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Fibroblast activation protein (FAP) has been extensively studied as a cancer biomarker for decades. Recently, small-molecule FAP inhibitors have been widely adopted as a targeting moiety of experimental theranostic radiotracers. Here we present a fast qPCR-based analytical method allowing FAP inhibition screening in a high-throughput regime. To identify clinically relevant compounds that might interfere with FAP-targeted approaches, we focused on a library of FDA-approved drugs. Using the DNA-linked Inhibitor Antibody Assay (DIANA), we tested a library of 2667 compounds within just a few hours and identified numerous FDA-approved drugs as novel FAP inhibitors. Among these, prodrugs of cephalosporin antibiotics and reverse transcriptase inhibitors, along with one elastase inhibitor, were the most potent FAP inhibitors in our dataset. In addition, by employing FAP DIANA in the quantification mode, we were able to determine FAP concentrations in human plasma samples. Together, our work expands the repertoire of FAP inhibitors, analyzes the potential interference of co-administered drugs with FAP-targeting strategies, and presents a sensitive and low-consumption ELISA alternative for FAP quantification with a detection limit of 50 pg/ml., Competing Interests: Declaration of competing interest DIANA technology is protected by patents US10718772 (B2) and US10718772 (B2) and ketoamide inhibitors are protected by patent US20230192647 (A1). The authors declare that they have no other conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Fibroblast activation protein-α (FAPα) is highly expressed in tumor-associated cells and has become one of the most attractive targeting sites in cancer diagnosis and therapy. To ameliorate the rapid metabolism of FAPα inhibitor (FAPI), here, a multifunctional binding agent was introduced to simultaneously achieve 211 At radiolabeling and tumor retention prolongation of corresponding radiolabeled drug. 211 At-APBA-FAPI was successfully synthesized by conjugating 211 At with the designed FAPI carrier in satisfactory radiochemical yield (>60 %). 211 At-APBA-FAPI exhibited excellent in vitro stability, significant tumor affinity and specific killing effect on FAPα-positive U87MG cells. Molecular docking reveals that FAPI decorated with albumin binder can bind with FAPα protein via multiple intermolecular interactions with a considerable binding energy of -9.66 kcal/mol 211 At-APBA-FAPI exhibits good targeting in murine xenograft models, showing obviously longer tumor retention than previously-reported radioastatinated compound. As a result, 211 At-APBA-FAPI presents pronounced therapeutic effect with ignorable normal organs/tissues biotoxicity. All these indicate that introducing a multifunctional binding agent can effectively enhance the availability of FAPI for 211 At conjugation and tumoricidal effect, providing vital hints for the translation of targeted-alpha therapy based on radiolabeled FAPI derivatives., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Novel nuclear medicine therapeutics are being developed by labeling medium-molecular-weight compounds with short-lived alpha-emitting radionuclides. Fibroblast activation protein α (FAPα) is recognized as a highly useful molecular target, and its inhibitor, FAPI, is a compound capable of theranostics , both therapeutic and diagnostic, for cancer treatment. In this study, we compared the functions of two compounds that target FAPα: 211 At-FAPI1 and 211 At-FAPI2. First, in vitro screening procedures are generally accepted because of the low endogenous expression of FAPα. We suggest the usefulness of this 3D culture system for in vitro screening. Second, when FAPIs are used therapeutically, the expected therapeutic effects are often not achieved. Therefore, we compared the accumulation and excretion in tumor tissues and the anti-tumor effects based on the length of the linker in the compounds. The compounds were rapidly labeled using the Shirakami reaction . Doubling the linker length increased tumor retention. Additionally, the excretion pathway was altered, suggesting a potential reduction in toxicity. Although no significant differences were observed in the anti-tumor effects of 211 At-FAPI1 and 211 At-FAPI2, it was confirmed that the linker length affects the biological half-life.

Fibroblast activation protein-α (FAP) has emerged as a promising target in the field of radiopharmaceuticals due to its selective expression in cancer-associated fibroblasts (CAFs) and other pathological conditions involving fibrosis and inflammation. Recent advancements have focused on developing FAP-specific radioligands for diagnostic imaging and targeted radionuclide therapy. This perspective summarized the latest progress in FAP radiopharmaceutical development, highlighting novel radioligands, preclinical evaluations, and potential clinical applications. Additionally, we analyzed the advantages and existing problems of targeted FAP radiopharmaceuticals, and discussed the key breakthrough directions of this target, so as to improve the development and conversion of FAP-targeted radiopharmaceuticals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zehui Wu reports financial support was provided by the National Natural Science Foundation of China (82372018 and 81701753). Zehui Wu reports financial support was provided by the Beijing Natural Science Foundation (7222299). Zehui Wu reports a relationship with Capital Medical University that includes: 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.)

Abstract: The fibroblast activation protein (FAP) is a biomarker that is selectively overexpressed on cancer-associated fibroblasts (CAFs) in various types of tumoral tissues and some nonmalignant diseases, including fibrosis, arthritis, cardiovascular, and metabolic diseases. FAP plays a critical role in tumor microenvironment through facilitating proliferation, invasion, angiogenesis, immunosuppression, and drug resistance. Recent studies reveal that FAP might be regarded as a promising target for cancer diagnosis and treatment. FAP-targeted imaging modalities, especially PET, have shown high sensitivity and specificity in detecting FAP-expressing tumors. FAP-targeted imaging can potentially enhance tumor detection, staging, and monitoring of treatment response, and facilitate the development of personalized treatment strategies. This study provides a comprehensive view of FAP and its function in the pathophysiology of cancer and nonmalignant diseases. It also will discuss the characteristics of radiolabeled FAP inhibitors, particularly those based on small molecules, their recent clinical implications in imaging and therapy, and the associated clinical challenges with them. In addition, we present the results of imaging and biodistribution radiotracer 68 Ga-FAPI-46 in patients with nonmalignant diseases, including interstitial lung disease, primary biliary cirrhosis, and myocardial infarction, who were referred to our department. Our results show that cardiac FAP-targeted imaging can provide a novel potential biomarker for managing left ventricle remodeling. Moreover, this study has been organized and presented in a manner that offers a comprehensive overview of the current status and prospects of FAPI inhibitors in the diagnosis and treatment of diseases., Competing Interests: Conflicts of interest and sources of funding: none declared., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Triple-negative breast cancer (TNBC) presents limited therapeutic options and is associated with poor prognosis. Early detection and the development of novel therapeutic agents are therefore imperative. Fibroblast activation protein (FAP) is a membrane protein expressed on cancer-associated fibroblasts (CAFs) that plays an essential role in TNBC proliferation, migration, and invasion. Consequently, it is hypothesized that the Astatine ( 211 At)-labeled FAP inhibitor (FAPI) selectively exerts anti-tumor effects through alpha-particle emission. In this study, we aimed to assess its theranostic capabilities by integrating [ 18 F]FAPI-74 PET imaging with targeted alpha therapy using [ 211 At]FAPI1 in TNBC models. Mice xenografts were established by transplanting MDA-MB-231 and HT1080 cells (control). As a parallel diagnostic method, [ 18 F]FAPI-74 was administered for PET imaging to validate FAP expression. A single dose of [ 211 At]FAPI1 (1.04 ± 0.10 MBq) was administered to evaluate the therapeutic efficacy. [ 18 F]FAPI-74 exhibited high accumulation in MDA-MB-231 xenografts, and FAP expression was pathologically confirmed via immunostaining. The group that received [ 211 At]FAPI1 (n = 11) demonstrated a significantly enhanced anti-tumor effect compared with the control group (n = 7) ( p = 0.002). In conclusion, [ 18 F]FAPI-74 PET imaging was successfully used to diagnose FAP expression, and as [ 211 At]FAPI1 showed promising therapeutic efficacy in TNBC models, it is expected to be a viable therapeutic option.

