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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 homogenous products obtained in the Gilbert-Seyferth homologation for C-C triple bond formation. 23 dipeptide alkynes and 12 analogous nitriles were synthesized and investigated for their inhibition of cathepsins B, L, S and K. Numerous combinations of residues in the P1 and P2 positions 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 more than three orders of magnitude (3-10,133 M-1s-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.

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.

Until recently, alkynes were considered bioinert. Thus, they are popular reaction partners in bioorthogonal click reactions in vitro and in vivo. Despite the virtual chemical inertness of the alkyne moiety, two research groups observed the irreversible inhibition of a cysteine protease by an alkyne functionalised substrate derivative: both EKKEBUS et al. and SOMMER et al. independently described the unexpected inactivation of de-ubiquitinating enzymes by propargylated ubiquitin or ubiquitin-like modifiers bearing propargylamine in place of C-terminal glycine [1, 2]. We intended to harness that finding for the design of inhibitor-based probes for the imaging of tumour-associated cysteine proteases. Cysteine cathepsins play an important role in tumour progression. In particular, cathepsin B is involved in a variety of tumour progression-related processes and an elevated extracellular levels are linked to increased malignancy and poor prognosis [3]. Therefore, this enzyme represents a promising target for the therapy and imaging of tumours. GREENSPAN et al. reported a potent and highly selective, dipeptidyl nitrile-based cathepsin B inhibitor (N-[2-[(3-Carboxyphenyl)methoxy]-1(S)-cyanoethyl]-3-methyl-N2-(2,4-difluorobenzoyl)-L-phenylalaninamide) [4]. Based on that lead compound, cathepsin B-targeting dipeptide alkynes were designed by isoelectronic replacement of the nitrile nitrogen atom by by a methine group and consecutive variation of the 2,4-difluorobenzoyl and (3-carboxybenzyl)oxymethyl residue. Formation of the C-C triple bond by reaction of the corresponding open-chain serine-derived aldehyde with the Bestmann-Ohira reagent was accompanied by partial enantiomerisation. Therefore, the synthesis was performed via Garner’s aldehyde, which accounted for high stereochemical purity of the final compounds. The inhibitory potential was investigated against cathepsins B, S, L and K. The most potent compound exhibited irreversible inhibition of cathepsin B with an inactivation constant (kinact/KI=771 M-1s-1). Values for cathepsins L, S and K were significantly lower; no irreverisible ihibition was observed for cathepsin K. In addition, inhibition of cathepsin B activity in human glioblastoma cell lysates and living cells has been demonstrated. Based on these promising results, dipeptidyl alkynes have the potential to become a valuable tool for imaging due to the expected low activity towards other cysteine proteases. In further studies, selected inhibitors for cathepsin B will be labelled with suitable radionuclides to obtain an inhibitor-based probe directed towards cathepsin B. [1] Ekkebus et al., J. Am. Chem. Soc., 2013, 135, 2867-2870 [2] Sommer et al., Bioorg. Med. Chem., 2013, 21, 2511-2517 [3] Löser and Pietzsch, Front. Chem., 2015, 3:37 [4] Greenspan et al., J. Med. Chem., 2001, 44, 4524-4534.

