Covalent activity-based probes for imaging of serine proteases.

Serine proteases are one of the largest mechanistic classes of proteases. They regulate a plethora of biochemical pathways inside and outside the cell. Aberrant serine protease activity leads to a wide variety of human diseases. Reagents to visualize these activities can be used to gain insight into the biological roles of serine proteases. Moreover, they may find future use for the detection of serine proteases as biomarkers. In this review, we discuss small molecule tools to image serine protease activity. Specifically, we outline different covalent activity-based probes and their selectivity against various serine protease targets. We also describe their application in several imaging methods.

Photopharmacology of Protease Inhibitors: Current Status and Perspectives.

Proteases are involved in many essential biological processes. Dysregulation of their activity underlies a wide variety of human diseases. Photopharmacology, as applied on various classes of proteins, has the potential to assist protease research by enabling spatiotemporal control of protease activity. Moreover, it may be used to decrease side-effects of protease-targeting drugs. In this review, we discuss the current status of the chemical design of photoactivatable proteases inhibitors and their biological application. Additionally, we give insight into future possibilities for further development of this field of research.

Cellular effects of BAPTA: Are they only about Ca2+ chelation?

Intracellular Ca2+ signals play a vital role in a broad range of cell biological and physiological processes in all eukaryotic cell types. Dysregulation of Ca2+ signaling has been implicated in numerous human diseases. Over the past four decades, the understanding of how cells use Ca2+ as a messenger has flourished, largely because of the development of reporters that enable visualization of Ca2+ signals in different cellular compartments, and tools that can modulate cellular Ca2+ signaling. One such tool that is frequently used is BAPTA; a fast, high-affinity Ca2+-chelating molecule. By making use of a cell-permeable acetoxymethyl ester (AM) variant, BAPTA can be readily loaded into the cytosol of cells (referred to as BAPTAi), where it is trapped and able to buffer changes in cytosolic Ca2+. Due to the ease of loading of the AM version of BAPTA, this reagent has been used in hundreds of studies to probe the role of Ca2+ signaling in specific processes. As such, for decades, researchers have almost universally attributed changes in biological processes caused by BAPTAi to the involvement of Ca2+ signaling. However, BAPTAi has often been used without any form of control, and in many cases has neither been shown to be retained in cells for the duration of experiments nor to buffer any Ca2+ signals. Moreover, increasing evidence points to off-target cellular effects of BAPTA that are clearly not related to Ca2+ chelation. Here, we briefly introduce Ca2+ signaling and the history of Ca2+ chelators and fluorescent Ca2+ indicators. We highlight Ca2+-independent effects of BAPTAi on a broad range of molecular targets and describe some of BAPTAi's impacts on cell functions that occur independently of its Ca2+-chelating properties. Finally, we propose strategies for determining whether Ca2+ chelation, the binding of other metal ions, or off-target interactions with cell components are responsible for BAPTAi's effect on a particular process and suggest some future research directions.

Metabolic support by macrophages sustains colonic epithelial homeostasis.

The intestinal epithelium has a high turnover rate and constantly renews itself through proliferation of intestinal crypt cells, which depends on insufficiently characterized signals from the microenvironment. Here, we showed that colonic macrophages were located directly adjacent to epithelial crypt cells in mice, where they metabolically supported epithelial cell proliferation in an mTORC1-dependent manner. Specifically, deletion of tuberous sclerosis complex 2 (Tsc2) in macrophages activated mTORC1 signaling that protected against colitis-induced intestinal damage and induced the synthesis of the polyamines spermidine and spermine. Epithelial cells ingested these polyamines and rewired their cellular metabolism to optimize proliferation and defense. Notably, spermine directly stimulated proliferation of colon epithelial cells and colon organoids. Genetic interference with polyamine production in macrophages altered global polyamine levels in the colon and modified epithelial cell proliferation. Our results suggest that macrophages act as "commensals" that provide metabolic support to promote efficient self-renewal of the colon epithelium.

