in part to the lack of suitable xenograft mouse models for preclinical studies. To overcome these limitations, we established and characterized a localized subcutaneous bioluminescent canine DLBCL xenograft mouse model. The canine CLBL-1 cell line stably expressing the luciferase and green fluorescent protein reporters was generated and used to establish the xenograft tumor model. A pilot study was first conducted with three different cell densities (0.1×10(6), 0.5×10(6) and 1×10(6) cells) in SCID mice. All mice presented homogeneous tumor induction within eight days after subcutaneous injection, with a 100% engraftment efficiency and no significant differences were observed among groups. The tumors were highly aggressive and localized at the site of inoculation and reproduced histological features and immunophenotype consistent with canine DLBCL. Importantly, xenograft tumors were detected and quantified by bioluminescent imaging. To assess response to therapy, a therapeutic study with a histone deacetylase inhibitor, panobinostat, was performed. The results demonstrated that panobinostat (20 mg/kg) efficiently inhibited tumor growth and that bioluminescent imaging allowed the monitorization and quantification of tumor response to therapy. In summary, this study provides a bioluminescence canine DLBCL model that offers high engraftment efficiency, preservation of tumor features, and noninvasive monitoring of tumor progression, validating the model as a promising preclinical tool for both veterinary and human medicine.

A straightforward synthetic route for a new multifunctional 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivative is described. To demonstrate the versatility of this pro-chelator for the preparation of radiolabeled hybrid compounds containing two different biological targeting moieties, an antitumor agent (e.g., a DNA-intercalating agent) and an estrogen receptor (ER) ligand (e.g., LXXLL-based peptide) were regiospecifically conjugated to the DOTA derivative. The bifunctional probe was radiolabeled with the auger electron emitter indium-111, and the resulting radioconjugate was demonstrated to induce DNA damage in vitro, which, along with the nuclear internalization exhibited in breast cancer cells, might enhance its therapeutic activity. This favorable in vitro performance suggests that these hybrid compounds could be attractive probes for theranostic applications.

In order to target platinum complexes to cancer cells, trans-Pt(II) or trans-Pt(IV) complexes were bioconjugated to the cyclic peptide cRGDfK (cRGD), with affinity for αvβ3 integrin receptors, through their 4-picolinic acid spectator ligands. To tackle this goal, the Pt(II) and Pt(IV) precursors were activated at their carboxylic acid function and futher reacted with the cRGDfK peptide, to afford the bioconjugates Pt(II)-cRGD and Pt(IV)-cRGD, respectively. Pt(II)-cRGD was studied by 195Pt-NMR that confirmed the presence of the Pt(II) center. In contrast, the characterization of Pt(IV)-cRGD was not possible due to the tendency of this complex to undergo reduction to Pt(II) in solution. Thus, only the Pt(II)-cRGD complex was used for further biological studies, and it exhibited some cytotoxic activity against the HUVEC cell line, with the highest levels of αvβ3 expression. However, no improved effects were observed with respect to the Pt(II)-pic precursor. Studies by ICP-MS showed enhanced intracellular accumulation for Pt(II)-cRGD with respect to Pt(II)-pic in cancer cells. Overall, these results show that while Pt(II) bioconjugation enhances compound's uptake, it did not translate into an increase in cytotoxicity.

Human Epidermal Growth Factor Receptor 2 (HER2) is, among EGFR family, one of the most relevant members as it remains overexpressed on tumor cells and provides resistance to well-studied anti-HER2 monoclonal antibody, Trastuzumab (Herceptin®), or tyrosine kinase inhibitor. Furthermore, HER2 plays a key role in the HER family due the interaction with other HER receptors via a complex signaling network to regulate cell growth, differentiation and survival. In this work, we have employed computational modelling and Molecular Dynamic (MD) simulations to attain a deeper understanding of the interaction of specific anti-HER2 antibodies and HER2. The dynamic behavior of HER2 receptor in complex with F0178 and scFv from Trastuzumab was investigated by two replicas of 0.5 µs MD simulations for each system as well as for the individual ones. A variety of structural, energetic and dynamic characteristics ranging from pairwise interactions formation to covariance analyses were performed to the 2 bundle complexes. Our aim was to understand the all-atom details of these intermolecular couplings, fundamental for the development of new therapies.