Fibroblast activation protein (FAP) is specifically expressed on cancer-associated fibroblasts in over 90% of tumors and is considered a promising target for cancer theranostics. Here, we developed a novel tracer, DOTA-FAPT, and labeled it with gallium-68 and lutetium-177 as a theranostic pair. [ 68 Ga]Ga/[ 177 Lu]Lu-FAPT exhibited high stability and hydrophilicity, as well as strong affinity to the FAP target. Micro-PET/CT imaging revealed that [ 68 Ga]Ga-FAPT exhibited significantly increased uptake in tumors and extended retention in A549-FAP and U87MG tumor xenografts as compared to [ 68 Ga]Ga-FAPI-04, demonstrating favorable pharmacokinetic characteristics in vivo. Therapeutic studies showed that [ 177 Lu]Lu-FAPT had higher tumor accumulation compared to [ 177 Lu]Lu-FAPI-04, leading to stronger tumor growth inhibition. The first-in-human evaluation also revealed that [ 68 Ga]Ga-FAPT has good in vivo distribution and superior diagnostic efficacy on primary and lymph node metastases in a patient with lung cancer. Our encouraging results suggest that 68 Ga/ 177 Lu-labeled DOTA-FAPT is a theranostic pair with broad application prospect.

Fibroblast activation protein (FAP) has been an attractive target for cancer imaging and therapy. Radiolabeled FAP-targeting ligands have shown promising results for clinical applications. However, further improvements are ongoing in pursuit of increasing tumor uptake, prolonging tumor residence, and maintenance good tumor-to-background contrast for extensive theranostic application. Achieving a higher affinity of the precursor is one of the ways in research. In this study, we designed a series of FAP inhibitors based on N-(4-quinolinoyl)-Gly-2-cyanopyrrolidine and found compound QI-18 with an IC 50 value of 0.50 nM, which is a 6.5-fold increase in potency over that of UAMC-1110 (IC 50 of 3.25 nM). QI-18 was then functionalized with a DOTA chelator to obtain the ligand CY03 for further radiolabeling with 68 Ga to obtain the radiotracer [ 68 Ga]Ga-CY03. In BALB/c nude mice bearing U87MG tumor models, [ 68 Ga]Ga-CY03 exhibited a high and specific uptake (10.30 ± 0.63 % ID/g at 1 h post-injection and 9.28 ± 1.60 % ID/g at 2 h post-injection), which represented 3.2- and 4.1-fold increases over those for [ 68 Ga]Ga-FAPI-04 (3.24 ± 0.53 % ID/g and 2.25 ± 0.33 % ID/g, respectively). [ 68 Ga]Ga-CY03 also showed higher tumor-to-blood and tumor-to-kidney ratios (7.62 ± 0.44 and 2.59 ± 0.27, respectively) than [ 68 Ga]Ga-FAPI-04 (2.38 ± 0.47 and 0.98 ± 0.19, respectively). The results indicate that [ 68 Ga]Ga-CY03 is a promising imaging agent to target FAP., Competing Interests: Declaration of competing interest The authors 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.)

Enterococcus faecalis is a troublesome nosocomial pathogen that acquired resistance to most available antimicrobial agents. Antivirulence agents represent an unconventional treatment approach. Here, transcription factor decoy (TFD)-loaded cationic liposomes (TLL) were developed as an inhibitor of the Fsr quorum-sensing system and its associated virulence traits, in E. faecalis. The consensus sequence of the FsrA binding site was found conserved among 651 E. faecalis annotated genomes. The TFD was synthesized as an 82 bp DNA duplex, containing the conserved binding sequence, and loaded onto cationic liposomes. The optimum loading capacity, mean particle size, and zeta potential of the TLL were characterized. The developed TLL lacked any effect on E. faecalis growth and significantly inhibited the in vitro production of the proteolytic enzymes controlled by the Fsr system; gelatinase and serine protease, in a concentration-dependent manner. This inhibition was accompanied by a significant reduction in the transcription levels of FsrA-regulated genes (fsrB, gelE, and sprE). The developed TLL were safe as evidenced by the nonhemolytic effect on human RBCs and the negligible cytotoxicity on human skin fibroblast cells. Moreover, in the larvae infection model, TLL displayed a significant abolish in the mortality rates of Galleria mellonella larvae infected with E. faecalis. In conclusion, the developed TLL offer a new safe strategy for combating E. faecalis infection through the inhibition of quorum-sensing-mediated virulence; providing a platform for the development of similar agents to combat many other pathogens., Competing Interests: Declaration of competing interest The authors 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 Ltd. All rights reserved.)

The advancement of theranostics, which combines therapeutic and diagnostic capabilities in oncology, has significantly impacted cancer management. This review explores fibroblast activation protein (FAP) expression in the tumor microenvironment (TME) and its association with various malignancies, highlighting its potential as a theranostic marker for PET/CT imaging using FAP-targeted tracers and for FAP-targeted radiopharmaceutical therapy. We examine the development and clinical applications of FAP inhibitors (FAPIs) and peptides, providing insights into their diagnostic accuracy, initial therapeutic efficacy, and clinical impact across diverse cancer types, as well as the synthesis of novel FAP-targeted ligands. This review aims to showcase the promising outcomes and challenges in integrating FAP-targeted approaches into cancer management., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Frederik L. Giesel is a FAPI co-inventor and is an advisor at Telix Pharma, SOFIE, ABX, and Alpha Fusion and receives royalties from iTheranostics and SOFIE Biosciences. Richard P. Baum is an advisor to 3B Pharmaceuticals (Berlin, Germany), ITM, Full Life Technologies, Sinotau, Jiangsu Huayi Technology, and Telix Pharmaceuticals, and cofounder of RadioVaxx GmbH., (Copyright © 2024. Published by Elsevier Inc.)

PET/CT using radiolabeled fibroblast activation protein inhibitors (FAPIs) is a promising diagnostic tool in oncology, especially when non-increased and/or physiologically high [ 18 F]FDG uptake (as in liver parenchyma) is observed. We aimed to review the role of PET/CT using radiolabeled FAPIs in primary and/or metastatic liver lesions, and to compare their performances with more "conventional" radiopharmaceuticals. A search algorithm based on the terms "FAPI" AND ("hepatic" OR "liver") was applied, with the last update on 1st January 2024. Out of 177 articles retrieved, 76 studies reporting on the diagnostic application of radiolabeled FAPI PET/CT in at least one patient harboring primary or metastatic liver lesion(s) were fully analyzed. Although there was some heterogeneity in clinical conditions and/or study methodology, PET/CT with radiolabeled FAPIs showed an excellent performance in common primary liver malignancies (hepatocarcinoma, intrahepatic cholangiocarcinoma) and liver metastases (mostly from the gastrointestinal tract and lungs). A higher tumor-to-background ratio for FAPIs than for [ 18 F]FDG was found in primary and metastatic liver lesions, due to lower background activity. Despite limited clinical evidence, radiolabeled FAPIs may be used to assess the suitability and effectiveness of FAPI-derived therapeutic agents such as [ 177 Lu]Lu-FAPI. However, future prospective research on a wider population is needed to confirm the excellent performance.

