Active site-directed probes targeting dipeptidyl peptidases 8 and 9.

Dipeptidyl peptidases (DPP) 8 and 9 are intracellular serine proteases that play key roles in various biological processes and recent findings highlight DPP8 and DPP9 as potential therapeutic targets for hematological and inflammasome-related diseases. Despite the substantial progress, the precise biological functions of these proteases remain elusive, and the lack of selective chemical tools hampers ongoing research. In this paper, we describe the synthesis and biochemical evaluation of the first active site-directed DPP8/9 probes which are derived from DPP8/9 inhibitors developed in-house. Specifically, we synthesized fluorescent inhibitors containing nitrobenzoxadiazole (NBD), dansyl (DNS) and cyanine-3 (Cy3) reporters to visualize intracellular DPP8/9. We demonstrate that the fluorescent inhibitors have high affinity and selectivity towards DPP8/9 over related S9 family members. The NBD-labeled DPP8/9 inhibitors were nominated as the best in class compounds to visualize DPP8/9 in human cells. Furthermore, a method has been developed for selective labeling and visualization of active DPP8/9 in vitro by fluorescence microscopy. A collection of potent and selective biotinylated DPP8/9-targeting probes was also prepared by replacing the fluorescent reporter with a biotin group. The present work provides the first DPP8/9-targeting fluorescent compounds as useful chemical tools for the study of DPP8 and DPP9's biological functions.

Druglike, 18F-labeled PET Tracers Targeting Fibroblast Activation Protein.

Fibroblast activation protein (FAP) is a very reliable biomarker for tissue remodeling. FAP has so far mainly been studied in oncology, but there is growing interest in the enzyme in other diseases like fibrosis. Recently, FAP-targeting diagnostics and therapeutics have emerged, of which the so-called FAPIs are among the most promising representatives. FAPIs typically have a relatively high molecular weight and contain very polar, multicharged chelator moieties. While this is not limiting the application of FAPIs in oncology, more druglike FAPIs could be required to optimally study diseases characterized by denser, less permeable tissue. In response, we designed the first druglike 18F-labeled FAPIs. We report target potencies, biodistribution, and pharmacokinetics and demonstrate FAP-dependent uptake in murine tumor xenografts. Finally, this paper puts forward compound 10 as a highly promising, druglike FAPI for 18F-PET imaging. This molecule is fit for additional studies in fibrosis and its preclinical profile warrants clinical investigation.

5-Aminothiazoles Reveal a New Ligand-Binding Site on Prolyl Oligopeptidase Which is Important for Modulation of Its Protein-Protein Interaction-Derived Functions.

A series of novel 5-aminothiazole-based ligands for prolyl oligopeptidase (PREP) comprise selective, potent modulators of the protein-protein interaction (PPI)-mediated functions of PREP, although they are only weak inhibitors of the proteolytic activity of PREP. The disconnected structure-activity relationships are significantly more pronounced for the 5-aminothiazole-based ligands than for the earlier published 5-aminooxazole-based ligands. Furthermore, the stability of the 5-aminothiazole scaffold allowed exploration of wider substitution patterns than that was possible with the 5-aminooxazole scaffold. The intriguing structure-activity relationships for the modulation of the proteolytic activity and PPI-derived functions of PREP were elaborated by presenting a new binding site for PPI modulating PREP ligands, which was initially discovered using molecular modeling and later confirmed through point mutation studies. Our results suggest that this new binding site on PREP is clearly more important than the active site of PREP for the modulation of its PPI-mediated functions.

Next generation fibroblast activation protein (FAP) targeting PET tracers - The tetrazine ligation allows an easy and convenient way to 18F-labeled (4-quinolinoyl)glycyl-2-cyanopyrrolidines.

Small-molecular fibroblast activation protein inhibitor (FAPI)-based tracer have been shown to be promising Positron Emission Tomography (PET) 68Ga-labeled radiopharmaceuticals to image a variety of tumors including pancreatic, breast, and colorectal cancers, among others. In this study, we developed a novel 18F-labeled FAPI derivative. [18F]6 was labeled using a synthon approach based on the tetrazine ligation. It showed subnanomolar affinity for the FAP protein and a good selectivity profile against known off-target proteases. Small animal PET studies revealed high tumor uptake and good target-to-background ratios. [18F]6 was excreted via the liver. Overall, [18F]6 showed promising characteristics to be used as a PET tracer and could serve as a lead for further development of halogen-based theranostic FAPI radiopharmaceuticals.

