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.

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.

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.

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.

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.

In Vitro and In Situ Activity-Based Labeling of Fibroblast Activation Protein with UAMC1110-Derived Probes.

Fibroblast activation protein (FAP) is a proline-selective protease that belongs to the S9 family of serine proteases. It is typically highly expressed in the tumor microenvironment (TME) and especially in cancer-associated fibroblasts, the main cell components of the tumor stroma. The exact role of its enzymatic activity in the TME remains largely unknown. Hence, tools that enable selective, activity-based visualization of FAP within the TME can help to unravel FAP's function. We describe the synthesis, biochemical characterization, and application of three different activity-based probes (biotin-, Cy3-, and Cy5-labeled) based on the FAP-inhibitor UAMC1110, an in-house developed molecule considered to be the most potent and selective FAP inhibitor available. We demonstrate that the three probes have subnanomolar FAP affinity and pronounced selectivity with respect to the related S9 family members. Furthermore, we report that the fluorescent Cy3- and Cy5-labeled probes are capable of selectively detecting FAP in a cellular context, making these chemical probes highly suitable for further biological studies. Moreover, proof of concept is provided for in situ FAP activity staining in patient-derived cryosections of urothelial tumors.

Fibroblast Activation Protein (FAP) targeting homodimeric FAP inhibitor radiotheranostics: a step to improve tumor uptake and retention time.

Several radiopharmaceuticals targeting fibroblast activation protein (FAP) based on the highly potent FAP inhibitor UAMC1110 are currently under investigation. Pre-clinical as well as clinical research exhibited the potential of these imaging agents. However, the monomeric small molecules seemed to have a short retention time in the tumor in combination with fast renal clearance. Therefore, our strategy was to develop homodimeric systems having two FAP inhibitors to improve residence time and tumor accumulation. The homodimers with two squaramide coupled FAP inhibitor conjugates DOTA.(SA.FAPi)2 and DOTAGA.(SA.FAPi)2 were synthesized and radiochemically evaluated with gallium-68. [68Ga]Ga-DOTAGA.(SA.FAPi)2 was tested for its in vitro stability, lipophilicity and affinity properties. In addition, human PET/CT scans were performed for [68Ga]Ga-DOTAGA.(SA.FAPi)2 with a head-to-head comparison with [68Ga]Ga-DOTA.SA.FAPi and [18F]FDG. Labeling with gallium-68 demonstrated high radiochemical yields. Inhibition measurements revealed excellent affinity and selectivity with low nanomolar IC50 values for FAP. In PET/CT human studies, significantly higher tumor uptake as well as longer tumor retention could be observed for [68Ga]Ga-DOTAGA.(SA.FAPi)2 compared to [68Ga]Ga-DOTA.SA.FAPi. Therefore, the introduction of the dimer led to an advance in human PET imaging indicated by increased tumor accumulation and prolonged retention times in vivo and thus, the use of dimeric structures could be the next step towards prolonged uptake of FAP inhibitors resulting in radiotherapeutic analogs of FAP inhibitors.

Targeting fibroblast activation protein (FAP): next generation PET radiotracers using squaramide coupled bifunctional DOTA and DATA5m chelators.

BACKGROUND: Fibroblast activation protein (FAP) is a proline selective serine protease that is overexpressed in tumor stroma and in lesions of many other diseases that are characterized by tissue remodeling. In 2014, a most potent FAP-inhibitor (referred to as UAMC1110) with low nanomolar FAP-affinity and high selectivity toward related enzymes such as prolyl oligopeptidase (PREP) and the dipeptidyl-peptidases (DPPs): DPP4, DPP8/9 and DPP2 were developed. This inhibitor has been adopted recently by other groups to create radiopharmaceuticals by coupling bifunctional chelator-linker systems. Here, we report squaric acid (SA) containing bifunctional DATA5m and DOTA chelators based on UAMC1110 as pharmacophor. The novel radiopharmaceuticals DOTA.SA.FAPi and DATA5m.SA.FAPi with their non-radioactive derivatives were characterized for in vitro inhibitory efficiency to FAP and PREP, respectively and radiochemical investigated with gallium-68. Further, first proof-of-concept in vivo animal study followed by ex vivo biodistribution were determined with [68Ga]Ga-DOTA.SA.FAPi. RESULTS: [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DATA5m.SA.FAPi showed high complexation > 97% radiochemical yields after already 10 min and high stability over a period of 2 h. Affinity to FAP of DOTA.SA.FAPi and DATA5m.SA.FAPi and its natGa and natLu-labeled derivatives were excellent resulting in low nanomolar IC50 values of 0.7-1.4 nM. Additionally, all five compounds showed low affinity for the related protease PREP (high IC50 with 1.7-8.7 µM). First proof-of-principle in vivo PET-imaging animal studies of the [68Ga]Ga-DOTA.SA.FAPi precursor in a HT-29 human colorectal cancer xenograft mouse model indicated promising results with high accumulation in tumor (SUVmean of 0.75) and low background signal. Ex vivo biodistribution showed highest uptake in tumor (5.2%ID/g) at 60 min post injection with overall low uptake in healthy tissues. CONCLUSION: In this work, novel PET radiotracers targeting fibroblast activation protein were synthesized and biochemically investigated. Critical substructures of the novel compounds are a squaramide linker unit derived from the basic motif of squaric acid, DOTA and DATA5m bifunctional chelators and a FAP-targeting moiety. In conclusion, these new FAP-ligands appear promising, both for further research and development as well as for first human application.

Dysregulated activities of proline-specific enzymes in septic shock patients (sepsis-2).