Background: FAP radiopharmaceuticals show promise for cancer diagnosis; however, their limited tumor residency hinders treatment. This study compared two FAPi derivatives, DOTA.SA.FAPi and DOTAGA.(SA.FAPi) 2 , labeled with gallium-68 and lutetium-177, aiming to determine an optimum combination for creating theranostic pairs., Methods: The radiotracers were studied for lipophilicity, binding to human serum proteins, and binding to human cancer-associated fibroblasts (CAFs) in vitro, including saturation and internalization/externalization studies. PET/SPECT/CT and biodistribution studies were conducted in PC3 and U87MG xenografts for [ 68 Ga]Ga-DOTA.SA.FAPi and [ 68 Ga]Ga-DOTAGA.(SA.FAPi) 2 . [ 177 Lu]Lu-DOTA.SA.FAPi and [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 , were evaluated in PC3 xenografts. Biodistribution studies of [ 68 Ga]Ga-DOTA.SA.FAPi were performed in healthy male and female mice., Results: All radiotracers exhibited strong binding to FAP. Their internalization rate was fast while only [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 was retained longer in CAFs. [ 68 Ga]Ga-DOTAGA.(SA.FAPi) 2 and [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 displayed elevated lipophilicity and affinity for human serum proteins compared to [ 68 Ga]Ga-DOTA.SA.FAPi and [ 177 Lu]Lu-DOTA.SA.FAPi. In vivo studies revealed slower washout of [ 68 Ga]Ga-DOTAGA.(SA.FAPi) 2 within 3 h compared to [ 68 Ga]Ga-DOTA.SA.FAPi. The tumor-to-tissue ratios of [ 68 Ga]Ga-DOTAGA.(SA.FAPi) 2 versus [ 68 Ga]Ga-DOTA.SA.FAPi did not exhibit any significant differences. [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 maintained a significant tumor uptake even after 96 h p.i. compared to [ 177 Lu]Lu-DOTA.SA.FAPi., Conclusions: Dimeric compounds hold promise for therapy, while monomers are better suited for diagnostics. Finding the right combination is essential for effective disease management.

Fibroblast activation protein-α (FAP) is often highly expressed by sarcoma cells and by sarcoma-associated fibroblasts in the tumor microenvironment. This makes it a promising target for imaging and therapy. The level of FAP expression and the diagnostic value of 68 Ga-FAP inhibitor (FAPI) PET for sarcoma subtypes are unknown. We assessed the diagnostic performance and accuracy of 68 Ga-FAPI PET in various bone and soft-tissue sarcomas. Potential eligibility for FAP-targeted radiopharmaceutical therapy (FAP-RPT) was evaluated. Methods: This prospective observational trial enrolled 200 patients with bone and soft-tissue sarcoma who underwent 68 Ga-FAPI PET/CT and 18 F-FDG PET/CT (186/200, or 93%) for staging or restaging. The number of lesions detected and the uptake (SUV max ) of the primary tumor, lymph nodes, and visceral and bone metastases were analyzed. The Wilcoxon test was used for semiquantitative assessment. The association of 68 Ga-FAPI uptake intensity, histopathologic grade, and FAP expression in sarcoma biopsy samples was analyzed using Spearman r correlation. The impact of 68 Ga-FAPI PET on clinical management was investigated using questionnaires before and after PET/CT. Eligibility for FAP-RPT was defined by an SUV max greater than 10 for all tumor regions. Results: 68 Ga-FAPI uptake was heterogeneous among sarcoma subtypes. The 3 sarcoma entities with the highest uptake (mean SUV max ± SD) were solitary fibrous tumor (24.7 ± 11.9), undifferentiated pleomorphic sarcoma (18.8 ± 13.1), and leiomyosarcoma (15.2 ± 10.2). Uptake of 68 Ga-FAPI versus 18 F-FDG was significantly higher in low-grade sarcomas (10.4 ± 8.5 vs. 7.0 ± 4.5, P = 0.01) and in potentially malignant intermediate or unpredictable sarcomas without a World Health Organization grade (not applicable [NA]; 22.3 ± 12.5 vs. 8.5 ± 10.0, P = 0.0004), including solitary fibrous tumor. The accuracy, as well as the detection rates, of 68 Ga-FAPI was higher than that of 18 F-FDG in low-grade sarcomas (accuracy, 92.2 vs. 80.0) and NA sarcomas (accuracy, 96.9 vs. 81.9). 68 Ga-FAPI uptake and the histopathologic FAP expression score ( n = 89) were moderately correlated (Spearman r = 0.43, P < 0.0002). Of 138 patients, 62 (45%) with metastatic sarcoma were eligible for FAP-RPT. Conclusion: In patients with low-grade and NA sarcomas, 68 Ga-FAPI PET demonstrates uptake, detection rates, and accuracy superior to those of 18 F-FDG PET. 68 Ga-FAPI PET criteria identified eligibility for FAP-RPT in about half of sarcoma patients., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

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.

Fibrosis is commonly associated with chronic rheumatic diseases, and causes substantial morbidity and mortality. Treatment of fibrosis is extremely challenging but is badly needed, as approved antifibrotic therapies fibrosis do not halt its progression, which will be discussed with a focus on pulmonary fibrosis. Findings from recent studies indicate several therapeutic targets for treating fibrosis. Interleukin-11 is emerging as a fibrogenic cytokine whose activity can be blocked with neutralizing monoclonal antibodies. Fibroblast activation protein (FAP) is highly expressed by activated fibroblasts in inflammatory and fibrotic tissues. Targeting FAP with different modalities has been extensively explored as adjunct treatment for cancer, which can also apply to treating fibrosis in rheumatic diseases., (Published by Elsevier Ltd.)

Abstract: Fibroblast activation protein inhibitor positron emission tomography (PET) has gained interest for its ability to demonstrate uptake in a diverse range of tumors. Its molecular target, fibroblast activation protein, is expressed in cancer-associated fibroblasts, a major cell type in tumor microenvironment that surrounds various types of cancers. Although existing literature on FAPI PET is largely from single-center studies and case reports, initial findings show promise for some cancer types demonstrating improved imaging when compared with the widely used 18F-fludeoxyglucose PET for oncologic imaging. As we expand our knowledge of the utility of FAPI PET, accurate understanding of noncancerous uptake seen on FAPI PET is crucial for accurate evaluation. In this review, we summarize potential diagnostic and therapeutic applications of radiolabeled FAP inhibitors in oncological and nononcological disease processes., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The field of nuclear medicine has witnessed significant advancements in recent years, particularly in the area of PET imaging. One such development is the use of Fibroblast Activation Protein Inhibitors (FAPI) as a novel radiotracer. FAPI PET imaging has shown promising results in various malignancies, including sarcomas, which are a diverse group of cancers originating from mesenchymal cells. This paper aims to explore the potential of FAPI PET imaging in the diagnosis, staging, and treatment monitoring of sarcomas. Several studies have demonstrated the potential of FAPI PET in sarcomas. Furthermore, FAPI PET imaging has shown potential in assessing treatment response, with changes in FAPI uptake correlating with treatment outcomes. However, there are challenges to be addressed. The heterogeneity of sarcomas, both inter- and intra-tumoral, may affect the uniformity of Fibroblast Activation Protein (FAP) expression and thus the effectiveness of FAPI PET imaging. Additionally, the optimal timing and dosage of FAPI for PET imaging in sarcomas need further investigation. In conclusion, the introduction of FAPI PET imaging represents a significant advancement in the field of nuclear medicine and oncology. The ability to target FAP, a protein overexpressed in the majority of sarcomas, offers new possibilities for the diagnosis and treatment of these complex and diverse tumors. Its potential applications in diagnosis, staging, and theranostics are vast, and on-going research continues to explore and address its limitations. As we continue to deepen our understanding of this novel imaging technique, it is hoped that FAPI PET imaging will play an increasingly important role in the fight against cancer. However, as with any new technology, further research is needed to fully understand the potential and limitations of FAPI PET imaging in the clinical setting., Competing Interests: Declaration of competing interest The authors 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 Inc. All rights reserved.)

