Sulfone-based human liver pyruvate kinase inhibitors - Design, synthesis and in vitro bioactivity.

Non-alcoholic fatty liver disease (NAFLD) is a prevalent pathological condition characterised by the accumulation of fat in the liver. Almost one-third of the global population is affected by NAFLD, making it a significant health concern. However, despite its prevalence, there is currently no approved drug specifically designed for the treatment of NAFLD. To address this critical gap, researchers have been investigating potential targets for NAFLD drug development. One promising candidate is the liver isoform of pyruvate kinase (PKL). In recent studies, Urolithin C, an allosteric inhibitor of PKL, has emerged as a potential lead compound for therapeutic intervention. Building upon this knowledge, our team has conducted a comprehensive structure-activity relationship of Urolithin C. In this work, we have employed a scaffold-hopping approach, modifying the urolithin structure by replacing the urolithin carbonyl with a sulfone moiety. Our structure-activity relationship analysis has identified the sulfone group as particularly favourable for potent PKL inhibition. Additionally, we have found that the presence of catechol moieties on the two aromatic rings further improves the inhibitory activity. The most promising inhibitor from this new series displayed nanomolar inhibition, boasting an IC50 value of 0.07 µM. This level of potency rivals that of urolithin D and significantly surpasses the effectiveness of urolithin C by an order of magnitude. To better understand the molecular interactions underlying this inhibition, we obtained the crystal structure of one of the inhibitors complexed with PKL. This structural insight served as a valuable reference point, aiding us in the design of inhibitors.

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

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

Exploration of Novel Urolithin C Derivatives as Non-Competitive Inhibitors of Liver Pyruvate Kinase.

The inhibition of liver pyruvate kinase could be beneficial to halt or reverse non-alcoholic fatty liver disease (NAFLD), a progressive accumulation of fat in the liver that can lead eventually to cirrhosis. Recently, urolithin C has been reported as a new scaffold for the development of allosteric inhibitors of liver pyruvate kinase (PKL). In this work, a comprehensive structure-activity analysis of urolithin C was carried out. More than 50 analogues were synthesized and tested regarding the chemical features responsible for the desired activity. These data could pave the way to the development of more potent and selective PKL allosteric inhibitors.

Pretargeted imaging beyond the blood-brain barrier.

Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines and reduce the radiation burden to healthy tissue. Pretargeting is based on bioorthogonal chemistry. The most attractive reaction for this purpose is currently the tetrazine ligation, which occurs between trans-cyclooctene (TCO) tags and tetrazines (Tzs). Pretargeted imaging beyond the blood-brain barrier (BBB) is challenging and has not been reported thus far. In this study, we developed Tz imaging agents that are capable of ligating in vivo to targets beyond the BBB. We chose to develop 18F-labeled Tzs as they can be applied to positron emission tomography (PET) - the most powerful molecular imaging technology. Fluorine-18 is an ideal radionuclide for PET due to its almost ideal decay properties. As a non-metal radionuclide, fluorine-18 also allows for development of Tzs with physicochemical properties enabling passive brain diffusion. To develop these imaging agents, we applied a rational drug design approach. This approach was based on estimated and experimentally determined parameters such as the BBB score, pretargeted autoradiography contrast, in vivo brain influx and washout as well as on peripheral metabolism profiles. From 18 initially developed structures, five Tzs were selected to be tested for their in vivo click performance. Whereas all selected structures clicked in vivo to TCO-polymer deposited into the brain, [18F]18 displayed the most favorable characteristics with respect to brain pretargeting. [18F]18 is our lead compound for future pretargeted neuroimaging studies based on BBB-penetrant monoclonal antibodies. Pretargeting beyond the BBB will allow us to image targets in the brain that are currently not imageable, such as soluble oligomers of neurodegeneration biomarker proteins. Imaging of such currently non-imageable targets will allow early diagnosis and personalized treatment monitoring. This in turn will accelerate drug development and greatly benefit patient care.

Ellagic Acid and Its Metabolites as Potent and Selective Allosteric Inhibitors of Liver Pyruvate Kinase.

