Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis

Wüstenhagen, Doreen A.; Lukas, Phil; Müller, Christian; Aubele, Simone A.; Hildebrandt, Jan‐Peter; Kubick, Stefan

doi:10.1038/s41598-020-76715-w

Cited by 8 publications

(8 citation statements)

References 79 publications

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“…Despite HLF1 has no antithrombotic activity in previous studies, Muller et al found that similar thrombin-inhibiting activity to hirudins could be observed in HLF1 after N-terminal modification. Additionally, analogous phenomena could be found in HLF3 and HLF4 through modifications within the N-termini in combination with an exchange of the central globular domain (Muller et al, 2020) . These results suggested that the N-and C-termini as well as the central globular domains are crucial for hirudin and HLFs to exert antithrombotic activity.…”

Section: Hirudin-like Factorsmentioning

confidence: 56%

“…In recent decades, the hosts for expressing rH are also increasing, which vary from Bacillus subtilis (Chen et al, 2011) to Lactococcus lactis (Lv et al, 2012). rH can be produced not only by bacterial or yeast expression systems, but also by a cell-free protein synthesis approach, which can express rH with higher antithrombin activity (Wustenhagen et al, 2020). Although these methods can increase the expression of recombinant hirudin, the thrombin inhibition constant is still lower than that of natural hirudin, mainly because it lacks the tyrosine residue sulfated at site 63 (Nowak and Schror, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review

et al. 2021

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Hirudin, an acidic polypeptide secreted by the salivary glands of Hirudo medicinalis (also known as “Shuizhi” in traditional Chinese medicine), is the strongest natural specific inhibitor of thrombin found so far. Hirudin has been demonstrated to possess potent anti-thrombotic effect in previous studies. Recently, increasing researches have focused on the anti-thrombotic activity of the derivatives of hirudin, mainly because these derivatives have stronger antithrombotic activity and lower bleeding risk. Additionally, various bioactivities of hirudin have been reported as well, including wound repair effect, anti-fibrosis effect, effect on diabetic complications, anti-tumor effect, anti-hyperuricemia effect, effect on cerebral hemorrhage, and others. Therefore, by collecting and summarizing publications from the recent two decades, the pharmacological activities, pharmacokinetics, novel preparations and derivatives, as well as toxicity of hirudin were systematically reviewed in this paper. In addition, the clinical application, the underlying mechanisms of pharmacological effects, the dose-effect relationship, and the development potential in new drug research of hirudin were discussed on the purpose of providing new ideas for application of hirudin in treating related diseases.

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Section: Hirudin-like Factorsmentioning

confidence: 56%

Section: Introductionmentioning

confidence: 99%

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review

et al. 2021

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“…Hirudin is the most potent natural thrombin inhibitor known [ 16 ]. It was found that the 3-kD part of leech has some shortening of APTT and PT, suggesting that the 3-kD part may have some hemostatic effects, which is an interesting phenomenon, indicating that some hemostatic components may exist in leech.…”

Section: Resultsmentioning

confidence: 99%

Research on ACEI of Low-Molecular-Weight Peptides from Hirudo nipponia Whitman

Zhao

Chen

2022

Molecules

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The renin-angiotensin system (RAS) is the primary pathway for regulating blood pressure in the body, and angiotensin-converting enzymes (ACEs) play a crucial role in it. Hirudo nipponia is an invertebrate that contains a variety of active peptides; however, there are no studies on the ACE inhibitory activity of hirudo. In the present study, our aim was to identify the active peptides in hirudo based on active peptide database analysis, unexpectedly filling the gap in hirudo ACE inhibitory activity research. Prep-HPLC was used to separate the part below 3 kD from hirudo. The peptide composition of the isolates was obtained based on Orbitrap LC-MS. The activity of each group of peptides was predicted by the database and the activity was determined by bioassay. Peptides with validation activity were screened through the database. In total, 337 peptides and 18 peptides matching the NCBI leech protein database were identified. All four fractions showed ACE inhibitory activity, and the IC50 was 0.8266, 0.2708, 0.4432, and 0.1764 mg/mL, respectively. Six screened peptides showed good affinity for ACE. This work reveals for the first time that low-molecular-weight peptides from H. nipponia have ACE inhibitory activity, which can provide a new explanation for leech treatment of hypertension.

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“…32 Nevertheless, the mentioned approaches are quite complex and give a low yield of hirudin, so the scalability of these technologies for future industrial applications is questionable and should be investigated. 33 Alternatively, recombinant hirudin without tyrosine sulfation could be produced on a large scale in prokaryotic 34,35 and eukaryotic expression systems [36][37][38] as well as by a cell-free protein synthesis approach, 39 because of the absence of required sulfotransferases in these expression systems. However, recombinant desulfo-hirudin exhibits a 10-fold decrease in inhibitory potency compared to wildtype hirudin, 24,40 that makes sulfo-hirudin more advantageous to use in the clinic.…”

Section: Introductionmentioning

confidence: 99%

Tyrosine phosphorylation of recombinant hirudin increases affinity to thrombin and antithrombotic activity

Volkova,

Semenyuk

2023

Proteins

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Thrombin is one of the key enzymes of the blood coagulation system and a promising target for the development of anticoagulants. One of the most specific natural thrombin inhibitors is hirudin, contained in the salivary glands of medicinal leeches. The medicinal use of recombinant hirudin is limited because of the lack of sulfation on Tyr63, resulting in a 10‐fold decrease in activity compared to native (sulfated) hirudin. In the present work, a set of hirudin derivatives was tested for affinity to thrombin: phospho‐Tyr63, Tyr63(carboxymethyl)Phe, and Tyr63Glu mutants, which mimic Tyr63 sulfation and Gln65Glu mutant and lysine‐succinylated hirudin, which enhance the overall negative charge of hirudin, as well as sulfo‐hirudin and desulfo‐hirudin as references. Using steered molecular dynamics simulations with subsequent umbrella sampling, phospho‐hirudin was shown to exhibit the highest affinity to thrombin among all hirudin analogs, including native sulfo‐hirudin; succinylated hirudin was also prospective. Phospho‐hirudin exhibited the highest antithrombotic activity in in vitro assay in human plasma. Taking into account the modern methods for obtaining phospho‐hirudin and succinylated hirudin, they are prospective as anticoagulants in clinical practice.

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Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis

Cited by 8 publications

References 79 publications

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review

Research on ACEI of Low-Molecular-Weight Peptides from Hirudo nipponia Whitman

Tyrosine phosphorylation of recombinant hirudin increases affinity to thrombin and antithrombotic activity

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