Fibroblast activation protein alpha (FAP-α, EC3.4.2. B28), a type II transmembrane proteolytic enzyme for the serine protease peptidase family. It is underexpressed in normal tissues but increased significantly in disease states, especially in neoplasm, which is a potential biomarker to turmor diagnosis. The inhibition of FAP-α activity will retard tumor formation, which is expected to be a promising tumor therapeutic target. At present, although the FAP-α expression detection methods has diversification, a superlative detection means is necessary for the clinical diagnosis. This review covers the discovery and the latest advances in FAP-α, as well as the future research prospects. The tissue distribution, structural characteristics, small-molecule ligands and structure-activity relationship of major inhibitors of FAP-α were summarized in this review. Furthermore, a variety of detection methods including traditional detection methods and emerging probes detection were classified and compared, and the design strategy and kinetic parameters of these FAP-α probe substrates were summarized. In addition, these comprehensive information provides a series of practical and reliable assays for the optimal design principles of FAP-α probes, promoting the application of FAP-α as a disease marker in diagnosis, and a drug target in drug design., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Our previous works produced a whey fermentation methodology that yielded antibacterial activity and potential inhibition of matrix metalloproteases (MMP)-2 and -9. Here, we evaluated if these activities were due to fermentation-produced peptides. Prolonged fermentation was carried out in the presence of our specific lactic acid bacteria (LAB) consortium. LAB fermentation yielded a total of 11 polypeptides, which were predominantly produced after 6 days of fermentation. One which was derived from beat casein presented a particularly high antibacterial activity against food pathogenic bacteria and was more effective than standard food disinfectants. This polypeptide was further studied and was also found to be active against several strains of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), in a dose-dependent manner. It also inhibited MMP-2 and MMP-9 whilst reducing HT29 cancer cell migration in vitro. Overall, this novel whey-derived polypeptide presents dual antibacterial and anti-inflammatory activity, revealing a strong potential to be used in functional foods or as a nutraceutical. Its identification and further characterization can open novel perspectives in the field of preventive/curative diets related to gut microbiota, gut inflammation, and cancer prevention, particularly if used in in vivo studies.

Purpose: Fibroblast activation protein (FAP) is a cell membrane-bound serine peptidase, overexpressed in cancer-associated fibroblasts and activated fibroblasts at wound healing/inflammatory sites. Recently, molecular PET/CT imaging with radiolabeled FAP inhibitor (FAPI) has been evaluated in different diseases. We aimed to assess its potential role based on the available literature., Patients and Methods: We conducted a comprehensive review of the available preclinical and clinical data on FAPI PET/CT in an attempt to summarize its current status and potential future role. Based on that, we have discussed the pathophysiology behind FAP-based imaging, followed by a discussion of FAPI radiopharmaceuticals including their synthesis, biodistribution, and dosimetry. Next, we have discussed studies evaluating FAPI PET/CT in different oncological and nononcological pathologies. The potential of FAPI PET/CT in theranostics has also been addressed., Results: Based on the early scientific evidence available, including preclinical and clinical studies, FAPI PET/CT seems to be a promising molecular imaging tool, especially in oncology. It can be used for imaging different types of cancers and outperforms 18F-FDG PET/CT in some of these. Its potential as a theranostic tool warrants special attention., Conclusions: Fibroblast activation protein inhibitor PET/CT has the potential to emerge as a powerful molecular imaging tool in the future. However, as of yet, the available evidence is limited, warranting further research and trials in this field., Competing Interests: Conflicts of interest and sources of funding: none declared., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

An investigation into the methanol extracts obtained from the stems of Dodonaea viscosa led to the isolation of one nor-clerodane diterpene (1) and two labdane diterpenes (2, 3), as well as 17 known compounds (4-20). The structures of these compounds were elucidated based on chemical and spectral evidence. The stereochemical structure of the nor-clerodane diterpene was confirmed via its circular dichroism spectrum and calculated electronic circular dichroism spectrum. Isolated compounds were evaluated for their inhibitory effects on collagenase and tyrosinase. Since 5,7,4'-trihydroxy-3'-(4-hydroxy-3-methylbutyl)-5'-(3-methylbut-2-enyl)-3,6-dimethoxyflavone (9) showed collagenase inhibitory activity and scopoletin (12) had significant tyrosinase inhibitory activity, they were considered to be good candidates for cosmetic agents.

Background: Aloe's reported bioactivities (anticancer, anti-inflammatory and wound healing) suggest they might inhibit a subgroup of matrix metalloproteinases (MMPs) called gelatinases (MMP-2 and MMP-9). The goal of the present study was to compare the MMP inhibitory potential of two Aloe species, A. vera and A. arborescens., Methods: Different types of extraction were tested and specific bioactive compounds were quantified. Cancer cell invasion inhibitory activities were measured in vitro using the wound healing assay in human colon cancer cells (HT29). Effects on gelatinase activities were further assessed by dye-quenched gelatin and gelatin zymography., Results: Different types of extraction yielded significantly different levels of bioactivities and of bioactive compounds, which might be due to a greater amount of extractable bioactive compounds such as anthraquinones. Both A. arborescens and A. vera have potential as inhibitory agents in cancer cell proliferation via MMP-9 and MMP-2 enzymatic activity inhibition, being able to reduce colon cancer cell proliferation and migration but A. arborescens showed to be a more effective inhibitor of cancer cell migration than A. vera., Conclusion: This work opens novel perspectives on the mode of action of Aloe species in cancer cell migration and may provide clues as to why there are so many conflicting results on Aloe's activities.

Fibroblast growth factor (FGF) 21 is a class of hepatokines that plays a protective role against obesity, insulin resistance, and liver damage. Despite this, protective effects of FGF21 in human appear to be minimal, possibly due to its proteolytic cleavage by the fibroblast activation protein (FAP). Here, we presented a novel FAP inhibitor, BR103354, and described its pharmacological activities as a potential therapeutic agent for the treatment of metabolic disorders. BR103354 inhibited FAP with an IC 50 value of 14 nM, showing high selectivity against dipeptidyl peptidase (DPP)-related enzymes and prolyl oligopeptidase (PREP). In differentiated 3T3/L1 adipocytes, the addition of FAP diminished hFGF21-induced Glut1 and phosphorylated levels of ERK, which were restored by BR103354. BR103354 exhibited good pharmacokinetic properties as evidenced by oral bioavailability of 48.4% and minimal hERG inhibition. Single co-administration of BR103354 with hFGF21 reduced nonfasting blood glucose concentrations, in association with increased intact form of hFGF21 in ob/ob mice. Additionally, chronic treatment of BR103354 for 4 weeks reduced nonfasting blood glucose concentrations with improved glucose tolerance and with reduced triglyceride (TG) content in liver of ob/ob mice. Consistently, BR103354 improved hepatic steatosis and fibrosis in a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-induced non-alcoholic steatohepatitis (NASH) mouse model. FAP inhibitory effects of BR103354 were confirmed in normal cynomolgus monkeys. Together, BR103354 acts as an effective FAP inhibitor in vitro and in vivo, thereby demonstrating its potential application as an anti-diabetic and anti-NASH agent.