Highly Selective Inhibitors of Dipeptidyl Peptidase 9 (DPP9) Derived from the Clinically Used DPP4-Inhibitor Vildagliptin.

Dipeptidyl peptidase 9 (DPP9) is a proline-selective serine protease that plays a key role in NLRP1- and CARD8-mediated inflammatory cell death (pyroptosis). No selective inhibitors have hitherto been reported for the enzyme: all published molecules have grossly comparable affinities for DPP8 and 9 because of the highly similar architecture of these enzymes' active sites. Selective DPP9 inhibitors would be highly instrumental to address unanswered research questions on the enzyme's role in pyroptosis, and they could also be investigated as therapeutics for acute myeloid leukemias. Compounds presented in this manuscript (42 and 47) combine low nanomolar DPP9 affinities with unprecedented DPP9-to-DPP8 selectivity indices up to 175 and selectivity indices >1000 toward all other proline-selective proteases. To rationalize experimentally obtained data, a molecular dynamics study was performed. We also provide in vivo pharmacokinetics data for compound 42.

Novel Generation of FAP Inhibitor-Based Homodimers for Improved Application in Radiotheranostics.

Radiopharmaceuticals based on the highly potent FAP inhibitor (FAPi) UAMC-1110 have shown great potential in molecular imaging, but the short tumor retention time of the monomers do not match the physical half-lives of the important therapeutic radionuclides 177Lu and 225Ac. This was improved with the dimer DOTAGA.(SA.FAPi)2, but pharmacological and radiolabeling properties still need optimization. Therefore, the novel FAPi homodimers DO3A.Glu.(FAPi)2 and DOTAGA.Glu.(FAPi)2. were synthesized and quantitatively radiolabeled with 68Ga, 90Y, 177Lu and 225Ac. The radiolabeled complexes showed high hydrophilicity and were generally stable in human serum (HS) and phosphate-buffered saline (PBS) at 37 °C over two half-lives, except for [225Ac]Ac-DOTAGA.Glu.(FAPi)2 in PBS. In vitro affinity studies resulted in subnanomolar IC50 values for FAP and high selectivity for FAP over the related proteases PREP and DPP4 for both compounds as well as for [natLu]Lu-DOTAGA.Glu.(FAPi)2. In a first proof-of-principle patient study (medullary thyroid cancer), [177Lu]Lu-DOTAGA.Glu.(FAPi)2 was compared to [177Lu]Lu-DOTAGA.(SA.FAPi)2. High uptake and long tumor retention was observed in both cases, but [177Lu]Lu-DOTAGA.Glu.(FAPi)2 significantly reduces uptake in non-target and critical organs (liver, colon). Overall, the novel FAPi homodimer DOTAGA.Glu.(FAPi)2 showed improved radiolabeling in vitro and pharmacological properties in vivo compared to DOTAGA.(SA.FAPi)2. [177Lu]Lu-DOTAGA.Glu.(FAPi)2 and [225Ac]Ac-DOTAGA.Glu.(FAPi)2 appear promising for translational application in patients.

Atorvastatin downregulates plasma procarboxypeptidase U concentrations and improves fibrinolytic potential dose-dependently in hyperlipidemic individuals.

BACKGROUND: Statins efficiently lower cholesterol and also exert pleiotropic effects that extend beyond lipid lowering. In a recent pilot study, valuable information on the carboxypeptidase U (CPU) system in hyperlipidemia and the effect of statin therapy was collected. It was shown that proCPU levels are increased in hyperlipidemic patients. Statins significantly decreased proCPU levels and improved plasma fibrinolysis. Furthermore, it was suggested that patients with high baseline proCPU levels are most likely to benefit from statin therapy. OBJECTIVES: We aimed to further substantiate the effect of hyperlipidemia and statin therapy on CPU-related parameters in a larger cohort of hyperlipidemic and statin-treated individuals. METHODS: Blood was collected from 141 individuals treated with different dosages of atorvastatin (10-80 mg), 38 normolipidemic, and 37 hyperlipidemic controls. Lipid parameters and markers of fibrinolysis (proCPU and clot lysis time) were determined and compared between the groups. RESULTS: Pilot study results of high proCPU concentrations in hyperlipidemic patients and the proCPU-reducing effect of atorvastatin were confirmed. Accordingly, an improvement in plasma fibrinolytic potential was seen under the influence of atorvastatin. High interindividual variation in proCPU concentrations was observed in the hyperlipidemic cohort, with up to 80% higher proCPU levels compared with normolipidemic controls. Furthermore, proCPU concentration and the dosage of atorvastatin were inversely correlated. CONCLUSIONS: This study clearly shows that plasma proCPU concentrations and its expected effect on the fibrinolytic rate (as measured by clot lysis time) are increased in hyperlipidemic patients and that these effects can be normalized (and even further reduced compared with normolipidemic patients) by atorvastatin treatment.

