Biology subjects

Drag, M.

Publications and source records attributed to Drag, M..

18 recordsLinked to original sources

bioRxiv2025

Engineering cathepsin S selective chemical probes and antibody-drug conjugates through substrate profiling with unnatural amino acids

Cysteine cathepsins, particularly cathepsin S, are important regulators of proteolytic signaling in health and disease, including cancer progression and immune modulation. Despite their therapeutic relevance, selective chemical tools to study individual cathepsins remain limited due to overlapping substrate preferences. Here we present design of cathepsin S selective chemical probes and cathepsin S-cleavable antibody-drug conjugates (ADCs) through substrate profiling with unnatural amino acids. First, Hybrid Combinatorial Substrate Library (HyCoSuL) technology incorporating a broad spectrum of unnatural amino acids was applied to comprehensively investigate the substrate specificity of cathepsin S. This approach enabled the identification of highly selective tetrapeptide motifs that served as scaffolds for the design of optimized fluorogenic substrates, irreversible inhibitors, and fluorescent activity-based probes (ABPs). These tools demonstrated high selectivity toward cathepsin S over closely related family members in both biochemical and cellular settings. We then translated these findings to develop cathepsin S-activated ADCs, incorporating the optimized peptide motifs as protease-cleavable linkers for targeted payload release. Using these linkers, we further generated MMAE-based antibody-drug conjugates directed against HER-2 and the TROP-2 proteins, demonstrating cathepsin S-dependent cytotoxicity in HER-2-positive as well as HER-2-negative/TROP-2-positive breast cancer models. Finally, we employed anti-cathepsin S antibodies in combination with mass cytometry (CyTOF) to assess the spatial distribution of cathepsin S expression in breast cancer patient samples. By correlating cathepsin S levels with tumor-associated markers such as TROP-2 and HER-2, we identified potential co-expression patterns that support the rationale for personalized therapy using antibody-drug conjugates selectively activated by cathepsin S. This integrative approach provides a comprehensive platform for profiling cathepsin S activity at the molecular, cellular, and tissue levels, with broad implications for the development of precision therapeutics and diagnostic strategies in oncology.

cancer biology↗

bioRxiv2025

Delivery of caspase inhibitors through GSDMD pores to inhibit pyroptosis

Caspase-1, -4, -5, and -11 activate Gasdermin D (GSDMD) to form pores in the plasma membrane. In addition to releasing interleukin (IL)-1{beta} and IL-18, GSDMD pores cause a lytic, proinflammatory form of cell death called pyroptosis. Blocking this pathway holds therapeutic promise for the treatment of inflammatory disorders, but clinical trials of cell permeable caspase inhibitors have been unsuccessful. Here, we describe covalent inhibitors of proinflammatory caspases that are impermeable to healthy cells but effectively block caspase-1 driven pyroptosis and IL-1{beta} secretion. Their failure to inhibit apoptosis implies inhibitor entry through GSDMD pores. Propidium iodide entered rescued cells, confirming transient membrane permeabilization via caspase-1 and GSDMD. Caspase-1 inhibition prevented rather than delayed cell death, likely due to the activation of membrane repair mechanisms neutralizing the initial GSDMD pores. Notably, inhibiting caspase-1 and -11 suppressed IL-1{beta} and IL-18 production in a mouse model of endotoxic shock. These findings underscore the therapeutic potential of exploiting GSDMD pores for the delivery of caspase inhibitors, offering a novel strategy for treating inflammatory diseases.

cell biology↗

bioRxiv2025

A Robust Fluorogenic Substrate for Chikungunya Virus Protease (nsP2) Activity

Chikungunya virus (CHIKV) is an emerging pathogen with pandemic potential. CHIKV infection in humans is transmitted by mosquitoes and induces common symptoms of high fever, arthralgia and myalgia. Because no specific antiviral drugs for treatment of CHIKV infection are available, drug development remains a central goal. The chikungunya virus protease nsP2 (CHIKVP) has emerged as a key drug target due to its indispensable role in viral replication via cleavage of the viral polyprotein. To date, effective tools for screening for CHIKVP inhibitors that reflect the most critical polyprotein cleavage sites have been lacking, hampering drug-development efforts. We found that the recognition ability of CHIKVP is sensitive to the length of peptide substrates. In this study, we report a robust fluorogenic substrate comprising a 15-mer peptide derived from the nsP3/4 junction from the CHIKV polyprotein. This peptide is flanked by an ACC-Lys(dnp) donor-quencher pair. Our new substrate acc-CHIK15-dnp shows a 20-fold improved signal-to-noise ratio as compared to the previously reported edab8 substrate, which is also based on the nsP3/4 junction. We found acc-CHIK15-dnp is recognized only by CHIKVP but not by other alphavirus proteases. This is surprising due to the high level of sequence conservation in the alpha virus polyprotein junctions and indicates that the P-side residues are more important than the P-side sequence for effective CHIKVP cleavage. The robust signal-to-noise ratio obtained using acc-CHIK15-dnp derived from the nsP3/4 cleavage site enabled much improved small molecule HTS on CHIKV relative to other fluorogenic reporters.

