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

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

Imaging the master regulator of the antioxidant response in non-small cell lung cancer with positron emission tomography.

Mutations in the NRF2-KEAP1 pathway are common in non-small cell lung cancer (NSCLC) and confer broad-spectrum therapeutic resistance, leading to poor outcomes. The cystine/glutamate antiporter, system xc-, is one of the >200 cytoprotective proteins controlled by NRF2, which can be non-invasively imaged by (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG) positron emission tomography (PET). Through genetic and pharmacologic manipulation, we show that [18F]FSPG provides a sensitive and specific marker of NRF2 activation in advanced preclinical models of NSCLC. We validate imaging readouts with metabolomic measurements of system xc- activity and their coupling to intracellular glutathione concentration. A redox gene signature was measured in patients from the TRACERx 421 cohort, suggesting an opportunity for patient stratification prior to imaging. Furthermore, we reveal that system xc- is a metabolic vulnerability that can be therapeutically targeted for sustained tumour growth suppression in aggressive NSCLC. Our results establish [18F]FSPG as predictive marker of therapy resistance in NSCLC and provide the basis for the clinical evaluation of both imaging and therapeutic agents that target this important antioxidant pathway.

The chicken chorioallantoic membrane as a low-cost, high-throughput model for cancer imaging.

Mouse models are invaluable tools for radiotracer development and validation. They are, however, expensive, low throughput, and are constrained by animal welfare considerations. Here, we assessed the chicken chorioallantoic membrane (CAM) as an alternative to mice for preclinical cancer imaging studies. NCI-H460 FLuc cells grown in Matrigel on the CAM formed vascularized tumors of reproducible size without compromising embryo viability. By designing a simple method for vessel cannulation it was possible to perform dynamic PET imaging in ovo, producing high tumor-to-background signal for both 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) and (4S)-4-(3-18F-fluoropropyl)-L-glutamate (18F-FSPG). The pattern of 18F-FDG tumor uptake were similar in ovo and in vivo, although tumor-associated radioactivity was higher in the CAM-grown tumors over the 60 min imaging time course. Additionally, 18F-FSPG provided an early marker of both treatment response to external beam radiotherapy and target inhibition in ovo. Overall, the CAM provided a low-cost alternative to tumor xenograft mouse models which may broaden access to PET and SPECT imaging and have utility across multiple applications.

ATG4B Inhibitor UAMC-2526 Potentiates the Chemotherapeutic Effect of Gemcitabine in a Panc02 Mouse Model of Pancreatic Ductal Adenocarcinoma.

Resistance against anti-cancer therapy is one of the major challenges during treatment of multiple cancers. Gemcitabine is a standard first-line chemotherapeutic drug, yet autophagy is highly activated in the hypoxic microenvironment of solid tumors and enhances the survival of tumor cells against gemcitabine chemotherapy. Recently, we showed the add-on effect of autophagy inhibitor UAMC-2526 to prevent HT-29 colorectal tumor growth in CD1-/- Foxn1nu mice treated with oxaliplatin. In this study, we aimed to investigate the potential beneficial effects of UAMC-2526 in a syngeneic Panc02 mouse model of pancreatic ductal adenocarcinoma (PDAC). Our data showed that UAMC-2526 combined with gemcitabine significantly reduced tumor growth as compared to the individual treatments. However, in contrast to in vitro experiments with Panc02 cells in culture, we were unable to detect autophagy inhibition by UAMC-2526 in Panc02 tumor tissue, neither via western blot analysis of autophagy markers LC3 and p62, nor by transmission electron microscopy. In vitro experiments revealed that UAMC-2526 enhances the potential of gemcitabine to inhibit Panc02 cell proliferation without obvious induction of cell death. Altogether, we conclude that although the combination treatment of UAMC-2526 with gemcitabine did not inhibit autophagy in the Panc02 mouse model, it has a beneficial effect on tumor growth inhibition.

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.

Synthesis and evaluation of novel benzotropolones as Atg4B inhibiting autophagy blockers.

