The Impact of Nε-Acryloyllysine Piperazides on the Conformational Dynamics of Transglutaminase 2.

In addition to the classic functions of proteins, such as acting as a biocatalyst or binding partner, the conformational states of proteins and their remodeling upon stimulation need to be considered. A prominent example of a protein that undergoes comprehensive conformational remodeling is transglutaminase 2 (TGase 2), the distinct conformational states of which are closely related to particular functions. Its involvement in various pathophysiological processes, including fibrosis and cancer, motivates the development of theranostic agents, particularly based on inhibitors that are directed toward the transamidase activity. In this context, the ability of such inhibitors to control the conformational dynamics of TGase 2 emerges as an important parameter, and methods to assess this property are in great demand. Herein, we describe the application of the switchSENSE® principle to detect conformational changes caused by three irreversibly binding Nε-acryloyllysine piperazides, which are suitable radiotracer candidates of TGase 2. The switchSENSE® technique is based on DNA levers actuated by alternating electric fields. These levers are immobilized on gold electrodes with one end, and at the other end of the lever, the TGase 2 is covalently bound. A novel computational method is introduced for describing the resulting lever motion to quantify the extent of stimulated conformational TGase 2 changes. Moreover, as a complementary biophysical method, native polyacrylamide gel electrophoresis was performed under similar conditions to validate the results. Both methods prove the occurrence of an irreversible shift in the conformational equilibrium of TGase 2, caused by the binding of the three studied Nε-acryloyllysine piperazides.

Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins.

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

Application of a Fluorescence Anisotropy-Based Assay to Quantify Transglutaminase 2 Activity in Cell Lysates.

Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates.

Solid-Phase Synthesis of Selectively Mono-Fluorobenz(o)ylated Polyamines as a Basis for the Development of 18F-Labeled Radiotracers.

Polyamines are highly attractive vectors for tumor targeting, particularly with regards to the development of radiolabeled probes for imaging by positron emission (PET) and single-photon emission computed tomography (SPECT). However, the synthesis of selectively functionalized derivatives remains challenging due to the presence of multiple amino groups of similar reactivity. In this work, we established a synthetic methodology for the selective mono-fluorobenz(o)ylation of various biogenic diamines and polyamines as lead compounds for the perspective development of substrate-based radiotracers for targeting polyamine-specific membrane transporters and enzymes such as transglutaminases. For this purpose, the polyamine scaffold was constructed by solid-phase synthesis of the corresponding oxopolyamines and subsequent reduction with BH3/THF. Primary and secondary amino groups were selectively protected using Dde and Boc as protecting groups, respectively, in orientation to previously reported procedures, which enabled the selective introduction of the reporter groups. For example, N1-FBz-spermidine, N4-FBz-spermidine, N8-FBz-spermidine, and N1-FBz-spermine and N4-FBz-spermine (FBz = 4-fluorobenzoyl) were obtained in good yields by this approach. The advantages and disadvantages of this synthetic approach are discussed in detail and its suitability for radiolabeling was demonstrated for the solid-phase synthesis of N1-[18F]FBz-cadaverine.

Solution-phase synthesis of the fluorogenic TGase 2 acyl donor Z-Glu(HMC)-Gly-OH and its use for inhibitor and amine substrate characterisation.

A reliable solution-phase synthesis of the water-soluble dipeptidic fluorogenic transglutaminase substrate Z-Glu(HMC)-Gly-OH is presented. The route started from Z-Glu-OH, which was converted into the corresponding cyclic anhydride. This building block was transformed into the regioisomeric α- and γ-dipeptides. The key step was the esterification of Z-Glu-Gly-OtBu with 4-methylumbelliferone. The final substrate compound was obtained in an acceptable yield and excellent purity without the need of purification by RP-HPLC. The advantage of this acyl donor substrate for the kinetic characterisation of inhibitors and amine-type acyl acceptor substrates is demonstrated by evaluating commercially available or literature-known irreversible inhibitors and the biogenic amines serotonin, histamine and dopamine, respectively.

Synthesis, 18F-labelling and radiopharmacological characterisation of the C-terminal 30mer of Clostridium perfringens enterotoxin as a potential claudin-targeting peptide.