Liver pyruvate kinase (PKL) has recently emerged as a new target for non-alcoholic fatty liver disease (NAFLD), and inhibitors of this enzyme could represent a new therapeutic option. However, this breakthrough is complicated by selectivity issues since pyruvate kinase exists in four different isoforms. In this work, we report that ellagic acid (EA) and its derivatives, present in numerous fruits and vegetables, can inhibit PKL potently and selectively. Several polyphenolic analogues of EA were synthesized and tested to identify the chemical features responsible for the desired activity. Molecular modelling studies suggested that this inhibition is related to the stabilization of the PKL inactive state. This unique inhibition mechanism could potentially herald the development of new therapeutics for NAFLD.

Synthesis and radiolabeling of a polar [125 I]I-1,2,4,5-tetrazine.

Pretargeting imaging has gained a lot of prominence, due to its excellent bioorthogonality and improved imaging contrast compared to conventional imaging. A new iodo tetrazine (Tz) derivative has been synthesized and further developed into the corresponding iodine-125 (125 I) analog (12), via the trimethylstannane precursor. Radiolabeling with either N-chlorosuccinimide or chloramine-T, in either MeCN or MeOH proceeded with a radiochemical conversion (RCC) of >80%. Subsequent deprotection only proved successful, among the tested conditions, when the radiolabeled Tz was stirred in 6-M HCl(aq.) at 60°C for 2.5 h. To the best of our knowledge, this is the first H-tetrazine labeled with iodine. In vivo investigations on the pretargeting ability of 12 are currently under way.

Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase.

Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50 =0.29 µM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.

Synthesis and in vivo evaluation of [11C]tucatinib for HER2-targeted PET imaging.

Tucatinib is a selective human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration (FDA) in April 2020 for HER2-positive lesions in metastatic breast cancer patients, including CNS metastases. In this article, we attempted to develop the first small molecule, blood-brain-barrier (BBB) penetrant HER2 PET imaging probe based on tucatinib. [11C]tucatinib was synthesized via a Stille-coupling from the respective trimethylstannyl precursor and its biodistribution was evaluated in NMRI nude mice bearing HER2-overexpressing human ovarian cancer cells (SKOV-3). No significant tumor accumulation was observed despite its high affinity for HER-2 receptors (IC50 = 6.9 nM). High liver and intestinal uptake indicate that [11C]tucatinib is too lipophilic to be used as a tumor targeting PET tracer. Therefore, chemical modifications of [11C]tucatinib are needed to increase the polarity for tumor imaging. Tucatinib as an FDA approved drug is still an interesting platform to develop the first small molecule HER2-selective PET tracer. The study highlights the differences between a drug, which needs to be effective, and an imaging agent, which is dependent on contrast.

Development of the First Tritiated Tetrazine: Facilitating Tritiation of Proteins.

Tetrazine (Tz)-trans-cyclooctene (TCO) ligation is an ultra-fast and highly selective reaction and it is particularly suited to label biomolecules under physiological conditions. As such, a 3 H-Tz based synthon would have wide applications for in vitro/ex vivo assays. In this study, we developed a 3 H-labeled Tz and characterized its potential for application to pretargeted autoradiography. Several strategies were explored to synthesize such a Tz. However, classical approaches such as reductive halogenation failed. For this reason, we designed a Tz containing an aldehyde and explored the possibility of reducing this group with NaBT4 . This approach was successful and resulted in [3 H]-(4-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)phenyl)methan-t-ol with a radiochemical yield of 22 %, a radiochemical purity of 96 % and a molar activity of 0.437 GBq/µmol (11.8 Ci/mmol). The compound was successfully applied to pretargeted autoradiography. Thus, we report the synthesis of the first 3 H-labeled Tz and its successful application as a labeling building block.

Evaluation of the α-synuclein PET radiotracer (d3)-[11C]MODAG-001 in pigs.

BACKGROUND: A positron emission tomography (PET) radiotracer to neuroimage α-synuclein aggregates would be a crucial addition for early diagnosis and treatment development in disorders such as Parkinson's disease, where elevated aggregate levels are a histopathological hallmark. The radiotracer (d3)-[11C]MODAG-001 has recently shown promise for visualization of α-synuclein pre-formed fibrils (α-PFF) in rodents. We here test the radiotracer in a pig model where proteins are intracerebrally injected immediately before scanning. Four pigs were injected in one hemisphere with 150 µg α-PFF, and in the other hemisphere, either 75 µg α-PFF or human brain homogenate from either dementia with Lewy bodies (DLB) or Alzheimer's disease (AD) was injected. All pigs underwent one or two (d3)-[11C]MODAG-001 PET scans, quantified with the non-invasive Logan graphical analysis using the occipital cortex as a reference region. RESULTS: The α-PFF and AD homogenate injected brain regions had high uptake of (d3)-[11C]MODAG-001 compared to the occipital cortex or cerebellum. BPND values in 150 µg α-PFF injected regions was 0.78, and in the AD homogenate injected regions was 0.73. By contrast, the DLB homogenate injected region did not differ in uptake and clearance compared to the reference regions. The time-activity curves and BPND values in the 150 µg and 75 µg injected regions of α-PFFs show a dose-dependent effect, and the PET signal could be blocked by pretreatment with unlabeled MODAG-001. CONCLUSION: We find that both α-PFF and AD brain homogenates give rise to increased binding of (d3)-[11C]MODAG-001 when injected into the pig brain. Despite its limited specificity for cerebral α-synuclein pathology, (d3)-[11C]MODAG-001 shows promise as a lead tracer for future radiotracer development.