Autophagy is an intracellular degradation/recycling pathway that provides nutrients and building blocks to cellular metabolism and keeps the cytoplasm clear of obsolete proteins and organelles. During recent years, dysregulated autophagy activity has been reported to be a characteristic of many different disease types, including cancer and neurodegenerative disorders. This has created a strong case for development of autophagy modulating compounds as potential treatments for these diseases. Inhibitors of autophagy have been proposed as a therapeutic intervention in, e.g., advanced cancer, and inhibiting the cysteine protease Atg4B has been put forward as a main strategy to block autophagy. We recently identified and demonstrated -both in vitro and in vivo - that compounds with a benzotropolone basic structure targeting Atg4B, can significantly slow down tumor growth and potentiate the effect of classical chemotherapy. In this study we report the synthesis and inhibition profile of new benzotropolone derivatives with additional structural modifications at 6 different positions. To obtain a solid inhibition profile, all compounds were evaluated on three levels, including two cell-based assays to confirm autophagy and intracellular Atg4B inhibition and an SDS-PAGE-based experiment to assess in vitro Atg4B affinity. Several molecules with a promising profile were identified.

Deciphering the Mechanism of Human Carbonic Anhydrases Inhibition with Sulfocoumarins: Computational and Experimental Studies.

The reaction mechanism of the carbonic anhydrase-mediated hydrolysis of sulfocoumarins to sulfonic acids has been investigated on an enzyme cluster model using the B3LYP hybrid density functional theory (DFT) and the QST procedure for the Transition State (TS) search. A multistep process was highlighted, with the rate-determining step identified in the initial dual nucleophilic/acidic attack of the zinc-bound hydroxide ion to the sulfocoumarin sulfur atom and to the C3=C4 double bond. The reported multi-step process, combined to SAR analysis on a new set of derivatives, highlighted unprecedented mechanistic aspects of the CA-mediated prodrug activation, which in turn possess relevant consequences to the isoforms-selective inhibition profiles reported by such a class of compounds.

Synthesis of novel 5-amino-1,3,4-thiadiazole-2-sulfonamide containing acridine sulfonamide/carboxamide compounds and investigation of their inhibition effects on human carbonic anhydrase I, II, IV and VII.

Herein, we report that acridine intermediates 5 were obtained from the reduction of nitro acridine derivatives 4, which were synthesized via condensation of dimedone, p-nitrobenzaldehyde with 4-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide, respectively. Then acridine sulfonamide/carboxamide (7a-i) compounds were synthesized by reaction of amino acridine 5 with sulfonyl chlorides and carbamoyl chlorides. The new compounds were characterized by melting points, FT-IR, 1H NMR, 13C NMR and HRMS analyzes. The evaluation of in vitro test of the synthesized compounds against hCA I, II, IV and VII showed that some of them are potent inhibitors. Among them, compound 7e showed the most potent activity against hCA II with a KI of 7.9 nM.

3-Hydroxy-1H-quinazoline-2,4-dione as a New Scaffold To Develop Potent and Selective Inhibitors of the Tumor-Associated Carbonic Anhydrases IX and XII.

In this paper, we describe the discovery of the 3-hydroxyquinazoline-2,4-dione as a useful scaffold to obtain potent inhibitors of the tumor-associated human carbonic anhydrases (hCAs) IX and XII. A set of derivatives (1-29), bearing different substituents on the fused benzo ring (Cl, NO2, NH2, CF3, ureido, amido, heterocycles), were synthesized, and several of them showed nanomolar activity in inhibiting the hCA IX and XII isoforms, while they were ineffective against the cytosolic enzymes hCAs I and II. Some selected compounds were tested for their antiproliferative activity against HT-29 colon cancer cell lines. After 48 h of treatment with the lower dose (30 µM), derivatives 12, 14, 15, and 19 were significantly active, inducing a mortality by about 50% in both normoxia and hypoxia. This finding led us to hypothesize for these compounds more than one mechanism of action involving both CAs IX and XII and other not yet identified target(s).

Effective Access to Multivalent Inhibitors of Carbonic Anhydrases Promoted by Peptide Bioconjugation.