ProCPU Is Expressed by (Primary) Human Monocytes and Macrophages and Expression Differs between States of Differentiation and Activation.

Carboxypeptidase U (CPU, TAFIa, CPB2) is a potent attenuator of fibrinolysis that is mainly synthesized by the liver as its inactive precursor proCPU. Aside from its antifibrinolytic properties, evidence exists that CPU can modulate inflammation, thereby regulating communication between coagulation and inflammation. Monocytes and macrophages play a central role in inflammation and interact with coagulation mechanisms resulting in thrombus formation. The involvement of CPU and monocytes/macrophages in inflammation and thrombus formation, and a recent hypothesis that proCPU is expressed in monocytes/macrophages, prompted us to investigate human monocytes and macrophages as a potential source of proCPU. CPB2 mRNA expression and the presence of proCPU/CPU protein were studied in THP-1, PMA-stimulated THP-1 cells and primary human monocytes, M-CSF-, IFN-γ/LPS-, and IL-4-stimulated-macrophages by RT-qPCR, Western blotting, enzyme activity measurements, and immunocytochemistry. CPB2 mRNA and proCPU protein were detected in THP-1 and PMA-stimulated THP-1 cells as well as in primary monocytes and macrophages. Moreover, CPU was detected in the cell medium of all investigated cell types and it was demonstrated that proCPU can be activated into functionally active CPU in the in vitro cell culture environment. Comparison of CPB2 mRNA expression and proCPU concentrations in the cell medium between the different cell types provided evidence that CPB2 mRNA expression and proCPU secretion in monocytes and macrophages is related to the degree to which these cells are differentiated. Our results indicate that primary monocytes and macrophages express proCPU. This sheds new light on monocytes and macrophages as local proCPU sources.

Prolyl Carboxypeptidase Activity Is Present in Human Adipose Tissue and Is Elevated in Serum of Obese Men with Type 2 Diabetes.

Prolyl carboxypeptidase (PRCP) is involved in metabolic disorders by hydrolyzing anorexigenic peptides. A link between serum PRCP activity and obesity has been reported, but its origin/source is still unclear. Previously proven correlations between human serum PRCP activity and the amount of adipose tissue may suggest that adipose tissue is an important source of circulating PRCP. We investigated PRCP activity in visceral, subcutaneous adipose tissue (VAT and SCAT), skeletal muscle tissue and serum of lean and obese men with or without type 2 diabetes (T2D). Correlations between PRCP activity, metabolic and biochemical parameters and immune cell populations were assessed. PRCP activity was the highest in VAT, compared to SCAT, and was very low in skeletal muscle tissue in the overall group. Serum PRCP activity was significantly higher in T2-diabetic obese men, compared to lean and obese non-diabetic men, and was positively correlated with glycemic control. A positive correlation was observed between serum PRCP activity and VAT immune cell populations, which might indicate that circulating PRCP activity is deriving rather from the immune fraction than from adipocytes. In conclusion, PRCP activity was observed in human adipose tissue for the first time and serum PRCP activity is correlated with T2D in obese men.

Vildagliptin-Derived Dipeptidyl Peptidase 9 (DPP9) Inhibitors: Identification of a DPP8/9-Specific Lead.

Vildagliptin is a marketed DPP4 inhibitor, used in the management of type 2 diabetes. The molecule also has notable DPP8/9 affinity, with some preference for DPP9. Therefore, we aimed to use vildagliptin as a starting point for selective DPP8/9 inhibitors, and to engineer out the parent compound's DPP4-affinity. In addition, we wanted to identify substructures in the obtained molecules that allow their further optimization into inhibitors with maximal DPP9 selectivity. Various 2S-cyanopyrrolidines and isoindoline were investigated as P1 residues of vildagliptin analogs. The obtained set was expanded with derivatives bearing O-substituted, N-(3-hydroxyadamantyl)glycine moieties at the P2 position. In this way, representatives were discovered with DPP8/9 potencies comparable to the parent molecule, but with overall selectivity towards DPP4, DPP2, FAP, and PREP. Furthermore, the most promising molecules in this series have a 4- to 7-fold preference for DPP9 over DPP8. Finally, a molecular dynamics study was carried out to maximize our insight into experimental selectivity data.