In our understanding of the many drivers of malignant progression and cancer metastasis, proteases are increasingly drawn into the spotlight as crucial agents in cancer angiogenesis, invasion, and metastasis [1]. Elevated activities of multiple members of the cathepsin family have been shown to correlate with increased metastasis and high therapy resistance [2, 3]. Especially high expression levels of extracellular cathepsin B (CTB) indicate poor prognosis in neoplastic diseases, making CTB an interesting target for activity-based molecular imaging in diagnostics as well as in therapy monitoring for personalised therapies. It is our aim to develop such a probe by combination of a polyarginine-based, activatable cell penetrating peptide (ACPP), as first described by R. Tsien, and an optimised endopeptidase substrate for CTB [4]. Substrate optimisation proofed to be challenging as two entirely opposite factors needed to be balanced – high stability against serum proteases to prevent premature cleavage of the activation sequence, while retaining efficient and specific endoproteolytic cleavability by CTB. We have generated a CTB-endoprotease substrate by C-terminally elongating the CTB carboxydipeptidase substrate Abz GIVR*AK(Dnp) OH (Abz - aminobenzoyl, Dnp – dinitrophenyl, * – cleavage site), described by Cotrin et al. in 2004, to the octapeptide Abz GIVR*AK(Dnp)GX CONH2, which could be used as activation site in the final ACPP [5]. Introduction of any amino acid other than glycine at the P4’ position resulted in hysteretic kinetics for the CTB-catalysed hydrolysis of the octapeptides, which might indicate the displacement of the occluding loop from the active site upon interaction with the substrates. Using LC-ES-MS-based analysis of serum-incubated substrates, the positions P1 and P3’ were determined to be primary determinants of serum stability. After suppression of the P3’ instability by Nα-methylation and optimisation within the positions P1-P3, we were able to increase serum half-life from < 5 min to > 1440 min under concomitant improvement of kinetic substrate efficiency towards CTB. Currently, the substrate is optimised towards CTB-specificity within the cathepsin protease family. Additionally, cell uptake studies of a fluorescently labelled ACPP using the optimised CTB-endoprotease substrate sequence are ongoing. Using this fluorescent probe, we plan to study cell uptake, in vivo stability and initial biodistribution. Furthermore, an ACPP conjugated to radiometal-chelating entities is currently prepared, which will allow for PET imaging in vivo. Literature (1) Yang et al., Cancer Growth Metastasis 2009, 2, 13-27 (2) Aggarwal and Sloane, Proteomics Clin. Appl. 2014, 8(5-6), 427-437 (3) Löser and Pietzsch, Front. Chem. 2015, 3, article 37 (4) Jiang et al., PNAS, 2004, 101(51), 17867-17872 (5) Cotrin et al., Anal. Biochem. 2004, 335, 244-252

Papain-like cysteine proteases bear an enormous potential as drug discovery targets for both infectious and systemic human diseases. The considerable progress in this field over the last two decades has also raised interest in the visualization of these enzymes in their native context, especially with regard to tumor imaging. After a short introduction to structure and general functions of human cysteine cathepsins, we highlight their importance for drug discovery and development and provide a critical update on the current state of knowledge towards their involvement in tumor progression, with a special emphasis on their role in therapy response. In accordance with a radiopharmaceutical point of view, the main focus of this review article will be the discussion of recently developed fluorescence and radiotracer-based imaging agents together with related molecular probes.

Considerable evidence for the implication of tissue transglutaminase (TGase 2) in a variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor progression, has been revealed over the recent years. This renders TGase 2 attractive for developing agents which allow the enzyme’s targeting for both therapeutic and imaging purposes. The development of such molecules requires the establishment of reliable methods to assess the interaction with TGase 2, which can be done most conveniently in continuous kinetic assays. Several assays have been published over the last decades to determine TGase 2 activity, with only very few using the method of fluorescence anisotropy. Measurement of fluorescence anisotropy offers a better signal to noise ratio than other techniques, such as those based solely on fluorescence emission and does not need washing or separation of unbound fluorescent substance. Here, we report a fluorescence anisotropy-based approach for the determination of TGase 2’s transamidase activity, established and validated by using fluorescein- and rhodamine B-labeled cadaverines as acyl acceptor substrates. The synthesis of the cadaverine derivatives has been accomplished in a solid-phase approach. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions have been introduced between the cadaverine and xanthenyl entities. The increase in fluorescence anisotropy resulting from covalent binding of the relatively small cadaverine derivatives to the much larger acyl donor substrate N,N-dimethylated casein was followed over time and enzyme activities were derived thereof. The assay was found to be highly reproducible and shows no background signal in the absence of the enzyme for all synthesized cadaverine derivatives. After characterization of the enzyme–substrate interaction by determination of the Michaelis constants, Km, and the maximum velocities of substrate conversion, Vmax, the assay was validated for screening of non-covalent and covalent inhibitors by using the literature-known substances GTP and iodoacetamide, respectively, as well as a recently reported L-lysine acrylamide derivative.