4-Oxo-ß-Lactams as Novel Inhibitors for Rhomboid Proteases.

Intramembrane serine proteases (rhomboid proteases) are involved in a variety of biological processes and are implicated in several diseases. Here, we report 4-oxo-ß-lactams as a novel scaffold for inhibition of rhomboids. We show that they covalently react with the active site and that the covalent bond is sufficiently stable for detection of the covalent rhomboid-lactam complex. 4-Oxo-ß-lactams may therefore find future use as both inhibitors and activity-based probes for rhomboid proteases.

Furin-targeting activity-based probes with phosphonate and phosphinate esters as warheads.

Activity-based probes (ABPs) are covalent chemical tools that are widely used to target proteases in chemical biology. Here, we report a series of novel ABPs for the serine protease furin with phosphonate and phosphinate esters as reactive electrophiles. We show that these probes covalently label furin and have nanomolar potencies, because of proposed interactions with the different recognition pockets around the active site of furin.

Chemical Probes for Profiling of MALT1 Protease Activity.

The paracaspase MALT1 is a key regulator of the human immune response. It is implicated in a variety of human diseases. For example, deregulated protease activity drives the survival of malignant lymphomas and is involved in the pathophysiology of autoimmune/inflammatory diseases. Thus, MALT1 has attracted attention as promising drug target. Although many MALT1 inhibitors have been identified, molecular tools to study MALT1 activity, target engagement and inhibition in complex biological samples, such as living cells and patient material, are still scarce. Such tools are valuable to validate MALT1 as a drug target in vivo and to assess yet unknown biological roles of MALT1. In this review, we discuss the recent literature on the development and biological application of molecular tools to study MALT1 activity and inhibition.

Mapping Peptide-Protein Interactions by Amine-Reactive Cleavable Photoaffinity Reagents.

Photoaffinity labeling followed by tandem mass spectrometry is an often used strategy to identify protein targets of small-molecule drugs or drug candidates, which, under ideal conditions, enables the identification of the actual drug binding site. In the case of bioactive peptides, however, identifying the distinct binding site is hampered because of complex fragmentation patterns during tandem mass spectrometry. We here report the development and use of small cleavable photoaffinity reagents that allow functionalization of bioactive peptides for light-induced covalent binding to their protein targets. Upon cleavage of the covalently linked peptide drug, a chemical remnant of a defined mass remains on the bound amino acid, which is then used to unambiguously identify the drug binding site. Applying our approach to known peptide-drug/protein pairs with reported crystal structures, such as the calmodulin-melittin interaction, we were able to validate the identified binding sites based on structural models. Overall, our cleavable photoaffinity labeling strategy represents a powerful tool to enable the identification of protein targets and specific binding sites of a wide variety of bioactive peptides in the future.

Ex situ Generation of Thiazyl Trifluoride (NSF3 ) as a Gaseous SuFEx Hub.

Sulfur(VI)-fluoride exchange (SuFEx) chemistry, an all-encompassing term for substitution events that replace fluoride at an electrophilic sulfur(VI), enables the rapid and flexible assembly of linkages around a SVI core. Although a myriad of nucleophiles and applications works very well with the SuFEx concept, the electrophile design has remained largely SO2 -based. Here, we introduce S≡N-based fluorosulfur(VI) reagents to the realm of SuFEx chemistry. Thiazyl trifluoride (NSF3 ) gas is shown to serve as an excellent parent compound and SuFEx hub to efficiently synthesize mono- and disubstituted fluorothiazynes in an ex situ generation workflow. Gaseous NSF3 was evolved from commercial reagents in a nearly quantitative fashion at ambient conditions. Moreover, the mono-substituted thiazynes could be extended further as SuFEx handles and be engaged in the synthesis of unsymmetrically disubstituted thiazynes. These results provide valuable insights into the versatility of these understudied sulfur functionalities paving the way for future applications.

Duodenal macrophages control dietary iron absorption via local degradation of transferrin.