The Effect of Serine Protease Inhibitors on Visceral Pain in Different Rodent Models With an Intestinal Insult.

Background: Serine proteases are believed to play a key role in the origin of abdominal pain in IBD and IBS. We previously demonstrated a reduction of visceral pain in a post-inflammatory IBS rat model after a single intraperitoneal or intracolonic administration of a serine protease inhibitor. The aim of this study was to investigate the efficacy of serine protease inhibition on visceral pain in two different animal models involving a colonic insult based either on acute inflammation or on neonatal irritation. Moreover, protease profiling was explored in the acute colitis model. Methods: An acute 2,4,6-trinitrobenzenesulphonic acid (TNBS) colitis rat model and a chronic neonatal acetic acid mouse model were used in this study. Visceral sensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30 min after intraperitoneal administration of the serine protease inhibitors nafamostat, UAMC-00050 or their vehicles. Colonic samples from acute colitis rats were used to quantify the mRNA expression of a panel of serine proteases and mast cell tryptase by immunohistochemistry. Finally, proteolytic activities in colonic and fecal samples were characterized using fluorogenic substrates. Key Results: We showed a significant and pressure-dependent increase in visceral hypersensitivity in acute colitis and neonatal acetic acid models. UAMC-00050 and nafamostat significantly reduced VMRs in both animal models. In acute colitis rats, the administration of a serine protease inhibitor did not affect the inflammatory parameters. Protease profiling of these acute colitis animals revealed an increased tryptase immunoreactivity and a downregulation of matriptase at the mRNA level after inflammation. The administration of UAMC-00050 resulted in a decreased elastase-like activity in the colon associated with a significantly increased elastase-like activity in fecal samples of acute colitis animals. Conclusion: In conclusion, our results suggest that serine proteases play an important role in visceral hypersensitivity in an acute TNBS colitis model in rats and a neonatal acetic acid model in mice. Moreover, we hypothesize a potential mechanism of action of UAMC-00050 via the alteration of elastase-like proteolytic activity in acute inflammation. Taken together, we provided fundamental evidence for serine protease inhibitors as a promising new therapeutic strategy for abdominal pain in gastrointestinal diseases.

Proline-specific peptidase activities (DPP4, PRCP, FAP and PREP) in plasma of hospitalized COVID-19 patients.

BACKGROUND: COVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospitalized COVID-19 patients. METHODS: Fifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longitudinal follow-up samples. RESULTS: Compared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 patients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge. CONCLUSION: The changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel.

Activation of the Carboxypeptidase U (CPU, TAFIa, CPB2) System in Patients with SARS-CoV-2 Infection Could Contribute to COVID-19 Hypofibrinolytic State and Disease Severity Prognosis.

Coronavirus disease 2019 (COVID-19) is a viral lower respiratory tract infection caused by the highly transmissible and pathogenic SARS-CoV-2 (severe acute respiratory-syndrome coronavirus-2). Besides respiratory failure, systemic thromboembolic complications are frequent in COVID-19 patients and suggested to be the result of a dysregulation of the hemostatic balance. Although several markers of coagulation and fibrinolysis have been studied extensively, little is known about the effect of SARS-CoV-2 infection on the potent antifibrinolytic enzyme carboxypeptidase U (CPU). Blood was collected longitudinally from 56 hospitalized COVID-19 patients and 32 healthy controls. Procarboxypeptidase U (proCPU) levels and total active and inactivated CPU (CPU+CPUi) antigen levels were measured. At study inclusion (shortly after hospital admission), proCPU levels were significantly lower and CPU+CPUi antigen levels significantly higher in COVID-19 patients compared to controls. Both proCPU and CPU+CPUi antigen levels showed a subsequent progressive increase in these patients. Hereafter, proCPU levels decreased and patients were, at discharge, comparable to the controls. CPU+CPUi antigen levels at discharge were still higher compared to controls. Baseline CPU+CPUi antigen levels (shortly after hospital admission) correlated with disease severity and the duration of hospitalization. In conclusion, CPU generation with concomitant proCPU consumption during early SARS-CoV-2 infection will (at least partly) contribute to the hypofibrinolytic state observed in COVID-19 patients, thus enlarging their risk for thrombosis. Moreover, given the association between CPU+CPUi antigen levels and both disease severity and duration of hospitalization, this parameter may be a potential biomarker with prognostic value in SARS-CoV-2 infection.