The proline-specific enzymes dipeptidyl peptidase 4 (DPP4), prolylcarboxypeptidase (PRCP), fibroblast activation protein α (FAP) and prolyl oligopeptidase (PREP) are known for their involvement in the immune system and blood pressure regulation. Only very limited information is currently available on their enzymatic activity and possible involvement in patients with sepsis and septic-shock. The activity of the enzymes was measured in EDTA-plasma of patients admitted to the intensive care unit (ICU): 40 septic shock patients (sepsis-2) and 22 ICU control patients after major intracranial surgery. These data were used to generate receiver operating characteristic (ROC) curves. A survival analysis (at 90 days) and an association study with other parameters was performed. PRCP (day 1) and PREP (all days) enzymatic activities were higher in septic shock patients compared to controls. In contrast, FAP and DPP4 were lower in these patients on all studied time points. Since large differences were found, ROC curves were generated and these yielded area under the curve (AUC) values for PREP, FAP and DPP4 of 0.88 (CI: 0.80-0.96), 0.94 (CI: 0.89-0.99) and 0.86 (CI: 0.77-0.95), respectively. PRCP had a lower predicting value with an AUC of 0.71 (CI: 0.58-0.83). A nominally significant association was observed between survival and the DPP4 enzymatic activity at day 1 (p<0.05), with a higher DPP4 activity being associated with an increase in survival. All four enzymes were dysregulated in septic shock patients. DPP4, FAP and PREP are good in discriminating between septic shock patients and ICU controls and should be further explored to see whether they are already dysregulated in earlier stages, opening perspectives for their further investigation as biomarkers in sepsis. DPP4 also shows potential as a prognostic biomarker. Additionally, the associations found warrant further research.

Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP).

Fibroblast activation protein (FAP) is a proline-selective serine protease. It is hardly expressed in healthy adult tissue but upregulated in tissue remodeling sites associated with several diseases including epithelial cancer types, atherosclerosis, arthritis and fibrosis. Ongoing research aims at clinical implementation of FAP as a biomarker for these diseases. Several immunochemical methods that quantify FAP expression have been reported. An alternative/complementary approach focuses on quantification of FAP's enzymatic activity. Developing an activity-based assay for FAP has nonetheless proven challenging because of selectivity issues with respect to prolyl oligopeptidase (PREP). Here, we present substrate-type FAP probes that are structurally derived from a FAP-inhibitor (UAMC1110) that we published earlier. Both cleavage efficiency and FAP-selectivity of the best compounds in the series equal or surpass the most advanced peptide-based FAP substrates reported to date. Finally, proof-of-concept is provided that 4-aminonaphthol containing probes can spatially localize FAP activity in biological samples.

The development and validation of a combined kinetic fluorometric activity assay for fibroblast activation protein alpha and prolyl oligopeptidase in plasma.

BACKGROUND: Fibroblast activiation protein alpha (FAP) is considered a diagnostic and prognostic biomarker for various types of cancer. FAP shares substrate specificity with prolyl oligopeptidase (PREP), studied in (neuro)inflammation and neurodegeneration as well as cancer. Current assays inadequately discriminate between FAP and PREP and there is need for an assay that reliably quantitates the FAP/PREP activity ratio in plasma. METHODS: FAP and PREP activities were measured in human EDTA-plasma in presence of well characterized PREP and FAP inhibitors. RESULTS: A combined kinetic assay was developed in conditions to optimally measure FAP as well as PREP activity with Z-Gly-Pro-AMC as substrate. Limit of detection was 0.009 U/L and limit of quantitation was 0.027 U/L for the combined FAP-PREP assay. Within-run coefficient of variation was 3% and 4% and between-run precision was 7% and 12% for PREP and FAP, respectively. Accuracy was demonstrated by comparison with established end-point assays. Hemolysis interferes with the assay with 1.5 g/L hemoglobin as cut-off value. PREP (but not FAP) activity can increase upon lysis of platelets and red blood cells during sample preparation. CONCLUSION: With this new assay, on average 67% of the Z-Gly-Pro-AMC converting activity in plasma can be attributed to FAP.

A search for molecular mechanisms underlying male idiopathic infertility.

Infertility affects approximately 15% of the couples wanting to conceive. In 30 - 40% of the cases the aetiology of male infertility remains unknown and is called idiopathic male infertility. When assisted reproductive technologies are used to obtain pregnancy, an adequate (epi)genetic diagnosis of male infertility is of major importance to evaluate if a genetic abnormality will be transmitted to the offspring. In addition, there is need for better diagnostic seminal biomarkers to assess the success rates of these assisted reproductive technologies. This review investigated the possible causes and molecular mechanisms underlying male idiopathic infertility by extensive literature searches of: (i) causal gene mutations; (ii) proteome studies of spermatozoa from idiopathic infertile men;(iii) the role of epigenetics; (iv) post-translational modifications; and (v) sperm DNA fragmentation in infertile men. In conclusion, male infertility is a complex, multi-factorial disorder and the underlying causes often remain unknown. Further research on the (epi)genetic and molecular defects in spermatogenesis and sperm function is necessary to improve the diagnosis and to develop more personalized treatments of men with idiopathic infertility.

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda.

Globins are among the best investigated proteins in biological and medical sciences and represent a prime tool for the study of the evolution of genes and the structure-function relationship of proteins. Here, we explore the recombinant expression of globins in three different expression systems: Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda. We expressed two different human globin types in these three expression systems: I) the well-characterized neuroglobin and II) the uncharacterized, circular permutated globin domain of the large chimeric globin androglobin. It is clear from the literature that E.coli is the most used expression system for expression and purification of recombinant globins. However, the major disadvantage of E. coli is the formation of insoluble aggregates. We experienced that, for more complex multi-domain globins, like the chimeric globin androglobin, it is recommended to switch to a higher eukaryotic expression system.