Iron is an essential cellular metal that is important for many physiological functions including erythropoiesis and host defense. It is absorbed from the diet in the duodenum and loaded onto transferrin (Tf), the main iron transport protein. Inefficient dietary iron uptake promotes many diseases, but mechanisms regulating iron absorption remain poorly understood. By assessing mice that harbor a macrophage-specific deletion of the tuberous sclerosis complex 2 (Tsc2), a negative regulator of mechanistic target of rapamycin complex 1 (mTORC1), we found that these mice possessed various defects in iron metabolism, including defective steady-state erythropoiesis and a reduced saturation of Tf with iron. This iron deficiency phenotype was associated with an iron import block from the duodenal epithelial cells into the circulation. Activation of mTORC1 in villous duodenal CD68+ macrophages induced serine protease expression and promoted local degradation of Tf, whereas the depletion of macrophages in mice increased Tf levels. Inhibition of mTORC1 with everolimus or serine protease activity with nafamostat restored Tf levels and Tf saturation in the Tsc2-deficient mice. Physiologically, Tf levels were regulated in the duodenum during the prandial process and Citrobacter rodentium infection. These data suggest that duodenal macrophages determine iron transfer to the circulation by controlling Tf availability in the lamina propria villi.

Pepstatin-Based Probes for Photoaffinity Labeling of Aspartic Proteases and Application to Target Identification.

Aspartic proteases are a small class of proteases implicated in a wide variety of human diseases. Covalent chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We here report a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin incorporating a minimal diazirine reactive group. The position of this group in the inhibitor determines the labeling efficiency. The most effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Moreover, through chemical proteomics experiments and deep learning algorithms, we identified sequestosome-1, an important player in autophagy, as a direct interaction partner and substrate of cathepsin D.

Azapeptide activity-based probes for the SARS-CoV-2 main protease enable visualization of inhibition in infected cells.

The COVID-19 pandemic has revealed the vulnerability of the modern, global society. With expected waves of future infections by SARS-CoV-2, treatment options for infected individuals will be crucial in order to decrease mortality and hospitalizations. The SARS-CoV-2 main protease is a validated drug target, for which the first inhibitor has been approved for use in patients. To facilitate future work on this drug target, we designed a solid-phase synthesis route towards azapeptide activity-based probes that are capped with a cysteine-reactive electrophile for covalent modification of the active site of Mpro. This design led to the most potent ABP for Mpro and one of the most potent inhibitors reported thus far. We demonstrate that this ABP can be used to visualize Mpro activity and target engagement by drugs in infected cells.

Clotrimazole-Based Modulators of the TRPM3 Ion Channel Reveal Narrow Structure-Activity Relationship.

TRPM3 is an ion channel that is highly expressed in nociceptive neurons and plays a key role in pain perception. In the presence of the endogenous TRPM3 ligand, pregnenolone sulfate (PS), the antifungal compound clotrimazole (Clt) augments Ca2+ signaling and opens a non-canonical pore, permeable to Na+, which aggravates TRPM3-induced pain. To date, little is known about structural features that govern the Clt modulatory effect of TRPM3. Here, we synthesized and evaluated several Clt analogues in order to gain insights into their structure-activity relationship. Our results reveal a tight SAR with the three phenyl rings on the trityl moiety being essential for the activity, as well as the presence of fluorine or chlorine substituents on the trityl group. Imidazole as a heterocycle is also necessary for activity. Interestingly, we identified a pentafluoro-trityl analogue (29a) that is able to act as a TRPM3 agonist in the absence of PS. The compounds we report in this work will be useful tools for the further study of TRPM3 modulation and its effect on pain perception.

Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System.

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2- and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than does ATP13A2. We also compared the uptake of new green fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure-activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed.

Synthesis, In Vitro Profiling, and In Vivo Evaluation of Benzohomoadamantane-Based Ureas for Visceral Pain: A New Indication for Soluble Epoxide Hydrolase Inhibitors.

The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field.