Transglutaminases and polyamine transporters are promising targets for functional imaging of tumours. Therefore, our aim is to synthesise polyamine-based radiotracers that allow the in vivo imaging of the aforementioned targets by positron emission tomography (PET). Labelling with the radionuclide fluorine-18 can be accomplished via attaching a [18F]fluorobenzoyl group with the prosthetic labelling reagent N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). To access the required non- radioactive analogues, a solid-phase synthesis was developed that enables selective fluorobenzoylation at distinct amino groups of various polyamines (e.g. cadaverine, spermidine, spermine) on the basis of a recently described synthetic concept for the selective functionalisation of polyamines. The established route can be directly applied to synthesise the 18F-labelled analogues. The mono-fluorobenzoylated polyamines were obtained by solidphase synthesis of the corresponding oxopolyamines and subsequent reduction of the amide bond with BH3-THF. By applying Dde and Boc as orthogonal protecting groups and taking advantage of the selective reaction of 2-acetyldimedone with primary amino groups in the presence of secondary amines, the selective fluorobenzoylation (FBz) of different amino groups becomes possible. Additionally, the selective mono-fluorobenzylation (FBn) of selected diamines by reaction with 4-fluorobenzaldehyde and subsequent reduction of the resulting imine using sodium triacetoxyborohydride was performed. Based on the established methodology, the following compounds among others were obtained in good yields: N-FBzcadaverine, N-FBn-cadaverine, N1-FBz- spermidine, N4-FBz-spermidine, N8-FBz-spermidine and N1-FBz-spermine. Furthermore, the naturally occurring diamine cadaverine was conjugated to different reporter groups such as biotin. The identity of the compounds was confirmed by NMR spectroscopy and mass spectrometry. The kinetic parameters towards transglutaminase 2-catalysed acyl transfer were determined for selected compounds with an in-house fluorimetric assay using the fluorogenic acyl donor Cbz–Glu(HMC)–Gly–OH.

Objectives: Lysyl oxidase (LOX, EC 1.4.3.13) and its family members LOX-like 1-4 are copper-dependent matrixmodifying enzymes [1]. The expression of LOX is elevated in many human cancers, including breast cancer and correlates with tissue hypoxia. The enzyme plays a critical role in breast cancer metastasis [2]. The goal of the current study was to target LOX with fluorescent and radiolabeled oligopeptides to visualize LOX in preclinical models of breast cancer. Methods: mRNA expression of all 5 LOX family members was analyzed by gene expression microarray analysis on samples from 176 breast cancer patients. The peptidic substrate GGGDPKGGGGG was selected to target LOX [3]. The peptide was labeled with either FITC for confocal microscopy experiments or with the positron emitter fluorine-18 for molecular imaging in vivo using positron emission tomography (PET) (Figure 1). The preclinical breast cancer models utilized were the murine breast cancer cell line EMT-6 and xenografts of MCF-7 and MDAMB-231. Results: Immunofluorescence with a LOX-specific antibody confirmed that LOX protein expression is enhanced in hypoxic EMT-6 cells. FITC-labeled oligopeptide binds to several cell compartments of EMT6 cells under hypoxic conditions. After injection of 18F-labeled oligopeptide, radioactivity uptake was visible in all three breast cancer models in vivo with SUV5min values of: 0.70±0.07 (n=3) in EMT-6, 0.57±0.01 (n=3) in MCF-7 and 0.68 (n=2) in MDA-MB-231. The following continuous washout of radioactivity led to SUV60min values of: 0.18±0.03 (n=3) in EMT-6, 0.14±0.02 (n=3) in MCF-7 and 0.13 (n=2) in MDA-MB-231. Tumor uptake was reduced by pre-dosing with the irreversible LOX inhibitor BAPN 4 h and 24 h prior to injection of the radiotracer. Conclusions: These data support further investigations towards the development of LOX-binding peptides as molecular probes for imaging of LOX expression in breast cancer. Acknowledgements: The access to the Alberta Cancer Foundation-supported CBCF Tumor Bank is well appreciated. References [1] Payne SL, et al (2007) J. Cell. Biochem.101, 1338-54. [2] Erler JT, et al (2006) Nature 440, 1222-6. [3] Nagan N and Kagan HM (1994) J. Biol. Chem. 269, 22366-71.

