On the size of the active site in proteases. I. Papain
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Cited by (4944)
Characterisation of ten NS2B-NS3 proteases: Paving the way for pan-flavivirus drugs
2024, Antiviral ResearchFlaviviruses can cause severe illness in humans. Effective and safe vaccines are available for some species; however, for many flaviviruses disease prevention or specific treatments remain unavailable. The viral replication cycle depends on the proteolytic activity of the NS2B-NS3 protease, which releases functional viral proteins from a non-functional polyprotein precursor, rendering the protease a promising drug target. In this study, we characterised recombinant NS2B-NS3 proteases from ten flaviviruses including three unreported proteases from the Usutu, Kyasanur forest disease and Powassan viruses. All protease constructs comprise a covalent Gly4-Ser-Gly4 linker connecting the NS3 serine protease domain with its cofactor NS2B. We conducted a comprehensive cleavage site analysis revealing areas of high conversion. While all proteases were active in enzymatic assays, we noted a 1000-fold difference in catalytic efficiency across proteases from different flaviviruses. Two bicyclic peptide inhibitors displayed anti-pan-flaviviral protease activity with inhibition constants ranging from 10 to 1000 nM.
Azapeptides with unique covalent warheads as SARS-CoV-2 main protease inhibitors
2024, Antiviral ResearchThe main protease (MPro) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target MPro's catalytic cysteine. Our characterization identified potent MPro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC50 value of 10 nM against SARS-CoV-2 infection in ACE2+ A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent MPro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19.
Thrombin has dual trypsin-like and chymotrypsin-like specificity
2024, Journal of Thrombosis and HaemostasisThe residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates.
To establish if thrombin can cleave at Trp residues.
The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage.
Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond.
The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.
An N terminomics toolbox combining 2-pyridinecarboxaldehyde probes and click chemistry for profiling protease specificity
2024, Cell Chemical BiologyProteomic profiling of protease-generated N termini provides key insights into protease function and specificity. However, current technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an N terminomics toolbox that combines selective N-terminal biotinylation using 2-pyridinecarboxaldehyde (2PCA) reagents with chemically cleavable linkers to enable efficient enrichment of protein N termini. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 (Proteomic Identification of Cleavage Sites with 2PCA) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER (chemical enrichment of protease substrates with purchasable, elutable reagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology.
Development, preclinical evaluation and preliminary dosimetry profiling of SB03178, a first-of-its-kind benzo[h]quinoline-based fibroblast activation protein-α-targeted radiotheranostic for cancer imaging and therapy
2024, European Journal of Medicinal ChemistryFibroblast activation protein-α (FAP) is a marker of cancer-associated fibroblasts (CAFs) that constitute a significant portion of most carcinomas. Since it plays a critical role in tumor growth and metastasis, its timely detection to identify tumor lesions in early developmental stages using targeted radiopharmaceuticals has gained significant impetus. In the present work, two novel FAP-targeted precursors SB03178 and SB04033 comprising of an atypical benzo[h]quinoline construct were synthesized and either chelated to diagnostic radionuclide gallium-68 or therapeutic radionuclide lutetium-177, with ≥90% radiochemical purities and 22–76% decay-corrected radiochemical yields. natGa-labeled complexes displayed dose-dependent FAP inhibition, with binding potency of natGa-SB03178 being ∼17 times higher than natGa-SB04033. To evaluate their pharmacokinetic profiles, PET imaging and ex vivo biodistribution analyses were executed in FAP-overexpressing HEK293T:hFAP tumor-bearing mice. While both tracers displayed clear tumor visualization that was primarily FAP-arbitrated, with negligible uptake in most peripheral tissues, [68Ga]Ga-SB03178 demonstrated higher tumor uptake and superior tumor-to-background contrast ratios than [68Ga]Ga-SB04033. 177Lu-labeled SB03178 was subjected to tumor retention studies, mouse dosimetry profiling and mouse-to-human dose extrapolations also using the HEK293T:hFAP tumor model. [177Lu]Lu-SB03178 exhibited a combination of high and sustained tumor uptake, with excellent tumor-to-critical organ uptake ratios resulting in a high radiation absorbed dose to the tumor and a low estimated whole-body dose to humans. Our preliminary findings are considerably encouraging to support clinical development of [68Ga]Ga-/[177Lu]Lu-SB03178 theranostic pair for use in a vast majority of FAP-overexpressing neoplasms, particularly carcinomas.
Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase–mediated proteolysis of corticosteroid-binding globulin
2024, Journal of Biological ChemistryCorticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure–function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344–Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modeling of a CBG–GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.