© 2017 American Chemical Society, The delivery of therapeutic molecules to the central nervous system is hampered by poor delivery across the blood-brain barrier (BBB). Several strategies have been proposed to enhance transport into the brain, including invasive techniques and receptor-mediated transport (RMT). Both approaches have several drawbacks, such as BBB disruption, receptor saturation, and off-target effects, raising safety issues. Herein, we show that specific domains of Dengue virus type 2 capsid protein (DEN2C) can be used as trans-BBB peptide vectors. Their mechanism of translocation is receptor-independent and consistent with adsorptive-mediated transport (AMT). One peptide in particular, named PepH3, reaches equilibrium distribution concentrations across the BBB in less than 24 h in a cellular in vitro assay. Importantly, in vivo biodistribution data with radiolabeled peptide derivatives show high brain penetration. In addition, there is fast clearance from the brain and high levels of excretion, showing that PepH3 is a very good candidate to be used as a peptide shuttle taking cargo in and out of the brain., The authors thank the Portuguese Funding Agency, Fundação para a Ciência e a Tecnologia, FCT IP, for financial support (grants SFRH/BPD/94466/2013; SFRH/BPD/109010/2015; IF/01010/2013; PTDC/BBBNAN/1578/2014; HIVERA/ 0002/2013) and Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE), call 20-MSCARISE-2014 (grant agreement H20 644167 − INPACT). M.M., L.G., C.F., and J.D.G.C. gratefully acknowledge FCT support through the UID/Multi/04349/2013 project.

Bone-seeking radiopharmaceuticals such as radiolabeled bisphosphonates (BPs) bind to bone matrix in areas of increased bone turnover, characteristic of metastization sites, delivering selectively γ radiation or β− particles for bone imaging or bone-pain palliation, respectively. Recent preclinical studies suggest that nitrogen-containing BPs may have also a direct anti-tumor activity. We have recently introduced a set of 99mTc-organometallic complexes that allow the simultaneous delivery of γ radiation and BPs to bone tissue. Aimed at finding complexes of the same family with improved bone-seeking properties and adequate tumor cell uptake profile, we have synthesized novel complexes of the type fac-[M(CO)3(k3-L)]+ (M = 99mTc/Re, Tc3/Re3–Tc5/Re5) stabilized by bifunctional chelators with different molecular weight, overall charge, (lipo)hydrophilic nature and different positions of BP attachment. Biodistribution studies in normal mice have shown that Tc3, where the BP unit (alendronate) is at position 4 of the pyrazolyl ring, presents the most favorable pharmacokinetic profile with fast blood clearance, high bone uptake (17.1 ± 3.6% I.A./g at 1 h p.i.), and higher bone-to-blood and bone-to-muscle ratios than the gold standard 99mTc-MDP. Cellular uptake studies in the MDAMB231 metastatic breast cancer cells showed a maximal uptake of 4–6% at 6 h incubation for all complexes. Interestingly, cell fragmentation studies have shown that Tc3 presents the highest accumulation in the cytosol (35%). The promising biological profile of the BP-containing complex Tc3 encourages further research towards evaluation of 188Re congeners for potential therapeutic applications.

We describe the synthesis and biological evaluation of the cationic (99m)Tc-tricarbonyl complex fac-[(99m)Tc(CO)3 (κ(3) -L1)](+) (Tc1) anchored by a pyrazole-diamine-methylbenzylguanidine-based ligand (L1), as potentially useful for myocardial imaging. The rhenium complex fac-[Re(CO)3 (κ(3)-L1)](+) (Re1) was prepared and characterized as a 'cold' surrogate of the radioactive complex. Cell uptake studies in a neuroblastoma cell line suggest that Tc1 uptake mechanism is related to the norepinephrine transporter (NET). Tissue distribution studies in CD1 mice showed that Tc1 presents high initial heart uptake and a slow washout from the heart (7.8 ± 1.3% injected dose per gram (ID/g), 30-min post-injection (p.i.); 6.3 ± 1.3% ID/g, 60-min p.i.), with heart to blood ratios of 11.8 and 9.0 at 30- and 60-min p.i., respectively. The uptake mechanism of Tc1 appears to be similar to that of metaiodobenzylguanidine (MIBG), as it can be reduced by coinjection with nonradioactive MIBG. The biodistribution profile of Tc2, where the benzylguanidine pharmacophore is absent, corroborates the fact that Tc1 does not accumulate in the heart by a simple diffusion mechanism but rather by a NET-mediated mechanism. The results confirm those obtained in the cell assays. Despite the persistent heart uptake found for Tc1, the high hepatic and renal uptake remains to be improved.