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Abstract A spontaneously occurring temperature increase in solid tumors has been reported sporadically, but is largely overlooked in terms of cancer biology. Here we show that temperature is increased in tumors of patients with pancreatic ductal adenocarcinoma (PDAC) and explore how this could affect therapy response. By mimicking this observation in PDAC cell lines, we demonstrate that through adaptive changes in lipid metabolism, the temperature increase found in human PDAC confers protection to lipid peroxidation and contributes to gemcitabine resistance. Consistent with the recently uncovered role of p38 MAPK in ferroptotic cell death, we find that the reduction in lipid peroxidation potential following adaptation to tumoral temperature allows for p38 MAPK inhibition, conferring chemoresistance. As an increase in tumoral temperature is observed in several other tumor types, our findings warrant taking tumoral temperature into account in subsequent studies related to ferroptosis and therapy resistance. More broadly, our findings indicate that tumoral temperature affects cancer biology.

Abstract Background Trans-cyclooctenes (TCOs) are highly strained alkenes with remarkable reactivity towards tetrazines (Tzs) in inverse electron-demand Diels–Alder reactions. Since their discovery as bioorthogonal reaction partners, novel TCO derivatives have been developed to improve their reactivity, stability, and hydrophilicity, thus expanding their utility in diverse applications. Main body TCOs have garnered significant interest for their applications in biomedical settings. In chemical biology, TCOs serve as tools for bioconjugation, enabling the precise labeling and manipulation of biomolecules. Moreover, their role in nuclear medicine is substantial, with TCOs employed in the radiolabeling of peptides and other biomolecules. This has led to their utilization in pretargeted nuclear imaging and therapy, where they function as both bioorthogonal tags and radiotracers, facilitating targeted disease diagnosis and treatment. Beyond these applications, TCOs have been used in targeted cancer therapy through a "click-to-release" approach, in which they act as key components to selectively deliver therapeutic agents to cancer cells, thereby enhancing treatment efficacy while minimizing off-target effects. However, the search for a suitable TCO scaffold with an appropriate balance between stability and reactivity remains a challenge. Conclusions This review paper provides a comprehensive overview of the current state of knowledge regarding the synthesis of TCOs, and its challenges, and their development throughout the years. We describe their wide ranging applications as radiolabeled prosthetic groups for radiolabeling, as bioorthogonal tags for pretargeted imaging and therapy, and targeted drug delivery, with the aim of showcasing the versatility and potential of TCOs as valuable tools in advancing biomedical research and applications.

ABSTRACTThe World Health Organization’s goal to combat tuberculosis (TB) is hindered by the emergence of anti-microbial resistance, therefore necessitating the exploration of new drug targets. Multidrug regimens are indispensable in TB therapy as they provide synergetic bactericidal effects, shorten treatment duration, and reduce the risk of resistance development. The research within our European RespiriTB consortium explores Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we describe the development and validation of a luminescence-coupled, target-based assay for the identification of novel compounds inhibiting Mycobacterium tuberculosis mycothione reductase (MtrMtb), an enzyme with a role in the protection against oxidative stress. Recombinant MtrMtb was employed for the development of a highly sensitive, robust high-throughput screening (HTS) assay by coupling enzyme activity to a bioluminescent readout. Its application in a semi-automated setting resulted in the screening of a diverse library of ~130,000 compounds, from which 19 hits were retained after an assessment of their potency, selectivity, and specificity. The selected hits formed two clusters and four fragment molecules, which were further evaluated in whole-cell and intracellular infection assays. The established HTS discovery pipeline offers an opportunity to deliver novel MtrMtb inhibitors and lays the foundation for future efforts in developing robust biochemical assays for the identification and triaging of inhibitors from high-throughput library screens.IMPORTANCEThe growing anti-microbial resistance poses a global public health threat, impeding progress toward eradicating tuberculosis. Despite decades of active research, there is still a dire need for the discovery of drugs with novel modes of action and exploration of combination drug regimens. Within the European RespiriTB consortium, we explore Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we present the development of a high-throughput screening pipeline that led to the identification of M. tuberculosis mycothione reductase inhibitors.