Optimization of Direct Aromatic 18F-Labeling of Tetrazines.

Radiolabeling of tetrazines has gained increasing attention due to their important role in pretargeted imaging or therapy. The most commonly used radionuclide in PET imaging is fluorine-18. For this reason, we have recently developed a method which enables the direct aromatic 18F-fluorination of tetrazines using stannane precursors through copper-mediated fluorinations. Herein, we further optimized this labeling procedure. 3-(3-fluorophenyl)-1,2,4,5-tetrazine was chosen for this purpose because of its high reactivity and respective limited stability during the labeling process. By optimizing parameters such as elution conditions, precursor amount, catalyst, time or temperature, the radiochemical yield (RCY) could be increased by approximately 30%. These conditions were then applied to optimize the RCY of a recently successfully developed and promising pretargeting imaging agent. This agent could be isolated in a decay corrected RCY of 14 ± 3% and Am of 201 ± 30 GBq/µmol in a synthesis time of 70 min. Consequently, the RCY increased by 27%.

Recent Advances in the Development of Tetrazine Ligation Tools for Pretargeted Nuclear Imaging.

Tetrazine ligation has gained interest as a bio-orthogonal chemistry tool within the last decade. In nuclear medicine, tetrazine ligation is currently being explored for pretargeted approaches, which have the potential to revolutionize state-of-the-art theranostic strategies. Pretargeting has been shown to increase target-to-background ratios for radiopharmaceuticals based on nanomedicines, especially within early timeframes. This allows the use of radionuclides with short half-lives which are more suited for clinical applications. Pretargeting bears the potential to increase the therapeutic dose delivered to the target as well as reduce the respective dose to healthy tissue. Combined with the possibility to be applied for diagnostic imaging, pretargeting could be optimal for theranostic approaches. In this review, we highlight efforts that have been made to radiolabel tetrazines with an emphasis on imaging.

Development of 18F-Labeled Bispyridyl Tetrazines for In Vivo Pretargeted PET Imaging.

Pretargeted PET imaging is an emerging and fast-developing method to monitor immuno-oncology strategies. Currently, tetrazine ligation is considered the most promising bioorthogonal reaction for pretargeting in vivo. Recently, we have developed a method to 18F-label ultrareactive tetrazines by copper-mediated fluorinations. However, bispyridyl tetrazines-one of the most promising structures for in vivo pretargeted applications-were inaccessible using this strategy. We believed that our successful efforts to 18F-label H-tetrazines using low basic labeling conditions could also be used to label bispyridyl tetrazines via aliphatic nucleophilic substitution. Here, we report the first direct 18F-labeling of bispyridyl tetrazines, their optimization for in vivo use, as well as their successful application in pretargeted PET imaging. This strategy resulted in the design of [18F]45, which could be labeled in a satisfactorily radiochemical yield (RCY = 16%), molar activity (Am = 57 GBq/µmol), and high radiochemical purity (RCP > 98%). The [18F]45 displayed a target-to-background ratio comparable to previously successfully applied tracers for pretargeted imaging. This study showed that bispyridyl tetrazines can be developed into pretargeted imaging agents. These structures allow an easy chemical modification of 18F-labeled tetrazines, paving the road toward highly functionalized pretargeting tools. Moreover, bispyridyl tetrazines led to near-instant drug release of iTCO-tetrazine-based 'click-to-release' reactions. Consequently, 18F-labeled bispyridyl tetrazines bear the possibility to quantify such release in vivo in the future.

[11 C]Carboxylated Tetrazines for Facile Labeling of Trans-Cyclooctene-Functionalized PeptoBrushes.