Validating Cell Surface Proteases as Drug Targets for Cancer Therapy: What Do We Know, and Where Do We Go?

Cell surface proteases (also known as ectoproteases) are transmembrane and membrane-bound enzymes involved in various physiological and pathological processes. Several members, most notably dipeptidyl peptidase 4 (DPP4/CD26) and its related family member fibroblast activation protein (FAP), aminopeptidase N (APN/CD13), a disintegrin and metalloprotease 17 (ADAM17/TACE), and matrix metalloproteinases (MMPs) MMP2 and MMP9, are often overexpressed in cancers and have been associated with tumour dysfunction. With multifaceted actions, these ectoproteases have been validated as therapeutic targets for cancer. Numerous inhibitors have been developed to target these enzymes, attempting to control their enzymatic activity. Even though clinical trials with these compounds did not show the expected results in most cases, the field of ectoprotease inhibitors is growing. This review summarizes the current knowledge on this subject and highlights the recent development of more effective and selective drugs targeting ectoproteases among which small molecular weight inhibitors, peptide conjugates, prodrugs, or monoclonal antibodies (mAbs) and derivatives. These promising avenues have the potential to deliver novel therapeutic strategies in the treatment of cancers.

Chemically diverse activity-based probes with unexpected inhibitory mechanisms targeting trypsin-like serine proteases.

Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with "clickable" affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC50 values, mechanism of inhibition, and kinetic constants were determined. The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery.

The Chemokine-Based Peptide, CXCL9(74-103), Inhibits Angiogenesis by Blocking Heparan Sulfate Proteoglycan-Mediated Signaling of Multiple Endothelial Growth Factors.

Growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) are important angiogenesis-mediating factors. They exert their effects not only through their respective receptor tyrosine kinases (RTKs), but they also require molecular pairing with heparan sulfate proteoglycans (HSPGs). Angiogenic growth factors and their signaling pathways are commonly targeted in current anti-angiogenic cancer therapies but have unfortunately insufficient impact on patient survival. Considering their obvious role in pathological angiogenesis, HS-targeting drugs have become an appealing new strategy. Therefore, we aimed to reduce angiogenesis through interference with growth factor-HS binding and downstream signaling using a CXCL9-derived peptide with a high affinity for glycosaminoglycans (GAGs), CXCL9(74-103). We showed that CXCL9(74-103) reduced EGF-, VEGF165- and FGF-2-mediated angiogenic processes in vitro, such as endothelial cell proliferation, chemotaxis, adhesion and sprouting, without exerting cell toxicity. CXCL9(74-103) interfered with growth factor signaling in diverse ways, e.g., by diminishing VEGF165 binding to HS and by direct association with FGF-2. The dependency of CXCL9(74-103) on HS for binding to HMVECs and for exerting its anti-angiogenic activity was also demonstrated. In vivo, CXCL9(74-103) attenuated neovascularization in the Matrigel plug assay, the corneal cauterization assay and in MDA-MB-231 breast cancer xenografts. Additionally, CXCL9(74-103) reduced vascular leakage in the retina of diabetic rats. In contrast, CXCL9(86-103), a peptide with low GAG affinity, showed no overall anti-angiogenic activity. Altogether, our results indicate that CXCL9(74-103) reduces angiogenesis by interfering with multiple HS-dependent growth factor signaling pathways.

Proteolytic Cleavage of Bioactive Peptides and Protease-Activated Receptors in Acute and Post-Colitis.

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of ß-endorphin and disarming of the TL-sequence of the PAR3-based peptide were observed in acute colitis and linked to chymotrypsin-like activity. Increased processing of the enkephalins points to the involvement of proteases with specificities different from trypsin- or chymotrypsin-like enzymes. In conclusion, our results suggest thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD.

The C-terminal cleavage of angiotensin II and III is mediated by prolyl carboxypeptidase in human umbilical vein and aortic endothelial cells.