The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumours and represents an important emerging target for both diagnosis and treatment of solid tumours of epithelial origin. The C-terminal fragment of the Clostridium perfringens enterotoxin cCPE290-319 appears as a suitable ligand for targeting Cld-4. The synthesis of this 30mer peptide was attempted via several approaches, which has revealed sequential SPPS using three pseudoproline dipeptide building blocks to be the most efficient one. Labelling with fluorine-18 was achieved on solid phase using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and 4-[18F]fluorobenzoyl chloride as 18F-acylating agents, which was the most advantageous when [18F]SFB was reacted with the resin-bound 30mer containing an N-terminal 6-aminohexanoic spacer. Binding to Cld-4 was demonstrated via surface plasmon resonance using a protein construct containing both extracellular loops of Cld-4. In addition, cell binding experiments were performed for 18F-labelled cCPE290-319 with the Cld-4 expressing tumour cell lines HT-29 and A431 that were complemented by fluorescence microscopy studies using the corresponding fluorescein isothiocyanate-conjugated peptide. The 30mer peptide proved to be sufficiently stable in blood plasma. Studying the in vivo behaviour of 18F-labelled cCPE290-319 in healthy mice and rats by dynamic PET imaging and radiometabolite analyses has revealed that the peptide is subject to substantial liver uptake and rapid metabolic degradation in vivo, which limits its suitability as imaging probe for tumour-associated Cld-4.

Synthesis and preliminary radiopharmacological characterisation of an 11 C-labelled azadipeptide nitrile as potential PET tracer for imaging of cysteine cathepsins.

An O-methyltyrosine-containing azadipeptide nitrile was synthesised and investigated for its inhibitory activity towards cathepsins L, S, K, and B. Labelling with carbon-11 was accomplished by reaction of the corresponding phenolic precursor with [11 C]methyl iodide starting from cyclotron-produced [11 C]methane. Radiopharmacological evaluation of the resulting radiotracer in a mouse xenograft model derived from a mammary tumour cell line by small animal PET imaging indicates tumour targeting with complex pharmacokinetics. Radiotracer uptake in the tumour region was considerably lower under treatment with the nonradioactive reference compound and the epoxide-based irreversible cysteine cathepsin inhibitor E64. The in vivo behaviour observed for this radiotracer largely confirms that of the corresponding 18 F-fluoroethylated analogue and suggests the limited suitability of azadipeptide nitriles for the imaging of tumour-associated cysteine cathepsins despite target-mediated uptake is evidenced.

A fluorescence anisotropy-based assay for determining the activity of tissue transglutaminase.

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2's transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein- and rhodamine B-conjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solid-phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published L-lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z' factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

Synthesis and Kinetic Characterisation of Water-Soluble Fluorogenic Acyl Donors for Transglutaminase†2.

Small glutamate-containing peptides bearing coumarin derivatives as fluorescent leaving groups attached to the γ-carboxylic acid group of the Glu residue were synthesised and investigated with regard to their potential to act as substrates for transglutaminase 2 (TGase 2). Their synthesis was accomplished by an efficient solid-phase approach. The excellent water solubility of the compounds enabled their extensive kinetic characterisation in the context of TGase 2-catalysed hydrolysis and aminolysis. The influence of the coumarin skeleton's substitution pattern on the kinetic properties was studied. Derivatives containing 7-hydroxy-4-methylcoumarin (HMC) revealed properties superior to those of their 7-hydroxycoumarin counterparts; analogous amides are not accepted as substrates. Z-Glu(HMC)-Gly-OH, which exhibited the best substrate properties out of the investigated derivatives, was selected for representative kinetic characterisation of acyl acceptor substrates and irreversible inhibitors.

Cyclopeptides containing the DEKS motif as conformationally restricted collagen telopeptide analogues: synthesis and conformational analysis.

The collagen telopeptides play an important role for lysyl oxidase-mediated crosslinking, a process which is deregulated during tumour progression. The DEKS motif which is located within the N-terminal telopeptide of the α1 chain of type I collagen has been suggested to adopt a ßI-turn conformation upon docking to its triple-helical receptor domain, which seems to be critical for lysyl oxidase-catalysed deamination and subsequent crosslinking by Schiff-base formation. Herein, the design and synthesis of cyclic peptides which constrain the DEKS sequence in a ß-turn conformation will be described. Lysine-side chain attachment to 2-chlorotrityl chloride-modified polystyrene resin followed by microwave-assisted solid-phase peptide synthesis and on-resin cyclisation allowed for an efficient access to head-to-tail cyclised DEKS-derived cyclic penta- and hexapeptides. An N(ε)-(4-fluorobenzoyl)lysine residue was included in the cyclopeptides to allow their potential radiolabelling with fluorine-18 for PET imaging of lysyl oxidase. Conformational analysis by (1)H NMR and chiroptical (electronic and vibrational CD) spectroscopy together with MD simulations demonstrated that the concomitant incorporation of a D-proline and an additional lysine for potential radiolabel attachment accounts for a reliable induction of the desired ßI-turn structure in the DEKS motif in both DMSO and water as solvents. The stabilised conformation of the cyclohexapeptide is further reflected by its resistance to trypsin-mediated degradation. In addition, the deaminated analogue containing allysine in place of lysine has been synthesised via the corresponding ε-hydroxynorleucine containing cyclohexapeptide. Both ε-hydroxynorleucine and allysine containing cyclic hexapeptides have been subjected to conformational analysis in the same manner as the lysine-based parent structure. Thus, both a conformationally restricted lysyl oxidase substrate and product have been synthetically accessed, which will enable their potential use for molecular imaging of these important enzymes.