Natural products and analogues are a source of antibacterial drug discovery. Considering drug resistance levels emerging for antibiotics, identification of bacterial metalloenzymes and the synthesis of selective inhibitors are interesting for antibacterial agent development. Peptide nucleic acids are attractive antisense and antigene agents representing a novel strategy to target pathogens due to their unique mechanism of action. Antisense inhibition and development of antisense peptide nucleic acids is a new approach to antibacterial agents. Due to the increased resistance of biofilms to antibiotics, alternative therapeutic options are necessary. To develop antimicrobial strategies, optimised in vitro and in vivo models are needed. In vivo models to study biofilm-related respiratory infections, device-related infections: ventilator-associated pneumonia, tissue-related infections: chronic infection models based on alginate or agar beads, methods to battle biofilm-related infections are discussed. Drug delivery in case of antibacterials often is a serious issue therefore this review includes overview of drug delivery nanosystems.

Herein, we report a series of 1,3-diarylpyrazoles that are analogues of compound 26/HIT 8. We previously identified this molecule as a ‘hit’ during a high-throughput screening campaign for autophagy inducers. A variety of synthetic strategies were utilized to modify the 1,3-diarylpyrazole core at its 1-, 3-, and 4-position. Compounds were assessed in vitro to identify their cytotoxicity properties. Of note, several compounds in the series displayed relevant cytotoxicity, which warrants scrutiny while interpreting biological activities that have been reported for structurally related molecules. In addition, antiparasitic activities were recorded against a range of human-infective protozoa, including Trypanosoma cruzi, T. brucei rhodesiense, and Leishmania infantum. The most interesting compounds displayed low micromolar whole-cell potencies against individual or several parasitic species, while lacking cytotoxicity against human cells.

Receptor-Interacting serine/threonine-Protein Kinase 1 (RIPK1) emerged as an important driver of inflammation and, consequently, inflammatory pathologies. The enzymatic activity of RIPK1 is known to indirectly promote inflammation by triggering cell death, in the form of apoptosis, necroptosis and pyroptosis. Small molecule Receptor-Interacting serine/threonine-Protein Kinase 1 inhibitors have therefore recently entered clinical trials for the treatment of a subset of inflammatory pathologies. We previously identified GSK2656157 (GSK’157), a supposedly specific inhibitor of protein kinase R (PKR)-like ER kinase (PERK), as a much more potent type II Receptor-Interacting serine/threonine-Protein Kinase 1 inhibitor. We now performed further structural optimisation on the GSK’157 scaffold in order to develop a novel class of more selective Receptor-Interacting serine/threonine-Protein Kinase 1 inhibitors. Based on a structure-activity relationship (SAR) reported in the literature, we anticipated that introducing a substituent on the para-position of the pyridinyl ring would decrease the interaction with PERK. Herein, we report a series of novel GSK’157 analogues with different para-substituents with increased selectivity for Receptor-Interacting serine/threonine-Protein Kinase 1. The optimisation led to UAMC-3861 as the best compound of this series in terms of activity and selectivity for Receptor-Interacting serine/threonine-Protein Kinase 1 over PERK. The most selective compounds were screened in vitro for their ability to inhibit RIPK1-dependent apoptosis and necroptosis. With this work, we successfully synthesised a novel series of potent and selective type II Receptor-Interacting serine/threonine-Protein Kinase 1 inhibitors based on the GSK’157 scaffold.

Catalytic iron is associated with intensive care unit mortality and is known to cause free radical-mediated cellular toxicity. Here the authors show that a soluble ferrostatin-analogue (a ferroptosis inhibitor) protects mice from injury and death in experimental iron overload induced and genetic models of organ dysfunction, but not sepsis-induced multiorgan dysfunction.

Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with “clickable” affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC50 values, mechanism of inhibition, and kinetic constants were determined. The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery.