The renin-angiotensin system, with the octapeptide angiotensin II as key player, is important in the renal, cardiac and vascular physiology. Prolyl carboxypeptidase (PRCP), prolyl endopeptidase (PREP) and angiotensin converting enzyme 2 (ACE2) are reported to be involved in the conversion of angiotensin II to angiotensin (1-7). Previous investigations showed that the processing of angiotensin II is cell- and species-specific and little is known about its conversion in human endothelial cells. Therefore, we aimed to investigate the C-terminal processing of angiotensin II and III in comparison to the processing of des-Arg9-bradykinin in human endothelial cells. To this end, human umbilical vein and aortic endothelial cells (HUVEC and HAoEC) were incubated with the peptides for different time periods. Mass spectrometry analysis was performed on the supernatants to check for cleavage products. Contribution of PRCP, ACE2 and PREP to the peptide cleavage was evaluated by use of the selective inhibitors compound 8o, DX600 and KYP-2047. The use of these selective inhibitors revealed that the C-terminal cleavage of angiotensin II and III was PRCP-dependent in HUVEC and HAoEC. In contrast, the C-terminal cleavage of des-Arg9-bradykinin was PRCP-dependent in HUVEC and PRCP- and ACE2-dependent in HAoEC. With this study, we contribute to a better understanding of the processing of peptides involved in the alternative renin-angiotensin system. We conclude that PRCP is the main enzyme for the C-terminal processing of angiotensin peptides in human umbilical vein and aortic endothelial cells. For the first time the contribution of PRCP was investigated by use of a selective PRCP-inhibitor.

In Vitro Evaluation of the Squaramide-Conjugated Fibroblast Activation Protein Inhibitor-Based Agents AAZTA5.SA.FAPi and DOTA.SA.FAPi.

Recently, the first squaramide-(SA) containing FAP inhibitor-derived radiotracers were introduced. DATA5m.SA.FAPi and DOTA.SA.FAPi with their non-radioactive complexes showed high affinity and selectivity for FAP. After a successful preclinical study with [68Ga]Ga-DOTA.SA.FAPi, the first patient studies were realized for both compounds. Here, we present a new squaramide-containing compound targeting FAP, based on the AAZTA5 chelator 1,4-bis-(carboxylmethyl)-6-[bis-(carboxymethyl)-amino-6-pentanoic-acid]-perhydro-1,4-diazepine. For this molecule (AAZTA5.SA.FAPi), complexation with radionuclides such as gallium-68, scandium-44, and lutetium-177 was investigated, and the in vitro properties of the complexes were characterized and compared with those of DOTA.SA.FAPi. AAZTA5.SA.FAPi and its derivatives labelled with non-radioactive isotopes demonstrated similar excellent inhibitory potencies compared to the previously published SA.FAPi ligands, i.e., sub-nanomolar IC50 values for FAP and high selectivity indices over the serine proteases PREP and DPPs. Labeling with all three radiometals was easier and faster with AAZTA5.SA.FAPi compared to the corresponding DOTA analogue at ambient temperature. Especially, scandium-44 labeling with the AAZTA derivative resulted in higher specific activities. Both DOTA.SA.FAPi and AAZTA5.SA.FAPi showed sufficiently high stability in different media. Therefore, these FAP inhibitor agents could be promising for theranostic approaches targeting FAP.

Prolyl Carboxypeptidase Mediates the C-Terminal Cleavage of (Pyr)-Apelin-13 in Human Umbilical Vein and Aortic Endothelial Cells.

The aim of this study was to investigate the C-terminal cleavage of (pyr)-apelin-13 in human endothelial cells with respect to the role and subcellular location of prolyl carboxypeptidase (PRCP). Human umbilical vein and aortic endothelial cells, pre-treated with prolyl carboxypeptidase-inhibitor compound 8o and/or angiotensin converting enzyme 2 (ACE2)-inhibitor DX600, were incubated with (pyr)-apelin-13 for different time periods. Cleavage products of (pyr)-apelin-13 in the supernatant were identified by mass spectrometry. The subcellular location of PRCP was examined via immunocytochemistry. In addition, PRCP activity was measured in supernatants and cell lysates of LPS-, TNFα-, and IL-1ß-stimulated cells. PRCP cleaved (pyr)-apelin-13 in human umbilical vein and aortic endothelial cells, while ACE2 only contributed to this cleavage in aortic endothelial cells. PRCP was found in endothelial cell lysosomes. Pro-inflammatory stimulation induced the secretion of PRCP in the extracellular environment of endothelial cells, while its intracellular level remained intact. In conclusion, PRCP, observed in endothelial lysosomes, is responsible for the C-terminal cleavage of (pyr)-apelin-13 in human umbilical vein endothelial cells, while in aortic endothelial cells ACE2 also contributes to this cleavage. These results pave the way to further elucidate the relevance of the C-terminal Phe of (pyr)-apelin-13.