Fibroblast activation protein (FAP) has emerged as an interesting molecular target used in the imaging and therapy of various types of cancers. 68 Ga-labeled chelator-linked FAP inhibitors (FAPIs) have been successfully applied to PET imaging of various tumor types. To broaden the spectrum of applicable PET tracers for extended imaging studies of FAP-dependent diseases, we herein report the radiosynthesis and preclinical evaluation of an 18 F-labeled glycosylated FAPI ([ 18 F]FGlc-FAPI). Methods: An alkyne-bearing precursor was synthesized and subjected to click chemistry-based radiosynthesis of [ 18 F]FGlc-FAPI by 2-step 18 F-fluoroglycosylation. FAP-expressing HT1080hFAP cells were used to study competitive binding to FAP, cellular uptake, internalization, and efflux of [ 18 F]FGlc-FAPI in vitro. Biodistribution studies and in vivo small-animal PET studies of [ 18 F]FGlc-FAPI compared with [ 68 Ga]Ga-FAPI-04 were conducted in nude mice bearing HT1080hFAP tumors or U87MG xenografts. Results: [ 18 F]FGlc-FAPI was synthesized with a 15% radioactivity yield and a high radiochemical purity of more than 99%. In HT1080hFAP cells, [ 18 F]FGlc-FAPI showed specific uptake, a high internalized fraction, and low cellular efflux. Compared with FAPI-04 (half maximal inhibitory concentration [IC 50 ] = 32 nM), the glycoconjugate, FGlc-FAPI (IC 50 = 167 nM), showed slightly lower affinity for FAP in vitro, whereas plasma protein binding was higher for [ 18 F]FGlc-FAPI. Biodistribution studies revealed significant hepatobiliary excretion of [ 18 F]FGlc-FAPI; however, small-animal PET studies in HT1080hFAP xenografts showed higher specific tumor uptake of [ 18 F]FGlc-FAPI (4.5 percentage injected dose per gram of tissue [%ID/g]) than of [ 68 Ga]Ga-FAPI-04 (2 %ID/g). In U87MG tumor-bearing mice, both tracers showed similar tumor uptake, but [ 18 F]FGlc-FAPI showed a higher tumor retention. Interestingly, [ 18 F]FGlc-FAPI demonstrated high specific uptake in bone structures and joints. Conclusion: [ 18 F]FGlc-FAPI is an interesting candidate for translation to the clinic, taking advantage of the longer half-life and physical imaging properties of 18 F. The availability of [ 18 F]FGlc-FAPI may allow extended PET studies of FAP-related diseases, such as cancer, but also arthritis, heart diseases, or pulmonary fibrosis., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Despite a high degree of structural similarity, it is known that MMP2 and MMP9 have distinct roles in the angiogenic switch and in cell migration, as they activate diverse signaling pathways. Indeed, inhibition of MMP2 and MMP9 can show beneficial or detrimental effects depending on the stage of tumor progression. Thus, the selective inhibition of gelatinases is of relevance for a successful drug lead, which has to be achieved despite the high structural similarity of the two gelatinases. Herein, the synthesis and evaluation of d-proline-derived hydroxamic acids containing amino appendages at C-4 as gelatinase inhibitors are reported. Inhibition assays enabled the identification of a > 200-fold selective MMP9 inhibitor when Lys was considered as a C-4 substituent, thus addressing gelatinase selectivity beyond the S1' subsite, which is a major driver for selectivity. Molecular docking studies revealed the basic moiety of Lys as detrimental for inhibition of MMP2 as compared to MMP9., Competing Interests: Declaration of Competing Interest The authors 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 © 2020 Elsevier Ltd. All rights reserved.)

Most epithelial tumors recruit fibroblasts and other nonmalignant cells and activate them into cancer-associated fibroblasts. This often leads to overexpression of the membrane serine protease fibroblast-activating protein (FAP). It has already been shown that DOTA-bearing FAP inhibitors (FAPIs) generate high-contrast images with PET/CT scans. Since SPECT is a lower-cost and more widely available alternative to PET, 99m Tc-labeled FAPIs represent attractive tracers for imaging applications in a larger number of patients. Furthermore, the chemically homologous nuclide 188 Re is available from generators, which allows FAP-targeted endoradiotherapy. Methods: For the preparation of 99m Tc-tricarbonyl complexes, a chelator was selected whose carboxylic acids can easily be converted into various derivatives in the finished product, enabling a platform strategy based on the original tracer. The obtained 99m Tc complexes were investigated in vitro by binding and competition experiments on FAP-transfected HT-1080 (HT-1080-FAP) or on mouse FAP-expressing (HEK-muFAP) and CD26-expressing (HEKCD26) HEK cells and characterized by planar scintigraphy and organ distribution studies in tumor-bearing mice. Furthermore, a first-in-humans application was done on 2 patients with ovarian and pancreatic cancer, respectively. Results: 99m Tc-FAPI-19 showed specific binding to recombinant FAP-expressing cells with high affinity. Unfortunately, liver accumulation, biliary excretion, and no tumor uptake were observed on planar scintigraphy for a HT-1080-FAP-xenotransplanted mouse. To improve the pharmacokinetic properties, hydrophilic amino acids were attached to the chelator moiety of the compound. The resulting 99m Tc-labeled FAPI tracers revealed excellent binding properties (≤45% binding; >95% internalization), high affinity (half-maximal inhibitory concentration, 6.4-12.7 nM), and significant tumor uptake (≤5.4% injected dose per gram of tissue) in biodistribution studies. The lead candidate 99m Tc-FAPI-34 was applied for diagnostic scintigraphy and SPECT of patients with metastasized ovarian and pancreatic cancer for follow-up to therapy with 90 Y-FAPI-46. 99m Tc-FAPI-34 accumulated in the tumor lesions, as also shown on PET/CT imaging using 68 Ga-FAPI-46. Conclusion: 99m Tc-FAPI-34 represents a powerful tracer for diagnostic scintigraphy, especially when PET imaging is not available. Additionally, the chelator used in this compound allows labeling with the therapeutic nuclide 188 Re, which is planned for the near future., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Using venom for predation often leads to the evolution of resistance in prey. Understanding individual variation in venom resistance is key to unlocking basic mechanisms by which antagonistic coevolution can sustain variation in traits under selection. For prey, the opposing challenges of predator avoidance and resource acquisition often lead to correlated levels of risk and reward, which in turn can favor suites of integrated morphological, physiological and behavioral traits. We investigate the relationship between risk-sensitive behaviors, physiological resistance to rattlesnake venom, and stress in a population of California ground squirrels. For the same individuals, we quantified foraging decisions in the presence of snake predators, fecal corticosterone metabolites (a measure of "stress"), and blood serum inhibition of venom enzymatic activity (a measure of venom resistance). Individual responses to snakes were repeatable for three measures of risk-sensitive behavior, indicating that some individuals were consistently risk-averse whereas others were risk tolerant. Venom resistance was lower in squirrels with higher glucocorticoid levels and poorer body condition. Whereas resistance failed to predict proximity to and interactions with snake predators, individuals with higher glucocorticoid levels and in lower body condition waited the longest to feed when near a snake. We compared alternative structural equation models to evaluate alternative hypotheses for the relationships among stress, venom resistance, and behavior. We found support for stress as a shared physiological correlate that independently lowers venom resistance and leads to squirrels that wait longer to feed in the presence of a snake, whereas we did not find evidence that resistance directly facilitates latency to forage. Our findings suggest that stress may help less-resistant squirrels avoid a deadly snakebite, but also reduces feeding opportunities. The combined lethal and non-lethal effects of stressors in predator-prey interactions simultaneously impact multiple key traits in this system, making environmental stress a potential contributor to geographic variation in trait expression of toxic predators and resistant prey.

