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

ATP13A3 is a major component of the enigmatic mammalian polyamine transport system

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG

Activity-Based Protein Profiling of Serine Proteases in Immune Cells

Multiple types of immune cells utilize serine proteases in their mechanisms of defense against pathogens or altered host cells. Dysregulation of the serine protease activity from these cells underlies different diseases. In the past, the technique of activity-based protein profiling proved to be especially useful for the study of proteases, and various studies have used small-molecule activity-based probes to covalently label and detect serine proteases from immune cells. In this review, we give an overview of the different activity-based probes that have been designed for serine proteases and how their selectivity can be steered. We also discuss how these have been utilized in the detection of various serine proteases from immune cells by different analysis methods (gel electrophoresis, microscopy and flow cytometry) and what biological insights these studies have produced. Overall, activity-based protein profiling has the potential to address functional aspects of serine proteases in the immune system and future efforts may bring translation into clinical application.status: publishe

Rapid Solid-Phase Construction of Serine Hydrolase Probes Results in Selective Activity-Based Probes for Acyl Protein Thioesterases-1/2.

Serine hydrolases (SHs) are a large, diverse family of enzymes that play various biomedically important roles. Their study has been substantially advanced by activity-based protein profiling, which makes use of covalent chemical probes for labeling the active site and detection by various methodologies. However, highly selective probes for individual SHs are scarce because probe synthesis usually takes place by time-consuming solution phase chemistry. We here report a general solid-phase synthesis toward SH chemical probes, which will speed up probe library synthesis. It involves the construction of a recognition element ending in a secondary amine followed by capping with different electrophiles. We illustrate the power of this approach by the discovery of selective chemical probes for the depalmitoylating enzymes APT-1/2. Overall, this study reports new methodologies to synthesize SH probes, while providing new reagents to study protein depalmitoylation.status: publishe

Phospholipids. In: Nollet, L. (ed.). . Marcel Dekker, New York

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Recent Advances in Activity-Based Protein Profiling of Proteases

The activity of proteases is tightly regulated, and dysregulation is linked to a variety of human diseases. For this reason, ABPP is a well-suited method to study protease biology and the design of protease probes has pushed the boundaries of ABPP. The development of highly selective protease probes is still a challenging task. After an introduction, the first section of this chapter discusses several strategies to enable detection of a single active protease species. These range from the usage of non-natural amino acids, combination of probes with antibodies, and engineering of the target proteases. A next section describes the different types of detection tags that facilitate the read-out possibilities including various types of imaging methods and mass spectrometry-based target identification. The power of protease ABPP is illustrated by examples for a selected number of proteases. It is expected that some protease probes that have been evaluated in animal models of human disease will find translation into clinical application in the near future.status: publishe

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 and structure-activity relationship studies of benzo[b][1,4]oxazin-3(4H)-one analogues as inhibitors of mycobacterial thymidylate synthase

Since the discovery of a flavin-dependent thymidylate synthase (ThyX or FDTS) that is absent in humans but crucial for DNA biosynthesis in a diverse group of pathogens, the enzyme has been pursued for the development of new antibacterial agents against Mycobacterium tuberculosis, the causative agent of the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous screening efforts and report herein an optimization campaign of a novel series of inhibitors with a unique inhibition profile. The inhibitors display competitive inhibition toward the methylene tetrahydrofolate cofactor of ThyX, enabling us to generate a model of the compounds bound to their target, thus offering insight into their structure-activity relationships.status: Published onlin

Synthesis and Structure–Activity Relationship Studies of Benzo[ b ][1,4]oxazin‐3(4 H )‐one Analogues as Inhibitors of Mycobacterial Thymidylate Synthase X

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