Investigation of the P1' and P2' sites of IQF substrates and their selectivity toward 20S proteasome subunits.

High levels of expression and activity of the 20S proteasome have been linked to many types of pathologies, including neoplasia, autoimmune disorders, neurodegenerative diseases and many more. Moreover, distinguishing between 20S proteasome catalytic subunits is neglected, although it may provide further insight into the development of pathologies. Several approaches have been developed to detect 20S proteasome activity, one of which is internally quenched fluorescent (IQF) substrates, which currently suffer from low efficiency and sensitivity. Previous reports focused on peptides including natural amino acids; therefore, in this report, we synthesized and analyzed IQF substrates with both natural and unnatural amino acids in the P1' and P2' positions to investigate their influences on selectivity toward 20S proteasome subunits. We found that elongation of the substrate by the P1' and P2' positions increased specificity in comparison to tetrapeptides. Moreover, we were able to obtain IQF substrates for the Ch-L subunit, which was characterized by higher selectivity than formerly used tetrapeptides. These findings may further contribute to the development of novel diagnostic tools for 20S proteasome-dependent disorders.

SARS-CoV-2 Mpro inhibitors and activity-based probes for patient-sample imaging.

In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed. Currently, there is no effective antiviral treatment for COVID-19. To address this emerging problem, we focused on the SARS-CoV-2 main protease that constitutes one of the most attractive antiviral drug targets. 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. On the basis of these findings, we designed and synthesized a potent SARS-CoV-2 inhibitor (Ac-Abu-DTyr-Leu-Gln-VS, half-maximal effective concentration of 3.7 µM) and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. We visualized active SARS-CoV-2 Mpro in nasopharyngeal epithelial cells of patients suffering from COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents and/or diagnostic tests.

Multiplexed Probing of Proteolytic Enzymes Using Mass Cytometry-Compatible Activity-Based Probes.

The subset of the proteome that contains enzymes in their catalytically active form can be interrogated by using probes targeted toward 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, multiplex analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry giving us the ability to monitor the activity of multiple proteases in parallel. Using these probes, we were able to identify the distribution of four proteases with different active site geometries in three cell lines and peripheral blood mononuclear cells. This provides a framework for the use of mass cytometry for multiplexed enzyme activity detection.

Small Molecule Active Site Directed Tools for Studying Human Caspases.

Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes.

Structure and substrate fingerprint of aminopeptidase P from Plasmodium falciparum.

Malaria is one of the world's most prevalent parasitic diseases, with over 200 million cases annually. Alarmingly, the spread of drug-resistant parasites threatens the effectiveness of current antimalarials and has made the development of novel therapeutic strategies a global health priority. Malaria parasites have a complicated lifecycle, involving an asymptomatic 'liver stage' and a symptomatic 'blood stage'. During the blood stage, the parasites utilise a proteolytic cascade to digest host hemoglobin, which produces free amino acids absolutely necessary for parasite growth and reproduction. The enzymes required for hemoglobin digestion are therefore attractive therapeutic targets. The final step of the cascade is catalyzed by several metalloaminopeptidases, including aminopeptidase P (APP). We developed a novel platform to examine the substrate fingerprint of APP from Plasmodium falciparum (PfAPP) and to show that it can catalyze the removal of any residue immediately prior to a proline. Further, we have determined the crystal structure of PfAPP and present the first examination of the 3D structure of this essential malarial enzyme. Together, these analyses provide insights into potential mechanisms of inhibition that could be used to develop novel antimalarial therapeutics.

Human 20S proteasome activity towards fluorogenic peptides of various chain lengths.

The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal.

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

Recent advances and concepts in substrate specificity determination of proteases using tailored libraries of fluorogenic substrates with unnatural amino acids.

Substrate specificity of proteases can be determined using several methods among which the most frequently used are positional scanning library, proteomics and phage display. Classic approaches can deliver information about preferences for natural amino acids in binding pockets of virtually all proteases. However, recent studies demonstrate the ability to obtain much more information by application of unnatural amino acids to positional scanning library approaches. This knowledge can be used for the design of more active and specific substrates, inhibitors and activity based probes. In this minireview we describe recent strategies and concepts for the design and application of fluorogenic substrates library tailored for exopeptidases and endopeptidases.

Differential specificity of SARS-CoV-2 main protease variants on peptide versus protein-based substrates.

The SARS-CoV-2 main protease (Mpro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.

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.

Design and Synthesis of Ubiquitin-Based Chemical Tools with Unnatural Amino Acids for Selective Detection of Deubiquitinases.