To facilitate the discovery of FAP inhibitors, a convenient cell-based fluorescent assay was developed by using a commonly available U87MG cell line and a FAP-specific substrate Suc-Gly-Pro-AMC. The assay enabled the fast determination of multiple IC 50 s by simply incubating a solution of phosphate-buffered saline in a 96-well plate within 30 min. The substrate specificity, cross-reaction and other related conditions were systematically optimized. This method was successfully applied to determine the IC 50 s of seven known inhibitors. The results are in consistence with the trend reported, which indicating that this practical assay is a valuable method to accelerate the discovery of FAP inhibitor., Competing Interests: Declaration of Competing Interest The authors 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 © 2020 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinases (MMPs), a family of zinc-containing endopeptidases involved in the degradation of the extracellular matrix, make a major contribution to the progression of a vast number of diseases, such cancer or epilepsy. Although several MMP inhibitors (MMPi) have been developed to date for the treatment of cancer, they have all failed in clinical trials due to lack of efficacy and, most importantly, the presence of severe side effects. The latter can be explained by their lack of selectivity of these inhibitors. In this regard, MMPs' family members have a high structural homology, which challenge the development of selective inhibitors for a specific MMP. Here, we have used in silico calculations and in vitro data to design MMPi that selectively target gelatinases (MMP-2 and MMP-9) and have the capacity to cross the blood-brain barrier. Following this approach, we obtained compound 40 that shows high proteolytic stability and low cytotoxicity. This compound may be of particular interest for the treatment of central nervous diseases such epilepsy or Alzheimer's disease, where gelatinase activity is increased. Our data show the specificity of compound 40 for recombinant MMP-9 and MMP-2 and endogenous MMP-9 from rat hippocampal cell cultures, and reveals its permeability across the blood-brain barrier in vivo., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fibroblast activation protein-α (FAP), as a crucial member of cell surface glycoprotein, highly expresses in reactive fibroblasts of tumors and several fibrosis diseases. It is a potential target for drug design and also reported as a prodrug strategy to increase the therapeutic window of some anticancer agents. In this work, we developed the first bioluminogenic probe for FAP with a limit-of-detection of 0.254 ng/mL, which could be applied to evaluate the FAP inhibitors in vitro. The experiments of transgenic mice and tumor-bearing nude mice validated our probe 1 could reflect the endogenous FAP level in vivo. Furthermore, this probe was successfully used to reflect FAP up-regulation in the lung homogenates of the bleomycin-induced idiopathic pulmonary fibrosis mice.

Matrix metalloproteinases (MMPs) play critical roles in a multiple number of autoimmunity diseases progression and metastasis of solid tumor. Gelatinases including MMP-2 and MMP-9 are extremely overexpressed in multiple pathological processes. MMP-9 and MMP-2 breakdown the extracellular matrix component gelatin very efficaciously. Therefore, designing and expansion of MMPs inhibitors can be an engrossing plan for therapeutic intermediacy. Anyway, a wide range of MMPs inhibitors face failure in several clinical trials. Due to sequence and structural conservation across the various MMPs, achieving specific and selective inhibitors is very demanding. In the current study, a phage-displayed peptide library was screened using active human recombinant MMP-9 protein and evaluated by enzyme-linked immunosorbent assay. Here, we isolate novel peptide sequence from phage display peptide libraries that can be a specific gelatinase inhibitor. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and some important residues of the MMP-9 and MMP-2 proteins at the fibronectin domain. A consensus peptide sequence was then synthesized (named as RSH-12) to evaluate its inhibitory potency by in vitro assays. Zymography assay was employed to evaluate the effect of RSH-12 on gelatinolysis activity of MMP-2 and MMP-9 secretion from the HT1080 cells using different concentrations of RSH-12 and inhibiting MMP-9- and MMP-2-driven gelatin proteolysis, measured by fluorescein isothiocyanate-gelatin degradation assay and HT1080 cell invasion assay on Matrigel (gelatinous protein mixture). The negative control peptide (CP) with the irrelevant sequence and no MMP inhibition properties and the positive control compound (GM6001) as a potent inhibitor of MMPs were used to assess the selectivity and specificity of gelatinases inhibition by RSH-12. Therefore, RSH-12 decreased the gelatin degradation by specifically preventing gelatin binding to MMP-9 and MMP-2. Selective gelatinase inhibitors may prove the usefulness of the new peptide discovered in tumor targeting and anticancer and anti-inflammation therapies., (© 2019 Wiley Periodicals, Inc.)

Prostate cancer is one of the most common malignant tumors in men, and inhibiting metastasis is a key event but still a major challenge in prostate cancer treatment. Cancer-associated fibroblasts (CAFs) play an important role in prostate tumor metastasis by shaping the malignant tumor microenvironment. Herein, we constructed a CAF-targeting siRNA delivery system by loading the fibroblast activation protein-α (FAP-α) antibody onto the cell-penetrating peptide (CPP)-based nanoparticles, which specifically downregulated C-X-C motif chemokine ligand 12 (CXCL12) expression in CAFs. This regulation generated a series of changes through inactivating CAFs so that the malignant prostate tumor microenvironment was reshaped. The tumor cell invasion, migration, and tumor angiogenesis were significantly inhibited, which all contributed to the suppression of the metastasis of an orthotopic prostate tumor. This tumor microenvironment reshaping strategy via CAF targeting and inactivation provides an alternative approach for malignant prostate tumor metastasis inhibition.

Over the past decade, there has been increasing interest in covalent inhibition as a drug design strategy. Our own interest in the development of prolyl oligopeptidase (POP) and fibroblast activation protein α (FAP) covalent inhibitors has led us to question whether these two serine proteases were equal in terms of their reactivity toward electrophilic warheads. To streamline such investigations, we exploited both computational and experimental methods to investigate the influence of different reactive groups on both potency and binding kinetics using both our own series of POP inhibitors and others' discovered hits. A direct correlation between inhibitor reactivity and residence time was demonstrated through quantum mechanics methods and further supported by experimental studies. This computational method was also successfully applied to FAP, as an overview of known FAP inhibitors confirmed our computational predictions that more reactive warheads (e.g., boronic acids) must be employed to inhibit FAP than for POP.

The effect of the GM6001 metalloproteinase inhibitor on the regeneration of ambulacral structures in Eupentacta fraudatrix has been investigated. Inhibition of proteinase activity exerts a marked effect on regeneration, being dependent on the time when GM6001 is injected. When administration of the inhibitor begins on day 3 post-injury, regeneration is completely abolished, and the animals die. This means that early activation of proteinases is crucial for triggering the regenerative process in holothurians. When GM6001 in first injected on day 7 post-injury, the regeneration rate decreases. However, this effect has proven to be reversible: when inhibition ceases, the regeneration resumes. The effect of the inhibitor is manifested as a retarded degradation of the extracellular matrix, the lack of cell dedifferentiation, and, probably, a slower cell migration. The gelatinase activity is detected in all the regenerating organs of E. fraudatrix. In the holothurian Cucumaria japonica, which is not capable of healing skin wounds and ambulacrum reparation, no gelatinase activity was observed at the site of damage. A suggestion is made that proteinases play an important role in regeneration in holothurians. The most probable morphogenesis regulators are matrix metalloproteinases with gelatinase activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The recent development of quinoline-based PET tracers that act as fibroblast-activation-protein inhibitors (FAPIs) demonstrated promising preclinical and clinical results. FAP is overexpressed by cancer-associated fibroblasts of several tumor entities. Here, we quantify the tumor uptake on 68 Ga-FAPI PET/CT of various primary and metastatic tumors to identify the most promising indications for future application. Methods: 68 Ga-FAPI PET/CT scans were requested by various referring physicians according to individual clinical indications that were considered insufficiently covered by 18 F-FDG PET/CT or other imaging modalities. All PET/CT was performed 1 h after injection of 122-312 MBq of 68 Ga-FAPI-04. We retrospectively identified 80 patients with histopathologically proven primary tumors or metastases or radiologically unequivocal metastatic lesions of histologically proven primary tumors. Tumor uptake was quantified by SUV max and SUV mean (60% isocontour). Results: Eighty patients with 28 different tumor entities (54 primary tumors and 229 metastases) were evaluated. The highest average SUV max (>12) was found in sarcoma, esophageal, breast, cholangiocarcinoma, and lung cancer. The lowest 68 Ga-FAPI uptake (average SUV max < 6) was observed in pheochromocytoma, renal cell, differentiated thyroid, adenoid cystic, and gastric cancer. The average SUV max of hepatocellular, colorectal, head-neck, ovarian, pancreatic, and prostate cancer was intermediate (SUV 6-12). SUV varied across and within all tumor entities. Because of low background in muscle and blood pool (SUV max < 2), the tumor-to-background contrast ratios were more than 3-fold in the intermediate and more than 6-fold in the high-intensity uptake group. Conclusion: Several highly prevalent cancers presented with remarkably high uptake and image contrast on 68 Ga-FAPI PET/CT. The high and rather selective tumor uptake may open up new applications for noninvasive tumor characterization, staging examinations, or radioligand therapy., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC., (Copyright © 2019. Published by Elsevier Inc.)

