Search

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 18 F-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 18 F-PET imaging. This molecule is fit for additional studies in fibrosis and its preclinical profile warrants clinical investigation.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Margarida Espadinha reports financial support was provided by European Commission Marie Sklodowska-Curie Actions. Joni De Loose reports financial support was provided by Research Foundation Flanders. Siham Benramdane reports financial support was provided by Research Foundation Flanders. Yentl Van Rymenant reports financial support was provided by Research Foundation Flanders. Sam Corthaut reports financial support was provided by Research Foundation Flanders. Ingrid De Meester reports financial support was provided by Research Foundation Flanders. Pieter Van der Veken reports financial support was provided by Research Foundation Flanders. Margarida Espadinha has patent #EP2023/064881 pending to University of Antwerp. Joni De Loose has patent #EP2023/064881 pending to University of Antwerp. Siham Benramdane has patent #EP2023/064881 pending to University of Antwerp. Ingrid De Meester has patent #EP2023/064881 pending to University of Antwerp. Pieter Van der Veken has patent #EP2023/064881 pending to University of Antwerp. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Small-molecular fibroblast activation protein inhibitor (FAPI)-based tracer have been shown to be promising Positron Emission Tomography (PET) 68 Ga-labeled radiopharmaceuticals to image a variety of tumors including pancreatic, breast, and colorectal cancers, among others. In this study, we developed a novel 18 F-labeled FAPI derivative. [ 18 F]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. [ 18 F]6 was excreted via the liver. Overall, [ 18 F]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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Matthias M. herth reports a relationship with Tetrakit Technologies that includes: board membership, consulting or advisory, and equity or stocks. Christian B. M. Poulie reports a relationship with Tetrakit Technologies that includes: board membership, employment, and equity or stocks. Vladimir Shalgunov reports a relationship with Tetrakit Technologies that includes: board membership, employment, and equity or stocks. Umberto Maria Battisti reports a relationship with Tetrakit technologies that includes: board membership, employment, and equity or stocks., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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 .

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 177 Lu and 225 Ac. 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 68 Ga, 90 Y, 177 Lu and 225 Ac. 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 [ 225 Ac]Ac-DOTAGA.Glu.(FAPi) 2 in PBS. In vitro affinity studies resulted in subnanomolar IC 50 values for FAP and high selectivity for FAP over the related proteases PREP and DPP4 for both compounds as well as for [ nat Lu]Lu-DOTAGA.Glu.(FAPi) 2 . In a first proof-of-principle patient study (medullary thyroid cancer), [ 177 Lu]Lu-DOTAGA.Glu.(FAPi) 2 was compared to [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 . High uptake and long tumor retention was observed in both cases, but [ 177 Lu]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 . [ 177 Lu]Lu-DOTAGA.Glu.(FAPi) 2 and [ 225 Ac]Ac-DOTAGA.Glu.(FAPi) 2 appear promising for translational application in patients.

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.

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., (© 2022 Wiley-VCH GmbH.)

Recently, the first squaramide-(SA) containing FAP inhibitor-derived radiotracers were introduced. DATA 5m .SA.FAPi and DOTA.SA.FAPi with their non-radioactive complexes showed high affinity and selectivity for FAP. After a successful preclinical study with [ 68 Ga]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 AAZTA 5 chelator 1,4-bis-(carboxylmethyl)-6-[bis-(carboxymethyl)-amino-6-pentanoic-acid]-perhydro-1,4-diazepine. For this molecule (AAZTA 5 .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. AAZTA 5 .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 IC 50 values for FAP and high selectivity indices over the serine proteases PREP and DPPs. Labeling with all three radiometals was easier and faster with AAZTA 5 .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 AAZTA 5 .SA.FAPi showed sufficiently high stability in different media. Therefore, these FAP inhibitor agents could be promising for theranostic approaches targeting FAP.