Several chemical approaches have been applied to develop Ub-based substrates and probes selective toward one or a narrow subset of deubiquitinases (DUBs). Since DUBs are highly specific toward ubiquitin and exhibit low activity toward shorter peptides, it is challenging to design truly selective chemical tools to investigate one DUB in biological samples. Incorporating amino acids other than canonical LRG at the P4-P2 positions in the Ub improves DUB activity and selectivity toward Ub derivatives. Here, we describe the protocol for identifying selective peptide sequences using a hybrid combinatorial substrate library (HyCoSuL) approach that can be introduced in the C-terminal motif of Ub. Furthermore, we describe the synthesis protocol of Ub-based probes and substrates containing unnatural amino acids and the application of Ub-based probes to detect DUBs in cell lysates.

Mapping the substrate-binding subsite specificity of a Porphyromonas gingivalis Tpr peptidase.

Calcium-dependent peptidases of the calpain family are widespread in eukaryotes but uncommon in prokaryotes. A few bacterial calpain homologs have been discovered but none of them have been characterized in detail. Here we present an in-depth substrate specificity analysis of the bacterial calpain-like peptidase Tpr from Porphyromonas gingivalis. Using the positional scanning hybrid combinatorial substrate library method, we found that the specificity of Tpr peptidase differs substantially from the papain family of cysteine proteases, showing a strong preference for proline residues at positions P2 and P3. Such a degree of specificity indicates that this P. gingivalis cell-surface peptidase has a more sophisticated role than indiscriminate protein degradation to generate peptide nutrients, and may fulfil virulence-related functions such as immune evasion.

SARS-CoV-2 Mpro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals.

The 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 the 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 (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the 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 a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a 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.

Ebselen derivatives inhibit SARS-CoV-2 replication by inhibition of its essential proteins: PLpro and Mpro proteases, and nsp14 guanine N7-methyltransferase.

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 antiviral assays. In this study, we screened a collection of 34 ebselen and ebselen diselenide 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. Independently, ebselen was shown to inhibit the N7-methyltransferase activity of SARS-CoV-2 nsp14 protein involved in viral RNA cap modification. Hence, selected compounds were also evaluated as nsp14 inhibitors. In the second part of our work, we employed 11 ebselen analogues-bis(2-carbamoylaryl)phenyl diselenides-in biological assays to evaluate their anti-SARS-CoV-2 activity in Vero E6 cells. We present their antiviral and cytoprotective activity and also low cytotoxicity. Our work shows that ebselen, its derivatives, and diselenide analogues constitute a promising platform for development of new antivirals targeting the SARS-CoV-2 virus.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Evaluation of the anti-SARS-CoV-2 properties of essential oils and aromatic extracts.

Essential oils and aromatic extracts (oleoresins, absolutes, concretes, resinoids) are often used as food flavorings and constituents of fragrance compositions. The flavor and fragrance industry observed significant growth in the sales of some natural materials during the COVID-19 outbreak. Some companies worldwide are making false claims regarding the effectiveness of their essential oils or blends (or indirectly point toward this conclusion) against coronaviruses, even though the available data on the activity of plant materials against highly pathogenic human coronaviruses are very scarce. Our exploratory study aimed to develop pioneering knowledge and provide the first experimental results on the inhibitory properties of hundreds of flavor and fragrance materials against SARS-CoV-2 main and papain-like proteases and the antiviral potential of the most active protease inhibitors. As essential oils are volatile products, they could provide an interesting therapeutic strategy for subsidiary inhalation in the long term.

A molecular sensor determines the ubiquitin substrate specificity of SARS-CoV-2 papain-like protease.

The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences. Using a combination of structural analyses and functional assays, we identify a molecular sensor within the S1 Ub-binding site of PLpro that serves as a key determinant of substrate specificity. Variations within the S1 sensor specifically alter cleavage of Ub substrates but not of the Ubl interferon-stimulated gene 15 protein (ISG15). Significantly, a variant of concern associated with immune evasion carries a mutation in the S1 sensor that enhances PLpro activity on Ub substrates. Collectively, our data identify the S1 sensor region as a potential hotspot of variability that could alter host antiviral immune responses to newly emerging SARS-CoV-2 lineages.

Re-emerging Aspartic Protease Targets: Examining Cryptococcus neoformans Major Aspartyl Peptidase 1 as a Target for Antifungal Drug Discovery.

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CLpro substrate degradome.

The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CLpro by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLpro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CLpro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.

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 lysate 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.