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic stroma with a poor lymphocyte infiltrate, in part driven by cancer-associated fibroblasts (CAFs). CAFs, which express fibroblast activation protein (FAP), contribute to immune escape via exclusion of anti-tumor CD8+ T cells from cancer cells, upregulation of immune checkpoint ligand expression, immunosuppressive cytokine production, and polarization of tumor infiltrating inflammatory cells. FAP is a post-proline peptidase selectively expressed during tissue remodeling and repair, such as with wound healing, and in the tumor microenvironment by cancer-associated fibroblasts. We targeted FAP function using a novel small molecule inhibitor, UAMC-1110, and mice with germline knockout of FAP and concomitant knock-in of E. coli beta-galactosidase. We depleted CAFs by adoptive transfer of anti-βgal T cells into the FAP knockout animals. Established syngeneic pancreatic tumors in immune competent mice were targeted with these 3 strategies, followed by focal radiotherapy to the tumor. FAP loss was associated with improved antigen-specific tumor T cell infiltrate and enhanced collagen deposition. However, FAP targeting alone or with tumor-directed radiation did not improve survival even when combined with anti-PD1 therapy. Targeting of CAFs alone or in combination with radiation did not improve survival. We conclude that targeting FAP and CAFs in combination with radiation is capable of enhancing anti-tumor T cell infiltrate and function, but does not result in sufficient tumor clearance to extend survival., Competing Interests: The authors have declared that no competing interests exist.

Background: Oncolytic virus (OV)-based therapies have an emerging role in the treatment of solid tumors, involving both direct cell lysis and immunogenic cell death. Nonetheless, tumor-associated stroma limits the efficacy of oncolytic viruses by forming a barrier that blocks efficient viral penetration and spread. The stroma also plays a critical role in progression, immunosuppression and invasiveness of cancer. Fibroblast activation protein-α (FAP) is highly overexpressed in cancer-associated fibroblasts (CAFs), the main cellular component of tumor stroma, and in this study we assessed whether arming oncolytic adenovirus (OAd) with a FAP-targeting Bispecific T-cell Engager (FBiTE) could retarget infiltrated lymphocytes towards CAFs, enhancing viral spread and T cell-mediated cytotoxicity against the tumor stroma to improve therapeutic activity., Methods: The bispecific T-cell Engager against FAP was constructed using an anti-human CD3 single-chain variable fragment (scFv) linked to an anti-murine and human FAP scFv. This FBiTE was inserted in the oncolytic adenovirus ICOVIR15K under the control of the major late promoter, generating the ICO15K-FBiTE. ICO15K-FBiTE replication and potency were assessed in HT1080 and A549 tumor cell lines. The expression of the FBiTE and the activation and proliferation of T cells that induced along with the T cell-mediated cytotoxicity of CAFs were evaluated by flow cytometry in vitro. In vivo, T-cell biodistribution and antitumor efficacy studies were conducted in NOD/scid/IL2rg - / - (NSG) mice., Results: FBiTE expression did not decrease the infectivity and replication potency of the armed virus. FBiTE-mediated binding of CD3 + effector T cells and FAP + target cells led to T-cell activation, proliferation, and cytotoxicity of FAP-positive cells in vitro. In vivo, FBiTE expression increased intratumoral accumulation of T cells and decreased the level of FAP, a marker of CAFs, in tumors. The antitumor activity of the FBiTE-armed adenovirus was superior to the parental virus., Conclusions: Combination of viral oncolysis of cancer cells and FBiTE-mediated cytotoxicity of FAP-expressing CAFs might be an effective strategy to overcome a key limitation of oncolytic virotherapy, encouraging its further clinical development.

Fibroblast activation protein (FAP) belongs to the dipeptidyl peptidase IV (DPP4; CD26) gene family. Other related genes in this family of enzyme include DPP4, 8 and 9. The FAP serine protease has the rare property of both dipeptidyl peptidase and endopeptidase activities capable of cleaving the post-proline bond at two or more residues from the N-terminus. FAP is involved in a variety of biological processes but its expression in healthy tissues is low. In contrast, FAP is significantly elevated in pathological conditions such as at sites of tissue remodelling and repair. Its differential pattern of expression in diseases supports the emerging concept for FAP as a potential disease biomarker as well as a useful therapeutic target for drug intervention. This review summarizes the current knowledge of FAP, particularly its diagnostic and pathological significance in liver fibrosis.

Cancer-associated fibroblasts (CAFs) are strongly implicated in tumor progression, including in the processes of tumorigenesis, invasion, and metastasis. The targeting of CAFs using various therapeutic approaches is a novel treatment strategy; however, the efficacy of such therapies remains limited. Recently, near-infrared photoimmunotherapy (NIR-PIT), which is a novel targeted therapy employing a cell-specific mAb conjugated to a photosensitizer, has been introduced as a new type of phototherapy. In this study, we have developed a novel NIR-PIT technique to target CAFs, by focusing on fibroblast activation protein (FAP), and we evaluate the treatment efficacy in vitro and in vivo . Esophageal carcinoma cells exhibited enhanced activation of fibroblasts, with FAP over-expressed in the cytoplasm and on the cell surface. FAP-IR700-mediated PIT showed induced rapid cell death specifically for those cells in vitro and in vivo , without adverse effects. This novel therapy for CAFs, designed as local control phototherapy, was safe and showed a promising inhibitory effect on FAP + CAFs. PIT targeting CAFs via the specific marker FAP may be a therapeutic option for CAFs in the tumor microenvironment in the future.

Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.