Preclinical evaluation of an 18F-labeled Nε-acryloyllysine piperazide for covalent targeting of transglutaminase 2.

BACKGROUND: Transglutaminase 2 (TGase 2) is a multifunctional protein and has a prominent role in various (patho)physiological processes. In particular, its transamidase activity, which is rather latent under physiological conditions, gains importance in malignant cells. Thus, there is a great need of theranostic probes for targeting tumor-associated TGase 2, and targeted covalent inhibitors appear to be particularly attractive as vector molecules. Such an inhibitor, equipped with a radionuclide suitable for noninvasive imaging, would be supportive for answering the general question on the possibility for functional characterization of tumor-associated TGase 2. For this purpose, the recently developed 18F-labeled Nε-acryloyllysine piperazide [18F]7b, which is a potent and selective irreversible inhibitor of TGase 2, was subject to a detailed radiopharmacological characterization herein. RESULTS: An alternative radiosynthesis of [18F]7b is presented, which demands less than 300 µg of the respective trimethylammonio precursor per synthesis and provides [18F]7b in good radiochemical yields (17 ± 7%) and high (radio)chemical purities (≥ 99%). Ex vivo biodistribution studies in healthy mice at 5 and 60 min p.i. revealed no permanent enrichment of 18F-activity in tissues with the exception of the bone tissue. In vivo pretreatment with ketoconazole and in vitro murine liver microsome studies complemented by mass spectrometric analysis demonstrated that bone uptake originates from metabolically released [18F]fluoride. Further metabolic transformations of [18F]7b include mono-hydroxylation and glucuronidation. Based on blood sampling data and liver microsome experiments, pharmacokinetic parameters such as plasma and intrinsic clearance were derived, which substantiated the apparently rapid distribution of [18F]7b in and elimination from the organisms. A TGase 2-mediated uptake of [18F]7b in different tumor cell lines could not be proven. Moreover, evaluation of [18F]7b in melanoma tumor xenograft models based on A375-hS100A4 (TGase 2 +) and MeWo (TGase 2 -) cells by ex vivo biodistribution and PET imaging studies were not indicative for a specific targeting. CONCLUSION: [18F]7b is a valuable radiometric tool to study TGase 2 in vitro under various conditions. However, its suitability for targeting tumor-associated TGase 2 is strongly limited due its unfavorable pharmacokinetic properties as demonstrated in rodents. Consequently, from a radiochemical perspective [18F]7b requires appropriate structural modifications to overcome these limitations.

Tissue transglutaminase: an emerging target for therapy and imaging.

Tissue transglutaminase (transglutaminase 2) is a multifunctional enzyme with many interesting properties resulting in versatile roles in both physiology and pathophysiology. Herein, the particular involvement of the enzyme in human diseases will be outlined with special emphasis on its role in cancer and in tissue interactions with biomaterials. Despite recent progress in unraveling the different cellular functions of transglutaminase 2, several questions remain. Transglutaminase 2 features in both confirmed and some still ambiguous roles within pathological conditions, raising interest in developing inhibitors and imaging probes which target this enzyme. One important prerequisite for identifying and characterizing such molecular tools are reliable assay methods to measure the enzymatic activity. This digest Letter will provide clarification about the various assay methods described to date, accompanied by a discussion of recent progress in the development of inhibitors and imaging probes targeting transglutaminase 2.

Comparative Saturation Binding Analysis of 64Cu-Labeled Somatostatin Analogues Using Cell Homogenates and Intact Cells.

The development of novel ligands for G-protein-coupled receptors (GPCRs) typically entails the characterization of their binding affinity, which is often performed with radioligands in a competition or saturation binding assay format. Since GPCRs are transmembrane proteins, receptor samples for binding assays are prepared from tissue sections, cell membranes, cell homogenates, or intact cells. As part of our investigations on modulating the pharmacokinetics of radiolabeled peptides for improved theranostic targeting of neuroendocrine tumors with a high abundance of the somatostatin receptor sub-type 2 (SST2), we characterized a series of 64Cu-labeled [Tyr3]octreotate (TATE) derivatives in vitro in saturation binding assays. Herein, we report on the SST2 binding parameters measured toward intact mouse pheochromocytoma cells and corresponding cell homogenates and discuss the observed differences taking the physiology of SST2 and GPCRs in general into account. Furthermore, we point out method-specific advantages and limitations.