Functionalization of macromolecules (antibodies, polymers, nanoparticles) with click-reactive groups greatly enhances the versatility of their potential applications. Click chemistry based on tetrazine - trans-cyclooctene (TCO) ligation is especially promising and is already widely applied for pretargeted imaging and therapy. Indirect radiolabeling of TCO-functionalized macromolecules with substoichiometric amounts of radioactive tetrazines is a convenient way to monitor the fate of those macromolecules by means of positron emission tomography (PET) imaging after their administration into the test subject. In this work, the preparation is reported of TCO-containing graft copolymers, namely PeptoBrushes (polyglutamic acid-graft-polysarcosine), novel [11 C]carboxylated tetrazines, and their combined use in radiolabeling the polymer by inverse electron demand Diels Alder reaction, to investigate it is potential for an application in pretarget imaging or injectable brachytherapy. The procedure for [11 C]tetrazine production is easy and scalable, while indirect TCO-PeptoBrushes labeling with these [11 C]tetrazines is mild, fast, and quantitative. This strategy allows facile 11 C-labeling of diverse TCO-functionalized macromolecules, so that their localization and distribution shortly after injection can be assessed by PET.

Insights into Elution of Anion Exchange Cartridges: Opening the Path toward Aliphatic 18F-Radiolabeling of Base-Sensitive Tracers.

Aliphatic nucleophilic substitution (SN2) with [18F]fluoride is the most widely applied method to prepare 18F-labeled positron emission tomography (PET) tracers. Strong basic conditions commonly used during 18F-labeling procedures inherently limit or prohibit labeling of base-sensitive scaffolds. The high basicity stems from the tradition to trap [18F]fluoride on anion exchange cartridges and elute it afterward with basic anions. This sequence is used to facilitate the transfer of [18F]fluoride from an aqueous to an aprotic organic, polar reaction medium, which is beneficial for SN2 reactions. Furthermore, this sequence also removes cationic radioactive contaminations from cyclotron-irradiated [18O]water from which [18F]fluoride is produced. In this study, we developed an efficient elution procedure resulting in low basicity that permits SN2 18F-labeling of base-sensitive scaffolds. Extensive screening of trapping and elution conditions (>1000 experiments) and studying their influence on the radiochemical yield (RCY) allowed us to identify a suitable procedure for this. Using this procedure, four PET tracers and three synthons could be radiolabeled in substantially higher RCYs (up to 2.5-fold) compared to those of previously published procedures, even from lower precursor amounts. Encouraged by these results, we applied our low-basicity method to the radiolabeling of highly base-sensitive tetrazines, which cannot be labeled using state-of-art direct aliphatic 18F-labeling procedures. Labeling succeeded in RCYs of up to 20%. We believe that our findings facilitate PET tracer development by opening the path toward simple and direct SN2 18F fluorination of base-sensitive substrates.

Alpha-Synuclein PET Tracer Development-An Overview about Current Efforts.

Neurodegenerative diseases such as Parkinson's disease (PD) are manifested by inclusion bodies of alpha-synuclein (α-syn) also called α-synucleinopathies. Detection of these inclusions is thus far only possible by histological examination of postmortem brain tissue. The possibility of non-invasively detecting α-syn will therefore provide valuable insights into the disease progression of α-synucleinopathies. In particular, α-syn imaging can quantify changes in monomeric, oligomeric, and fibrillic α-syn over time and improve early diagnosis of various α-synucleinopathies or monitor treatment progress. Positron emission tomography (PET) is a non-invasive in vivo imaging technique that can quantify target expression and drug occupancies when a suitable tracer exists. As such, novel α-syn PET tracers are highly sought after. The development of an α-syn PET tracer faces several challenges. For example, the low abundance of α-syn within the brain necessitates the development of a high-affinity ligand. Moreover, α-syn depositions are, in contrast to amyloid proteins, predominantly localized intracellularly, limiting their accessibility. Furthermore, another challenge is the ligand selectivity over structurally similar amyloids such as amyloid-beta or tau, which are often co-localized with α-syn pathology. The lack of a defined crystal structure of α-syn has also hindered rational drug and tracer design efforts. Our objective for this review is to provide a comprehensive overview of current efforts in the development of selective α-syn PET tracers.

Synthesis and biological evaluation of 1,3-dioxolane-based 5-HT1A receptor agonists for CNS disorders and neuropathic pain.