Multivalency has impressive effects on (bio)molecular recognition, through the simultaneous presentation of multiple copies of a ligand, which can change a weak millimolar binder into a potent nanomolar one. The implementation of multivalency in enzyme inhibition is rather recent, being exemplified by few serendipitous discoveries, and hitherto relying on the random exploration of new multivalent structures as potential enzyme inhibitors. Here, a straightforward and versatile method is reported that enables the construction of multivalent systems for the inhibition of carbonic anhydrases (CA), widespread enzymes that catalyze a fundamental biochemical reaction. Oxime and hydrazone click-type bioconjugation techniques were successfully used for the preparation of tetravalent peptide conjugates tethered with sulfonamide CA inhibitors. The enzyme inhibition assays show that multivalent effects were present with these novel compounds, but also reveal various structural effects provided by the scaffolds. The versatility of this approach may facilitate the exploration of structure-activity relationships for other types of enzyme inhibitors.

Human carbonic anhydrase inhibitory profile of mono- and bis-sulfonamides synthesized via a direct sulfochlorination of 3- and 4-(hetero)arylisoxazol-5-amine scaffolds.

Three distinct series of isoxazole-based primary mono- and bis-sulfonamides have been synthesized via direct sulfochlorination, each of them delivering nanomolar inhibitors of human carbonic anhydrase. Certain pronounced SAR trends have been established and rationalized by in silico docking. These findings expand the structure-activity knowledge base for heterocycle-containing sulfonamide carbonic anhydrase inhibitors and further validate the power of direct electrophilic sulfochlorination as a means of introducing the pharmacophoric primary sulfonamide group into structurally diverse aromatic precursors.

Microwave assisted synthesis of novel tetrazole/sulfonamide derivatives based on octahydroacridine, xanthene and chromene skeletons as inhibitors of the carbonic anhydrases isoforms I, II, IV and VII.

The synthesis of novel tetrazole/sulfonamide derivatives based on octahydroacridine, xanthene and chromene scaffold by using microwave (MW) assisted techniques is reported in this study. These synthesized hybrid compounds were assayed for the inhibition of carbonic anhydrase (CA, EC 4.2.1.1). The inhibitory activities were determined against three cytosolic human isoforms (hCA I, II and VII) and one membrane-associated (hCA IV) isoform. Some of the newly synthesized sulfonamides showed micromolar to nanomolar inhibitory activity against these enzymes.

A class of carbonic anhydrase I - selective activators.

A series of ureido and bis-ureido derivatives were prepared by reacting histamine with alkyl/aryl-isocyanates or di-isocyanates. The obtained derivatives were assayed as activators of the enzyme carbonic anhydrase (CA, EC 4.2.1.1), due to the fact that histamine itself has this biological activity. Although inhibition of CAs has pharmacological applications in the field of antiglaucoma, anticonvulsant, anticancer, and anti-infective agents, activation of these enzymes is not yet properly exploited pharmacologically for cognitive enhancement or Alzheimer's disease treatment, conditions in which a diminished CA activity was reported. The ureido/bis-ureido histamine derivatives investigated here showed activating effects only against the cytosolic human (h) isoform hCA I, having no effect on the widespread, physiologically dominant isoform hCA II. This is the first report in which CA I-selective activators were identified. Such compounds may constitute interesting tools for better understanding the physiological/pharmacological effects connected to activation of this widespread CA isoform, whose physiological function is not fully understood.

Synthesis and bioactivity studies of 1-aryl-3-(2-hydroxyethylthio)-1-propanones.

A series of Mannich bases having piperidine moiety were reacted with 2-mercaptoethanol, leading to 1-aryl-3-piperidine-4-yl-1-propanone hydrochlorides. The cytotoxicity and carbonic anhydrase inhibitory activities of these new compounds were evaluated. Among the compounds, only one derivative, nitro substituent bearing EU9, showed an effective cytotoxicity, although weak tumor specificity against human oral malignant versus nonmalignant cells. The compound induced apoptosis in HSC-2 oral squamous cell carcinoma cells, but not in human gingival fibroblast. Chemical modifications of this lead are thus necessary to further investigate it as a drug candidate and to obtain compounds with a better activity profile.

Microwave assisted synthesis of novel acridine-acetazolamide conjugates and investigation of their inhibition effects on human carbonic anhydrase isoforms hCA I, II, IV and VII.