biochemistry↗

bioRxiv2024

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating interferon-inducible ubiquitin-like (Ubl) modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) capable of selectively detecting USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) by incorporating unnatural amino acids into the C-terminal tail of ISG15. Combining with a ubiquitin-based DUB ABP, the selective USP18 ABP is employed in a chemoproteomic screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.

molecular biology↗

bioRxiv2023

Thermoprotection by a cell membrane-localized metacaspase in a green alga

Caspases are restricted to animals, while other organisms, including plants possess metacaspases (MCAs), a more ancient and broader class of structurally-related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis expressing nine MCAs with partly redundant activities. In contrast to streptophytes, most chlorophytes contain only one or two hitherto uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigate CrMCA-II, a single type II MCA from a model chlorophyte Chlamydomonas reinhardtii. Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlates with the dimerization. Most of CrMCA-II in the cell is present as a zymogen attached to the plasma membrane (PM). Deletion of CrMCA-II by CRISPR/Cas9 compromises thermotolerance leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restores thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we link the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

plant biology↗

bioRxiv2023

SARS-CoV-2 Mpro protease variants of concern display altered viral and host target processing but retain potency towards antivirals

Main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOC) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and subsequent significant decrease in cytokine secretion, providing evidence for alterations in escape of host-antiviral mechanisms. Accordingly, mutations associated with the highly virulent Delta VOC resulted in significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

biochemistry↗

bioRxiv2020

Mapping the substrate sequence and length of the Plasmodium M1 and M17 aminopeptidases

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dualtarget anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. When tested with a panel of peptides of increasing length, each aminopeptidase exhibited unique catalytic behavioral responses to the increase in peptide length, although all six aminopeptidases exhibited an increase in cooperativity as peptide length increased. Overall the P. vivax and P. berghei enzymes were generally faster than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.

biochemistry↗

bioRxiv2020

Ebselen derivatives are very potent dual inhibitors of SARS-CoV-2 proteases - PLpro and Mpro in in vitro studies

Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage - a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and anti-viral assays. In this study, we screened a collection of 23 ebselen derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Our work shows that ebselen constitutes a promising platform for development of new antiviral agents targeting both SARS-CoV-2 PLpro and Mpro.

biochemistry↗

bioRxiv2020

Design of optical imaging probes by screening of diverse substrate libraries directly in disease tissue extracts

Fluorescently-quenched probes that are specifically activated in the cancer microenvironment have great potential application for diagnosis, early detection and surgical guidance. These probes are often designed to target specific enzymes associated with disease by direct optimization using single purified targets. However, this can result in painstaking chemistry efforts to produce a probe with suboptimal performance when applied in vivo. We describe here an alternate, unbiased activity-profiling approach in which whole tissue extracts are used to directly identify optimal peptide sequences for probe design. Screening of mouse mammary tumor extracts with a hybrid combinatorial substrate library (HyCoSuL) identified a combination of natural and non-natural amino acid residues that could be used to generate highly efficient tumor-specific fluorescently quenched substrate probes. The most effective probe is significantly brighter than any of our previously reported tumor imaging probes designed for specific proteases and robustly discriminates tumor tissue from adjacent healthy tissue in a mouse model of cancer. Importantly, although the probes were developed by screening mouse mammary tumor tissues, they are able to effectively distinguish human ductal carcinomas from normal breast tissue with similar reactivity profiles to those observed in mouse tissues. This new strategy simplifies and enhances the process of probe optimization by direct screening in a tissue of interest without any a priori knowledge of enzyme targets. It has the potential to be applied to advance the development of probes for diverse disease states for which clinical or animal model tissues are available.

bioengineering↗

bioRxiv2020

Activity profiling of SARS-CoV-2-PLpro protease provides structural framework for anti-COVID-19 drug design

In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning.

biochemistry↗

bioRxiv2020

Substrate specificity profiling of SARS-CoV-2 Mpro protease provides basis for anti-COVID-19 drug design

In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed in Wuhan, China. Due to international travel and human-to-human transmission, the virus spread rapidly inside and outside of China. Currently, there is no effective antiviral treatment for coronavirus disease 2019 (COVID-19); therefore, research efforts are focused on the rapid development of vaccines and antiviral drugs. The SARS-CoV-2 main protease constitutes one of the most attractive antiviral drug targets. To address this emerging problem, we have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases, using natural and a large panel of unnatural amino acids. On the basis of these findings, we designed and synthesized an inhibitor and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. Using this approach we visualized SARS-CoV-2 active Mpro within nasopharyngeal epithelial cells of a patient with active COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents or diagnostic tests.