AIM: Targeting 5-HT1A receptor (5-HT1AR) as a strategy for CNS disorders and pain control. METHODOLOGY: A series of 1,3-dioxolane-based 2-heteroaryl-phenoxyethylamines was synthesized by a convergent approach and evaluated at α1-adrenoceptors and 5-HT1AR by binding and functional experiments. Absorption, distribution, metabolism, excretion and toxicity prediction studies were performed to explore the drug-likeness of the compounds. RESULTS & CONCLUSION: The most promising compound, the pyridin-4-yl derivative, emerged as a potent and selective 5-HT1AR agonist (pKi = 9.2; pD2 = 8.83; 5-HT1A/α1 = 135). In vitro it was able to permeate by passive diffusion MDCKII-MDR1 monolayer mimicking the blood-brain barrier and showed promising neuroprotective activity.

A Metabolomic Approach Applied to a Liquid Chromatography Coupled to High-Resolution Tandem Mass Spectrometry Method (HPLC-ESI-HRMS/MS): Towards the Comprehensive Evaluation of the Chemical Composition of Cannabis Medicinal Extracts.

INTRODUCTION: Cannabis sativa L. is a powerful medicinal plant and its use has recently increased for the treatment of several pathologies. Nonetheless, side effects, like dizziness and hallucinations, and long-term effects concerning memory and cognition, can occur. Most alarming is the lack of a standardised procedure to extract medicinal cannabis. Indeed, each galenical preparation has an unknown chemical composition in terms of cannabinoids and other active principles that depends on the extraction procedure. OBJECTIVE: This study aims to highlight the main differences in the chemical composition of Bediol® extracts when the extraction is carried out with either ethyl alcohol or olive oil for various times (0, 60, 120 and 180 min for ethyl alcohol, and 0, 60, 90 and 120 min for olive oil). METHODOLOGY: Cannabis medicinal extracts (CMEs) were analysed by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS) using an untargeted metabolomics approach. The data sets were processed by unsupervised multivariate analysis. RESULTS: Our results suggested that the main difference lies in the ratio of acid to decarboxylated cannabinoids, which dramatically influences the pharmacological activity of CMEs. Minor cannabinoids, alkaloids, and amino acids contributing to this difference are also discussed. The main cannabinoids were quantified in each extract applying a recently validated LC-MS and LC-UV method. CONCLUSIONS: Notwithstanding the use of a standardised starting plant material, great changes are caused by different extraction procedures. The metabolomics approach is a useful tool for the evaluation of the chemical composition of cannabis extracts. Copyright © 2017 John Wiley & Sons, Ltd.

Structure-Activity Relationships within a Series of σ1 and σ2 Receptor Ligands: Identification of a σ2 Receptor Agonist (BS148) with Selective Toxicity against Metastatic Melanoma.

A new series of spirocyclic σ receptor (σR) ligands were prepared and studied. Most were found to have a high affinity and selectivity for σ1 R; three compounds were shown to be σ1 R agonists, while another proved to be the only σ1 R antagonist. Only one of the σ1 R agonists (BS148) also exhibited σ2 R selectivity and was able to inhibit the growth of metastatic malignant melanoma cell lines without affecting normal human melanocytes. The antiproliferative activity of this compound suggested an σ2 R agonist profile. Further, preliminary investigations indicated that the mechanism of metastatic malignant melanoma cell death induced by BS148 is due, at least in part, to apoptosis.

Scouting new sigma receptor ligands: Synthesis, pharmacological evaluation and molecular modeling of 1,3-dioxolane-based structures and derivatives.

Herein we report the synthesis and biological activity of new sigma receptor (σR) ligands obtained by combining different substituted five-membered heterocyclic rings with appropriate σR pharmacophoric amines. Radioligand binding assay, performed on guinea pig brain membranes, identified 25b (1-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)-4-benzylpiperazine) as the most interesting compound of the series, displaying high affinity and selectivity for σ1R (pKiσ1 = 9.13; σ1/σ2 = 47). The ability of 25b to modulate the analgesic effect of the κ agonist (-)-U-50,488H and µ agonist morphine was evaluated in vivo by radiant heat tail-flick test. It exhibited anti-opioid effects on both κ and µ receptor-mediated analgesia, suggesting an agonistic behavior at σ1R. Docking studies were performed on the theoretical σ1R homology model. The present work represents a new starting point for the design of more potent and selective σ1R ligands.