Lysyl Oxidases as Targets for Cancer Therapy and Diagnostic Imaging.

The understanding of the contribution of the tumour microenvironment to cancer progression and metastasis, in particular the interplay between tumour cells, fibroblasts and the extracellular matrix has grown tremendously over the last years. Lysyl oxidases are increasingly recognised as key players in this context, in addition to their function as drivers of fibrotic diseases. These insights have considerably stimulated drug discovery efforts towards lysyl oxidases as targets over the last decade. This review article summarises the biochemical and structural properties of theses enzymes. Their involvement in tumour progression and metastasis is highlighted from a biochemical point of view, taking into consideration both the extracellular and intracellular action of lysyl oxidases. More recently reported inhibitor compounds are discussed with an emphasis on their discovery, structure-activity relationships and the results of their biological characterisation. Molecular probes developed for imaging of lysyl oxidase activity are reviewed from the perspective of their detection principles, performance and biomedical applications.

Enzymological Characterization of 64Cu-Labeled Neprilysin Substrates and Their Application for Modulating the Renal Clearance of Targeted Radiopharmaceuticals.

The applicability of radioligands for targeted endoradionuclide therapy is limited due to radiation-induced toxicity to healthy tissues, in particular to the kidneys as primary organs of elimination. The targeting of enzymes of the renal brush border membrane by cleavable linkers that permit the formation of fast eliminating radionuclide-carrying cleavage fragments gains increasing interest. Herein, we synthesized a small library of 64Cu-labeled cleavable linkers and quantified their substrate potentials toward neprilysin (NEP), a highly abundant peptidase at the renal brush border membrane. This allowed for the derivation of structure-activity relationships, and selected cleavable linkers were attached to the somatostatin receptor subtype 2 ligand [Tyr3]octreotate. Radiopharmacological characterization revealed that a substrate-based targeting of NEP in the kidneys with small peptides entails their premature cleavage in the blood circulation by soluble and endothelium-derived NEP. However, for a kidney-specific targeting of NEP, the additional targeting of albumin in the blood is highlighted.

Dipeptide-Derived Alkynes as Potent and Selective Irreversible Inhibitors of Cysteine Cathepsins.

The potential of designing irreversible alkyne-based inhibitors of cysteine cathepsins by isoelectronic replacement in reversibly acting potent peptide nitriles was explored. The synthesis of the dipeptide alkynes was developed with special emphasis on stereochemically homogeneous products obtained in the Gilbert-Seyferth homologation for C≡C bond formation. Twenty-three dipeptide alkynes and 12 analogous nitriles were synthesized and investigated for their inhibition of cathepsins B, L, S, and K. Numerous combinations of residues at positions P1 and P2 as well as terminal acyl groups allowed for the derivation of extensive structure-activity relationships, which were rationalized by computational covalent docking for selected examples. The determined inactivation constants of the alkynes at the target enzymes span a range of >3 orders of magnitude (3-10 133 M-1 s-1). Notably, the selectivity profiles of alkynes do not necessarily reflect those of the nitriles. Inhibitory activity at the cellular level was demonstrated for selected compounds.

Preparation of 18F-Labeled Tracers Targeting Fibroblast Activation Protein via Sulfur [18F]Fluoride Exchange Reaction.

Early detection and treatment of cancers can significantly increase patient prognosis and enhance the quality of life of affected patients. The emerging significance of the tumor microenvironment (TME) as a new frontier for cancer diagnosis and therapy may be exploited by radiolabeled tracers for diagnostic imaging techniques such as positron emission tomography (PET). Cancer-associated fibroblasts (CAFs) within the TME are identified by biomarkers such as fibroblast activation protein alpha (FAPα), which are expressed on their surfaces. Targeting FAPα using small-molecule 18F-labeled inhibitors (FAPIs) has recently garnered significant attention for non-invasive tumor visualization using PET. Herein, two potent aryl-fluorosulfate-based FAPIs, 12 and 13, were synthetically prepared, and their inhibition potency was determined using a fluorimetric FAP assay to be IC50 9.63 and 4.17 nM, respectively. Radiofluorination was performed via the sulfur [18F]fluoride exchange ([18F]SuFEx) reaction to furnish [18F]12 and [18F]13 in high activity yields (AY) of 39-56% and molar activities (Am) between 20-55 GBq/µmol. In vitro experiments focused on the stability of the radiolabeled FAPIs after incubation with human serum, liver microsomes and liver cytosol. Preliminary PET studies of the radioligands were performed in healthy mice to investigate the in vivo biodistribution and 18F defluorination rate. Fast pharmacokinetics for the FAP-targeting tracers were retained and considerable bone uptake, caused by either 18F defluorination or radioligand accumulation, was observed. In summary, our findings demonstrate the efficiency of [18F]SuFEx as a radiolabeling method as well as its advantages and limitations with respect to PET tracer development.