biochemistry↗

bioRxiv2020

Engineered unnatural ubiquitin for optimal detection of deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) are responsible for removing ubiquitin (Ub) from its protein conjugates. DUBs have been implicated as attractive therapeutic targets in the treatment of viral diseases, neurodegenerative disorders and cancer. The lack of selective chemical tools for the exploration of these enzymes significantly impairs the determination of their roles in both normal and pathological states. Commercially available fluorogenic substrates are based on the C-terminal Ub motif or contain Ub coupled to a fluorophore (Z-LRGG-AMC, Ub-AMC); therefore, these substrates suffer from lack of selectivity. By using a hybrid combinatorial substrate library (HyCoSuL) and a defined P2 library containing a wide variety of nonproteinogenic amino acids, we established a full substrate specificity profile for two DUBs--MERS PLpro and human UCH-L3. Based on these results, we designed and synthesized Ub-based substrates and activity-based probes (ABPs) containing selected unnatural amino acids located in the C-terminal Ub motif. Biochemical analysis and cell-based experiments confirmed the activity and selectivity of engineered Ub-based substrates and probes. Using this approach, we propose that for any protease that recognizes Ub and Ub-like substrates, a highly active and selective unnatural substrate or probe can be engineered.

biochemistry↗

bioRxiv2019

Noninvasive optical detection of Granzyme B from natural killer cells using enzyme-activated fluorogenic probes

Despite many studies on the cytotoxic protease granzyme B, key aspects of its function remain unexplored due to the lack of selective probes for its activity. In this study, we fully mapped the substrate preferences of GrB using a set of unnatural amino acids, demonstrating previously unknown GrB substrate preferences that we then used to design novel substrate-based inhibitors and a GrB-activatable activity-based probe. We showed that our GrB probes react poorly with caspases, making them ideal for the in-depth analysis of GrB localization and function in cells. With our quenched fluorescence substrate, we determined GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with the GrB attack MDA-MB-231 target cells, and active GrB influences its target cell killing efficiency.

biochemistry↗

bioRxiv2019

Detection of active Granzyme A in NK92 cells with fluorescent activity-based probe

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation; and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.

biochemistry↗

bioRxiv2019

Tissue-ABPP enables high-resolution confocal fluorescence imaging of serine hydrolase activity in cryosections - Application to glioma brain unveils activity hotspots originating from tumor-associated neutrophils

Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. The ABPP approach utilizes cell/tissue proteomes and features the FP warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Here, we advance the ABPP methodology to glioma brain cryosections, enabling high-resolution confocal fluorescence imaging of SH activity in different cell types of the tumor microenvironment, identified by using extensive immunohistochemistry on activity probe labeled sections. We name this technique tissue-ABPP to distinguish it from conventional gel-based ABPP. We show heightened SH activity in glioma vs. normal brain and unveil activity hotspots originating from tumor-associated neutrophils. Thorough optimization and validation is provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Tissue-ABPP enables a wide range of applications for confocal imaging of SH activity in any type of tissue or animal species.

cancer biology↗

bioRxiv2019

The Activome: multiplexed probing of activity of proteolytic enzymes using mass cytometry-compatible activity-based probes (TOF-probes)

The activome can be considered as a subset of the proteome that contains enzymes in their catalytically active form and can be interrogated by using probes targeted towards individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, simultaneous analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry. Using lanthanide-based tags instead of fluorophores gives us the ability to monitor the activity of multiple proteases in parallel. As proof of concept we developed a panel of cathepsin and legumain specific probes and showed that we were able to identify an activome of these proteases in two cell lines and peripheral blood mononuclear cells, providing a framework for the use of mass cytometry for multiplexed enzyme activity detection.

biochemistry↗

bioRxiv2019

Leveraging peptide substrate libraries to design inhibitors of bacterial Lon protease

Lon is a widely-conserved housekeeping protease found in all domains of life. Bacterial Lon is involved in the recovery from various types of stress, including tolerance to fluoroquinolone antibiotics, and is linked to pathogenesis in a number of organisms. However, detailed functional studies of Lon have been limited by the lack of selective, cell-permeable inhibitors. Here we describe the use of positional scanning libraries of hybrid peptide substrates to profile the primary sequence specificity of bacterial Lon. In addition to identifying optimal natural amino acid binding preferences, we identified several non-natural residues that were leveraged to develop optimal peptide substrates as well as a potent peptidic boronic acid inhibitor of Lon. Treatment of E. coli with this inhibitor promotes UV-induced filamentation and reduces tolerance to ciprofloxacin, phenocopying established lon-deletion phenotypes. It is also non-toxic to mammalian cells due to its increased selectivity for Lon over the proteasome. Our results provide new insight into the primary substrate specificity of Lon and identify substrates and an inhibitor that will serve as useful tools for dissecting the diverse cellular functions of Lon.

microbiology↗

bioRxiv2018

Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies different amino acid preferences between peptide-based substrates and inhibitors.

Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of non-optimal peptide substrates. Importantly, analysis of previously published data about the specificity of other proteases also unveiled significant discrepancies in the amino acid preference between substrates and inhibitors. In this article, we also discuss important structural and theoretical reasons that might account for these discrepancies. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds.

biochemistry↗