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Acute lymphoblastic leukaemia is currently treated with bacterial L-asparaginase; however, its side effects raise the need for the development of improved and efficient novel enzymes. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice treated with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, through in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. In addition, our single mutants were highly stable in biological environment simulation, unlike the double-mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have a higher cytotoxic effect than WT on T-cell-derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological responses and to study the enzymes' pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C elicited lower physiological changes and immune/allergenic responses. In relation to the pharmacokinetic profile, S206C exhibited twofold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and fewer side effects. [ABSTRACT FROM AUTHOR]

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) plays an important role in viral replication and transcription and received great attention as a vital target for drug/peptide development. Therapeutic agents such as small‐molecule drugs or peptides that interact with the Cys–His present in the catalytic site of Mpro are an efficient way to inhibit the protease. Although several emergency‐approved vaccines showed good efficacy and drastically dropped the infection rate, evolving variants are still infecting and killing millions of people globally. While a small‐molecule drug (Paxlovid) received emergency approval, small‐molecule drugs have low target specificity and higher toxicity. Besides small‐molecule drugs, peptide therapeutics are thus gaining increasing popularity as they are easy to synthesize and highly selective and have limited side effects. In this study, we investigated the therapeutic value of 67 peptides targeting Mpro using molecular docking. Subsequently, molecular dynamics (MD) simulations were implemented on eight protein–peptide complexes to obtain molecular‐level information on the interaction between these peptides and the Mpro active site, which revealed that temporin L, indolicidin, and lymphocytic choriomeningitis virus (LCMV) GP1 are the best candidates in terms of stability, interaction, and structural compactness. These peptides were synthesized using the solid‐phase peptide synthesis protocol, purified by reversed‐phase high‐performance liquid chromatography (RP‐HPLC), and authenticated by mass spectrometry (MS). The in vitro fluorometric Mpro activity assay was used to validate the computational results, where temporin L and indolicidin were observed to be very active against SARS‐CoV‐2 Mpro with IC50 values of 38.80 and 87.23 μM, respectively. A liquid chromatography–MS (LC–MS) assay was developed, and the IC50 value of temporin L was measured at 23.8 μM. The solution‐state nuclear magnetic resonance (NMR) structure of temporin L was determined in the absence of sodium dodecyl sulfate (SDS) micelles and was compared to previous temporin structures. This combined investigation provides critical insights and assists us to further develop peptide inhibitors of SARS‐CoV‐2 Mpro through structural guided investigation. [ABSTRACT FROM AUTHOR]

Platelets are necessary for maintaining haemostasis. Separately, platelets are important for the propagation of inflammation during the host immune response against infection. The activation of platelets also causes inappropriate inflammation in various disease pathologies, often in the absence of changes to haemostasis. The separate functions of platelets during inflammation compared with haemostasis are therefore varied and this will be reflected in distinct pathways of activation. The activation of platelets by the nucleotide adenosine diphosphate (ADP) acting on P2Y1 and P2Y12 receptors is important for the development of platelet thrombi during haemostasis. However, P2Y1 stimulation of platelets is also important during the inflammatory response and paradoxically in scenarios where no changes to haemostasis and platelet aggregation occur. In these events, Rho‐GTPase signalling, rather than the canonical phospholipase Cβ (PLCβ) signalling pathway, is necessary. We describe our current understanding of these differences, reflecting on recent advances in knowledge of P2Y1 structure, and the possibility of biased agonism occurring from activation via other endogenous nucleotides compared with ADP. Knowledge arising from these different pathways of P2Y1 stimulation of platelets during inflammation compared with haemostasis may help therapeutic control of platelet function during inflammation or infection, while preserving essential haemostasis. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non‐thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc [ABSTRACT FROM AUTHOR]

Background and Purpose: Platelet function during inflammation is dependent on activation by endogenous nucleotides. Non‐canonical signalling via the P2Y1 receptor is important for these non‐thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+, ADP‐ribose, and Up4A on platelet functions contributing to inflammation or haemostasis. Experimental Approach: Platelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+, ADP‐ribose, or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed. Key Results: Platelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+, ADP‐ribose, and Up4A. However, these endogenous nucleotides induced P2Y1‐dependent platelet chemotaxis, an effect that required RhoA and Rac‐1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket for Ap3A, NAD+, ADP‐ribose, or Up4A compared with ADP. Conclusion and Implications: Platelet function (aggregation vs motility) can be differentially modulated by biased‐agonist activation of P2Y1 receptors. This may be due to the character of the ligand‐binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant‐platelet drugs. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non‐thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc [ABSTRACT FROM AUTHOR]