Background: Serine proteases are believed to play a key role in the origin of abdominal pain in IBD and IBS. We previously demonstrated a reduction of visceral pain in a post-inflammatory IBS rat model after a single intraperitoneal or intracolonic administration of a serine protease inhibitor. The aim of this study was to investigate the efficacy of serine protease inhibition on visceral pain in two different animal models involving a colonic insult based either on acute inflammation or on neonatal irritation. Moreover, protease profiling was explored in the acute colitis model.Methods: An acute 2,4,6-trinitrobenzenesulphonic acid (TNBS) colitis rat model and a chronic neonatal acetic acid mouse model were used in this study. Visceral sensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30 min after intraperitoneal administration of the serine protease inhibitors nafamostat, UAMC-00050 or their vehicles. Colonic samples from acute colitis rats were used to quantify the mRNA expression of a panel of serine proteases and mast cell tryptase by immunohistochemistry. Finally, proteolytic activities in colonic and fecal samples were characterized using fluorogenic substrates.Key Results: We showed a significant and pressure-dependent increase in visceral hypersensitivity in acute colitis and neonatal acetic acid models. UAMC-00050 and nafamostat significantly reduced VMRs in both animal models. In acute colitis rats, the administration of a serine protease inhibitor did not affect the inflammatory parameters. Protease profiling of these acute colitis animals revealed an increased tryptase immunoreactivity and a downregulation of matriptase at the mRNA level after inflammation. The administration of UAMC-00050 resulted in a decreased elastase-like activity in the colon associated with a significantly increased elastase-like activity in fecal samples of acute colitis animals.Conclusion: In conclusion, our results suggest that serine proteases play an important role in visceral hypersensitivity in an acute TNBS colitis model in rats and a neonatal acetic acid model in mice. Moreover, we hypothesize a potential mechanism of action of UAMC-00050 via the alteration of elastase-like proteolytic activity in acute inflammation. Taken together, we provided fundamental evidence for serine protease inhibitors as a promising new therapeutic strategy for abdominal pain in gastrointestinal diseases.

Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy.

The research on new treatments for dry eye diseases (DED) has exponentially grown over the past decades. The increased prevalence of dry eye conditions, particularly in the younger population, has received much attention. Therefore, it is of utmost importance to identify novel therapeutical targets. Regulated cell death (RCD) is an essential process to control the biological homeostasis of tissues and organisms. The identification of different mechanisms of RCD stimulated the research on their involvement in different human pathologies. Whereas apoptosis has been widely studied in DED and included in the DED vicious cycle, the role of RCD still needs to be completely elucidated. In this review, we will explore the potential roles of different types of RCD in DED and ocular surface dysfunction. Starting from the evidence of oxidative stress and inflammation in dry eye pathology, we will analyse the potential therapeutic applications of the following principal RCD mechanisms: ferroptosis, necroptosis, and pyroptosis.

Corneal wound, associated with pain, impaired vision, and even blindness, is the most common ocular injury. In this study, we investigated the effect of a novel ferroptosis inhibitor, UAMC-3203 (10 nM–50 µM), in corneal epithelial wound healing in vitro in human corneal epithelial (HCE) cells and ex vivo using alkali-induced corneal wounded mice eye model. We evaluated in vivo acute tolerability of the compound by visual inspection, optical coherence tomography (OCT), and stereomicroscope imaging in rats after its application (100 µM drug solution in phosphate buffer pH 7.4) twice a day for 5 days. In addition, we studied the partitioning of UAMC-3203 in corneal epithelium and corneal stroma using excised porcine cornea. Our study demonstrated that UAMC-3203 had a positive corneal epithelial wound healing effect at the optimal concentration of 10 nM (IC50 value for ferroptosis) in vitro and at 10 µM in the ex vivo study. UAMC-3203 solution (100 µM) was well tolerated after topical administration with no signs of toxicity and inflammation in rats. Ex-vivo distribution study revealed significantly higher concentration (~12–38-fold) and partition coefficient (Kp) (~52 times) in corneal epithelium than corneal stroma. The UAMC-3203 solution (100 µM) was stable for up to 30 days at 4 °C, 37 °C, and room temperature. Overall, UAMC-3203 provides a new prospect for safe and effective therapy for corneal wounds.

Dry eye disease (DED) is a multifactorial disorder that leads to ocular discomfort, visual disturbance, and tear film instability. DED is accompanied by an increase in tear osmolarity and ocular surface inflammation. The diagnosis and treatment of DED still present significant challenges. Therefore, novel biomarkers and treatments are of great interest. Proteases are present in different tissues on the ocular surface. In a healthy eye, proteases are highly regulated. However, dysregulation occurs in various pathologies, including DED. With this review, we provide an overview of the implications of different families of proteases in the development and severity of DED, along with studies involving protease inhibitors as potential therapeutic tools. Even though further research is needed, this review aims to give suggestions for identifying novel biomarkers and developing new protease inhibitors.