4-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide was condensed with cyclic-1,3-diketones (dimedone and cyclohexane-1,3-dione) and aromatic aldehydes under microwave irradiation, leading to a series of acridine-acetazolamide conjugates. The new compounds were investigated as inhibitors of carbonic anhydrases (CA, EC 4.2.1.1), and more precisely cytosolic isoforms hCA I, II, VII and membrane-bound one hCA IV. All investigated isoforms were inhibited in low micromolar and nanomolar range by the new compounds. hCA IV and VII were inhibited with KIs in the range of 29.7-708.8nM (hCA IV), and of 1.3-90.7nM (hCA VII). For hCA I and II the KIs were in the range of 6.7-335.2nM (hCA I) and of 0.5-55.4nM (hCA II). The structure-activity relationships (SAR) for the inhibition of these isoforms with the acridine-acetazolamide conjugates reported here were delineated.

9,10-Dibromo-N-aryl-9,10-dihydro-9,10-[3,4]epipyrroloanthracene-12,14-diones: Synthesis and Investigation of Their Effects on Carbonic Anhydrase Isozymes I, II, IX, and XII.

N-substituted maleimides were synthesized from maleic anhydride and primary amines. 1,4-Dibromo-dibenzo[e,h]bicyclo-[2,2,2]octane-2,3-dicarboximide derivatives (4a-f) were prepared by the [4+2] cycloaddition reaction of dibromoanthracenes with the N-substituted maleimide derivatives. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the new derivatives were assayed against the human (h) isozymes hCA I, II, IX, and XII. All tested bicyclo dicarboximide derivatives exhibited excellent inhibitory effects in the nanomolar range, with Ki values in the range of 117.73-232.87 nM against hCA I and of 69.74-111.51 nM against hCA II, whereas they were low micromolar inhibitors against hCA IX and XII.

Isoform-selective inhibitory profile of 2-imidazoline-substituted benzene sulfonamides against a panel of human carbonic anhydrases.

A series of novel benzene sulfonamides (previously evaluated as selective cyclooxygenase-2 inhibitors) has been profiled against human carbonic anhydrases I, II, IV and VII in an attempt to observe the manifestation of the well established "tail" approach for designing potent, isoform-selective inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1). The compounds displayed an excellent (pKi 7-8) inhibitory profile against CA II (a cytosolic anti-glaucoma and anti-edema biological target) and CA VII (also a cytosolic target believed to be involved in epilepsy and neuropathic pain) and a marked (1-2 orders of magnitude) selectivity against cytosolic isoform CA I and membrane-bound isoform CA IV. The separation of the CA II and CA IV (both of which are catalytically active isoforms, highly sensitive to sulfonamide-type inhibitors) is particularly remarkable and is adding significantly to the global body of data on the chemical biology of carbonic anhydrases.

Inhibitory effects of benzimidazole containing new phenolic Mannich bases on human carbonic anhydrase isoforms hCA I and II.

New phenolic mono and bis Mannich bases incorporating benzimidazole, such as 2-(aminomethyl)-4-(1H-benzimidazol-2-yl)phenol and 2,6-bis(aminomethyl)-4-(1H-benzimidazol-2-yl)phenol were synthesized starting from 4-(1H-benzimidazol-2-yl)phenol. Amines used for the synthesis included dimethylamine, pyrrolidine, piperidine, N-methylpiperazine and morpholine. The CA inhibitory properties of these compounds were tested on the human carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I and hCA II. These new compounds, as many phenols show moderate CA inhibitory properties.

Carbonic anhydrase inhibition and cytotoxicity studies of Mannich base derivatives of thymol.

Mannich bases of thymol were synthesized. The aminomethylation reaction was realised in the ortho position of the phenol for compounds 2 (dipropylamine), 3 (benzylamine), and 4 (dibenzylamine) while it was from para position for 1 (dimethylamine), 5 (piperidine), 6 (morpholine) and 7 (N-methylpiperazine). The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the compounds were asssessed against hCA I and hCA II. All compounds moderately inhibited hCA I and hCA II. The cytotoxicity of the compounds against four human oral squamous cell carcinoma cell lines were compared those against three normal oral cells. Tumor specificity values were about 2 or slightly more for the compounds 2, 3, 4, 5 and 6. Compound 2 showed cytostatic activity against OSCC cell lines at 16 to 32-fold lower concentrations as compared with normal cells. This suggests that compound 2 can be considered as cytotoxicity enhancing drug candidate for further investigations.