Combinatorial Optimization of Activity-Based Probes for Acyl Protein Thioesterases 1 and 2.

Acyl protein thioesterases hydrolyze fatty acid thioesters on cysteine residues of proteins. The two protein depalmitoylases APT1 and APT2 have a very high degree of similarity and show substantial overlap in substrate utility. Potent, selective, and cell-permeable activity-based probes are needed to study the role of these enzymes. Here, we employ solid-phase synthesis to create a library of covalent probes based on a triazole urea-reactive electrophile, leading to several potent and cell-permeable probes of human APT1/2. We demonstrate that inhibition of APT1/2 in cells does not have an effect on steady-state levels of protein palmitoylation, implying that substrates hydrolyzed by APT1/2 can also be hydrolyzed by other protein depalmitoylases.

Correction: SuFEx-enabled, chemoselective synthesis of triflates, triflamides and triflimidates.

[This corrects the article DOI: 10.1039/D1SC06267K.].

SuFEx-enabled, chemoselective synthesis of triflates, triflamides and triflimidates.

Sulfur(vi) Fluoride Exchange (SuFEx) chemistry has emerged as a next-generation click reaction, designed to assemble functional molecules quickly and modularly. Here, we report the ex situ generation of trifluoromethanesulfonyl fluoride (CF3SO2F) gas in a two chamber system, and its use as a new SuFEx handle to efficiently synthesize triflates and triflamides. This broadly tolerated protocol lends itself to peptide modification or to telescoping into coupling reactions. Moreover, redesigning the SVI-F connector with a S[double bond, length as m-dash]O → S[double bond, length as m-dash]NR replacement furnished the analogous triflimidoyl fluorides as SuFEx electrophiles, which were engaged in the synthesis of rarely reported triflimidate esters. Notably, experiments showed H2O to be the key towards achieving chemoselective trifluoromethanesulfonation of phenols vs. amine groups, a phenomenon best explained-using ab initio metadynamics simulations-by a hydrogen bonded termolecular transition state for the CF3SO2F triflylation of amines.

Stress-induced inflammation evoked by immunogenic cell death is blunted by the IRE1α kinase inhibitor KIRA6 through HSP60 targeting.

Mounting evidence indicates that immunogenic therapies engaging the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress favor proficient cancer cell-immune interactions, by stimulating the release of immunomodulatory/proinflammatory factors by stressed or dying cancer cells. UPR-driven transcription of proinflammatory cytokines/chemokines exert beneficial or detrimental effects on tumor growth and antitumor immunity, but the cell-autonomous machinery governing the cancer cell inflammatory output in response to immunogenic therapies remains poorly defined. Here, we profiled the transcriptome of cancer cells responding to immunogenic or weakly immunogenic treatments. Bioinformatics-driven pathway analysis indicated that immunogenic treatments instigated a NF-κB/AP-1-inflammatory stress response, which dissociated from both cell death and UPR. This stress-induced inflammation was specifically abolished by the IRE1α-kinase inhibitor KIRA6. Supernatants from immunogenic chemotherapy and KIRA6 co-treated cancer cells were deprived of proinflammatory/chemoattractant factors and failed to mobilize neutrophils and induce dendritic cell maturation. Furthermore, KIRA6 significantly reduced the in vivo vaccination potential of dying cancer cells responding to immunogenic chemotherapy. Mechanistically, we found that the anti-inflammatory effect of KIRA6 was still effective in IRE1α-deficient cells, indicating a hitherto unknown off-target effector of this IRE1α-kinase inhibitor. Generation of a KIRA6-clickable photoaffinity probe, mass spectrometry, and co-immunoprecipitation analysis identified cytosolic HSP60 as a KIRA6 off-target in the IKK-driven NF-κB pathway. In sum, our study unravels that HSP60 is a KIRA6-inhibitable upstream regulator of the NF-κB/AP-1-inflammatory stress responses evoked by immunogenic treatments. It also urges caution when interpreting the anti-inflammatory action of IRE1α chemical inhibitors.