Objectives: Thiol-dependent cathepsins are a class of cysteine proteases that have been shown to be linked to the progression of cancer in multiple ways. Particularly crucial is their involvement in proteolytic pathways that are related to tumour invasion and metastasis [1]. The aim of this study was the design of cathepsin inhibitors based on the structure of the azadipeptide nitrile chemotype [2] that permit the labelling with the positron emitters fluorine-18 and carbon-11 and to evaluate the potential of this inhibitor class for functional tumour imaging by PET. Additionally, a first insight into the pharmacokinetic behaviour of these inhibitors should be gained. Methods: The fluorine atom was linked by an ethylene bridge to the inhibitor core structure. Labelling with fluorine-18 was achieved by fluoroethylation of 1 with different substituted [18F]2-fluoroethyl benzenesulfonates [3]. In addition, the introduction of radiofluorine into 2 was attempted by direct fluorination of the corresponding tosyl and nosyl precursors. The stability of the tracer against chemical and enzymatic degradation as well as its metabolic fate in rat blood was investigated and its biodistribution was studied in vivo by small animal PET. To obtain an 11C-based tracer, the fluoroethyl group of 2 was replaced by methyl. Introduction of carbon-11 was done by conversion of the phenolic precursor 1 with [11C]methyl iodide generated by the gas-phase method. The affinities of the compounds 1-3 to their targets were determined in kinetic enzyme assays. Results: The azadipeptide nitriles 1-3 exhibited inhibition constants in the single-digit to subnanomolar range against the oncologically relevant cathepsins L, S, and B. Among the various 18F-fluoroethylating agents tested, [18F]2-fluoroethyl nosylate revealed as the most efficient one. This enabled the two-step radiosynthesis of [18F]2 in an average RCY (d.c.) of 27±5% (n = 13). The direct radiofluorination of the corresponding sulfonate precursors could provide [18F]2 in only low labelling yields. PET studies in rats together with in vitro investigations indicated the trapping of the tracer in erythrocytes, which could be attributed to its inherent thiol reactivity. The tracer [11C]3 could be obtained in labelling yields of 35-42% depending on the employed base. Its radiopharmacological behaviour is under current investigation. Conclusions: With the azadipeptide nitriles 2 and 3 highly potent cathepsin inhibitors were found and their labelling with fluorine-18 and carbon-11 could be successfully established. The compounds’ suitability as PET tracers for functional tumour imaging seems to be limited due to their thiol reactivity. The radiolabelling of further cathepsin inhibitors is underway. References: [1] Mohamed & Sloane (2006) Nat. Rev. Cancer 6, 764-775, [2] Löser et al. (2008) Angew. Chem. Int. Ed. 47, 4331-4334, [3] Musachio et al. (2005) J. Label. Compd. Radiopharm. 48, 735-747

Potent and noncovalent inhibitors of caspase-1 were produced by incorporating a secondary amine (reduced amide) isostere in place of the conventional electrophile (e.g., aldehyde) that normally confers high potency to cysteine protease inhibitors. Benzyl- or cyclohexylamines produced potent, reversible, and competitive inhibitors that were selective for caspase-1 (e.g., Ki=47 nM) over caspases 3 and 8with minimal cytotoxicity. Unlike most cysteine protease inhibitors, these compounds do not react covalently and indiscriminately with thiols.

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.

Introduction: Lysyl oxidase (LOX; ExPASy ENZYME entry: EC 1.4.3.13) and members of the LOX-like family, LOXL1–LOXL4, are copper-dependent enzymes that can modify proteins of the extracellular matrix. Expression of LOX is elevated in many human cancers, including breast cancer. LOX expression correlates with the level of tissue hypoxia, and it is known to play a critical role in breast cancer metastasis. The goal of the present study was to target LOX with (1) molecular probe fluorescent labeling to visualize LOX in vitro and (2) a radiolabeled peptide to target LOX in vivo in three different preclinical models of breast cancer. Methods: Gene expression of all five members of the LOX family was analyzed at the transcript level via microarray analysis using tissue biopsy samples from 176 patients with breast cancer. An oligopeptide sequence (GGGDPKGGGGG) was selected as a substrate-based, LOX-targeting structure. The peptide was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy experiments with the murine breast cancer cell line EMT-6. In vivo molecular imaging experiments were performed using a C-terminal amidated peptide, GGGDPKGGGGG, labeled with a short-lived positron emitter, fluorine-18 (18F), for positron emission tomography (PET) in three different breast cancer models: EMT6, MCF-7 and MDA-MB-231. The PET experiments were carried out in the presence or absence of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX. Results: Immunostaining experiments using a LOX-specific antibody on EMT-6 cells cultured under hypoxic conditions confirmed the elevation of LOX expression in these cells. An FITC-labeled oligopeptide, FITC-Ava- GGGDPKGGGGG-NH2, was found to be localized in different cellular compartments under these conditions. After injection of [18F]fluorobenzoate-GGGDPKGGGGG-NH2, radioactivity uptake was visible in all three breast cancer models in vivo. Tumor uptake was reduced by predosing the animals with 2 mg of BAPN 4 h or 24 h before injection of the radiotracer. Conclusions: The present data support further investigation into the development of LOX-binding radiolabeled peptides as molecular probes for molecular imaging of LOX expression in cancer.