Investigation of Radiotracer Metabolic Stability In Vitro with CYP-Overexpressing Hepatoma Cell Lines.

The characterization of novel radiotracers toward their metabolic stability is an essential part of their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained on cytochrome P450 (CYP) enzyme and isoform-specific contribution to the metabolic fate of individual radiotracers. Herein, we investigated recently established CYP-overexpressing hepatoblastoma cell lines (HepG2) for their suitability to study the metabolic stability of radiotracers in general and to gain insight into CYP isoform specificity. Wildtype HepG2 and CYP1A2-, CYP2C19-, and CYP3A4-overexpressing HepG2 cells were incubated with radiotracers, and metabolic turnover was analyzed. The optimized protocol, covering cell seeding in 96-well plates and analysis of supernatant by radio thin-layer-chromatography for higher throughput, was transferred to the evaluation of three 18F-labeled celecoxib-derived cyclooxygenase-2 inhibitors (coxibs). These investigations revealed time-dependent degradation of the intact radiotracers, as well as CYP isoform- and substrate-specific differences in their metabolic profiles. HepG2 CYP2C19 proved to be the cell line showing the highest metabolic turnover for each radiotracer studied here. Comparison with human and murine liver microsome assays showed good agreement with the human metabolite profile obtained by the HepG2 cell lines. Therefore, CYP-overexpressing HepG2 cells provide a good complement for assessing the metabolic stability of radiotracers and allow the analysis of the CYP isoform-specific contribution to the overall radiotracer metabolism.

The Role of Transglutaminase 2 in the Radioresistance of Melanoma Cells.

Transglutaminase 2 (TG2) is a protein expressed in many tissues that exerts numerous, sometimes contradictory, intra- and extracellular functions, under both physiological and pathophysiological conditions. In the context of tumor progression, it has been found to be involved in cell adhesion, DNA repair mechanisms, induction of apoptosis, and mesenchymal transdifferentiation, among others. Here, we hypothesized that TG2 also contributes to the radioresistance of two human melanoma cell lines, A375 and MeWo, which can be seen to differ in their basal TG2 biosynthesis by examining their proliferation and clonal expansion after irradiation. For this purpose, cellular TG2 biosynthesis and TG2 activity were modulated by transfection-induced overexpression or TG2 knock-out and application of TG2-selective inhibitors. Proliferation and clonal expansion of TG2-overexpressing cells was not enhanced over wildtype cells, suggesting that increased TG2 biosynthesis does not further enhance the radioresistance of melanoma cells. Conversely, TG2 knock-out in A375 cells reduced their proliferation, as well as clonal and spheroidal expansion after irradiation, which indicates a contribution of TG2 to the radioresistance of melanoma cells. Since TG1, TG3, and partly also, TG6 biosynthesis was detectable in A375 and MeWo cells, it can be assumed that these other members of the TG family may exert a partially compensatory effect.

"Clickable" Albumin Binders for Modulating the Tumor Uptake of Targeted Radiopharmaceuticals.

The intentional binding of radioligands to albumin gains increasing attention in the context of radiopharmaceutical cancer therapy as it can lead to an enhanced radioactivity uptake into the tumor lesions and, thus, to a potentially improved therapeutic outcome. However, the influence of the radioligand's albumin-binding affinity on the time profile of tumor uptake has been only partly addressed so far. Based on the previously identified Nε-4-(4-iodophenyl)butanoyl-lysine scaffold, we designed "clickable" lysine-derived albumin binders (cLABs) and determined their dissociation constants toward albumin by novel assay methods. Structure-activity relationships were derived, and selected cLABs were applied for the modification of the somatostatin receptor subtype 2 ligand (Tyr3)octreotate. These novel conjugates were radiolabeled with copper-64 and subjected to a detailed in vitro and in vivo radiopharmacological characterization. Overall, the results of this study provide an incentive for further investigations of albumin binders for applications in endoradionuclide therapies.