Targeting matrix metalloproteinases (MMPs) is a pursued strategy for treating several pathological conditions, such as multiple sclerosis and cancer. Herein, a series of novel tetrahydro-β-carboline derivatives with outstanding inhibitory activity toward MMPs are present. In particular, compounds 9 f, 9 g, 9 h and 9 i show sub-nanomolar IC 50 values. Interestingly, compounds 9 g and 9 i also provide remarkable selectivity toward gelatinases; IC 50 =0.15 nm for both toward MMP-2 and IC 50 =0.63 and 0.58 nm, respectively, toward MMP-9. Molecular docking simulations, performed by employing quantum mechanics based partial charges, shed light on the rationale behind binding involving specific interactions with key residues of S1' and S3' domains. Taken together, these studies indicate that tetrahydro-β-carboline represents a promising scaffold for the design of novel inhibitors able to target MMPs and selectively bias gelatinases, over the desirable range of the pharmacokinetics spectrum., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Acute ischemic stroke (AIS) is caused by clotting in the cerebral arteries, leading to brain oxygen deprivation and cerebral infarction. Recombinant human tissue plasminogen activator (tPA) is currently the only Food and Drug Administration-approved drug for ischemic stroke. However, tPA has to be administered within 4.5 h from the disease onset and delayed treatment of tPA can increase the risk of neurovascular impairment, including neuronal cell death, blood-brain barrier (BBB) disruption, and hemorrhagic transformation. A key contributing factor for tPA-induced neurovascular impairment is activation of matrix metalloproteinase-9 (MMP-9). We used a clinically-relevant mouse embolic model of focal-cerebral ischemia by insertion of a single embolus of blood clot to block the right middle cerebral artery. We showed that administration of the potent and highly selective gelatinase inhibitor SB-3CT extends the time window for administration of tPA, attenuating infarct volume, mitigating BBB disruption, and antagonizing the increase in cerebral hemorrhage induced by tPA treatment. We demonstrated that SB-3CT attenuates tPA-induced expression of vascular MMP-9, prevents gelatinase-mediated cleavage of extracellular laminin, rescues endothelial cells, and reduces caveolae-mediated transcytosis of endothelial cells. These results suggest that abrogation of MMP-9 activity mitigates the detrimental effects of tPA treatment, thus the combination treatment holds great promise for extending the therapeutic window for tPA thrombolysis, which opens the opportunity for clinical recourse to a greater number of patients.

Fibroblast activation protein (FAP, seprase) is a serine protease with post-proline dipeptidyl peptidase and endopeptidase enzymatic activity. FAP is upregulated in several tumor types, while its expression in healthy adult tissues is scarce. FAP molecule itself and FAP+ stromal cells play an important although probably context-dependent and tumor type-specific pathogenetic role in tumor progression. We provide an overview of FAP expression under both physiological and pathological conditions with focus on human malignancies. We also review and critically analyze the results of studies which used various strategies for the therapeutic targeting of FAP including the use of low molecular weight inhibitors, FAP activated prodrugs, anti-FAP antibodies and their conjugates, FAP-CAR T cells, and FAP vaccines. A unique enzymatic activity and selective expression in tumor microenvironment make FAP a promising therapeutic target. A better understanding of its role in individual tumor types, careful selection of patients, and identification of suitable combinations with currently available anticancer treatments will be critical for a successful translation of preclinically tested approaches of FAP targeting into clinical setting.

Purpose: Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines. Experimental Design: We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data. Results: This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8 + and CD4 + immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen-specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8 + T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8 + T-cell infiltration. Conclusions: These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. Clin Cancer Res; 24(5); 1190-201. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Exposure to UV radiation to human skin up-regulates the synthesis of matrix metalloproteinase (MMP) family. Gelatinases are member of MMPs which have been suggested to play an important role in photoaging such as wrinkle formation. To inhibit gelatinase activity is regarded to be very important to keep healthy skin and to protect wrinkle formation. On the other hand, anti-photoaging agents are expected to be derived from natural resources, especially plants. Plant extracts having gelatinase-inhibitory effect that might be used as safe anti-photoaging ingredient were widely screened. An extract of rhizomes of Curcuma longa L. showed inhibitory effect of gelatinase activity. Curcuminoids and slight amount of compound, 6,11-dihydroxy-3-(4-hydroxy-3-mthoxyphenethyl)-7-[(E)-4-(4-hydroxy-3-methoxyphenyl)-2-oxo-3-butenyl]-10-methoxy-2-oxabicyclo[6.3.1.]dodeca-1(11),8(12),9-trien-5-yl (E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate (curcuminoid D) were isolated as the gelatinase-inhibitory components from methanol extract of rhizomes. The structure of curcuminoid D was determined by means of spectral data including 1 H- and 13 C-NMR, and IR. Curcumin exerted the enhancing effect on deposition of basement membrane component at dermal-epidermal junction in skin equivalent model. Topical application of cream containing turmeric extract significantly improved facial skin elasticity and decreased the number of gelatinase-positive stratum corneum clusters in human facial skins. These results indicated that turmeric is an effective ingredient to improve skin condition and to prevent skin from photoaging by suppressing activation of gelatinase chronically caused by UV., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP which triggers intracellular signaling and regulates extracellular matrix proteolysis, two functions that are critical for tumor-associated angiogenesis and inflammation. While green tea catechins, particularly epigallocatechin gallate (EGCG), are considered very effective in preventing MT1-MMP-mediated functions, lack of structure-function studies and evidence regarding their direct interaction with MT1-MMP-mediated biological activities remain. Here, we assessed the impact in both cellular and biophysical assays of four ungallated catechins along with their gallated counterparts on MT1-MMP-mediated functions and molecular binding partners. Concanavalin-A (ConA) was used to trigger MT1-MMP-mediated proMMP-2 activation, expression of MT1-MMP and of endoplasmic reticulum stress biomarker GRP78 in U87 glioblastoma cells. We found that ConA-mediated MT1-MMP induction was inhibited by EGCG and catechin gallate (CG), that GRP78 induction was inhibited by EGCG, CG, and gallocatechin gallate (GCG), whereas proMMP-2 activation was inhibited by EGCG and GCG. Surface plasmon resonance was used to assess direct interaction between catechins and MT1-MMP interactors. We found that gallated catechins interacted better than their ungallated analogs with MT1-MMP as well as with MT1-MMP binding partners MMP-2, TIMP-2, MTCBP-1 and LRP1-clusterIV. Overall, current structure-function evidence supports a role for the galloyl moiety in both direct and indirect interactions of green tea catechins with MT1-MMP-mediated oncogenic processes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Crohn's disease (CD) is a chronic bowel inflammation that ultimately leads to fibrosis, for which medical therapy is currently unavailable. Fibrotic strictures in CD are characterized by excessive extracellular matrix (ECM) deposition, altered balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), and overexpression of fibroblast activation protein (FAP), a marker of active fibroblasts. Here we investigated the role of FAP-targeted therapy in ECM remodeling in CD strictures ex vivo., Methods: Bowel specimens were obtained from stenotic and nonstenotic ileal segments from 30 patients with fibrostenotic CD undergoing surgery. FAP expression was evaluated in isolated mucosal myofibroblasts by immunoblotting and flow cytometry. Bowel tissue cultures were treated with anti-FAP antibody, and soluble collagen, TIMP-1, and MMPs were measured in tissue culture supernatants by immunoblotting. Anti-FAP-treated myofibroblasts were analyzed for TIMP-1 expression by immunoblotting, for migratory potential by wound healing assay, and for apoptosis by Annexin V staining., Results: Myofibroblasts from stenotic CD mucosa showed upregulation of FAP expression when compared with nonstenotic mucosa. Treatment of stenotic tissues with anti-FAP antibody induced a dose-dependent decrease in collagen production, particularly affecting type I collagen. The treatment also reduced TIMP-1 production in CD strictures, without altering MMP-3 and MMP-12 secretion. Accordingly, anti-FAP treatment inhibited TIMP-1 expression in stenotic CD myofibroblasts and enhanced myofibroblast migration without affecting survival., Conclusions: FAP inhibition reduced type I collagen and TIMP-1 production by CD strictures ex vivo without compromising uninvolved bowel areas. These results suggest that targeting FAP could reconstitute ECM homeostasis in fibrostenotic CD., (© 2018 Crohn’s & Colitis Foundation of America. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)