<i>Cis</i> autocatalytic cleavage of glycine‐linked Zika virus NS2B‐NS3 protease constructs

Hammerstein, F; Lauth, Luca M; Hammerschmidt, Stefan; Wagner, Annika; Schirmeister, Tanja; Hellmich, Ute A.

doi:10.1002/1873-3468.13507

Cited by 14 publications

(19 citation statements)

References 24 publications

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“…11 A). The protein melting point was 49.60 °C and 50.01 °C, respectively, for both instruments, very close to the literature value ( Table 1 ) [35] . The ZIKV protease is known to adopt multiple conformations due to the dynamic interaction of both NS2B and NS3 proteins.…”

Section: Validation and Characterizationsupporting

confidence: 88%

“… Protein Melting point [°C] N (replicates) Lit. melting point [°C] S/N [dB] M pro 57.09 ± 0.08 12 55.74 [31] 27.1 ± 0.32 SrtA 49.84 ± 0.13 16 50.50 [32] 31.1 ± 0.32 Cruzain 64.90 ± 0.14 8 66.40 [33] 30.5 ± 0.29 Thrombin 52.10 ± 0.08 4 58.30 [30] 28.8 ± 0.29 Lysozyme 67.58 ± 0.10 4 71.90 [29] 27.3 ± 0.27 BSA 58.72 ± 0.13 4 56.00 [3] 21.6 ± 0.53 Calpain I 44.60 ± 0.11 4 47.00 [34] 20.5 ± 0.16 NS2B/NS3 50.01 ± 0.07 4 49.00 [35] 27.9 ± 0.26 …”

Section: Validation and Characterizationmentioning

confidence: 99%

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A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments

et al. 2022

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Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications to drug discovery and protein biochemistry. DSF experiments are commonly performed in commercial real-time polymerase chain reaction (qPCR) thermal cyclers or nanoDSF instruments. Here, we report the construction, validation, and example applications of an open-source DSF system for 176 €, which, in addition to protein-DSF experiments, also proved to be a versatile biophysical instrument for less conventional RNA-DSF experiments. Using 3D-printed parts made of polyoxymethylene, we were able to fabricate a thermostable machine chassis for protein-melting experiments. The combination of blue high-power LEDs as the light source and stage light foil as filter components was proven to be a reliable and affordable alternative to conventional optics equipment for the detection of SYPRO Orange or Sybr Gold fluorescence. The ESP32 microcontroller is the core piece of this openDSF instrument, while the in-built I 2 S interface was found to be a powerful analog-to-digital converter for fast acquisition of fluorescence and temperature data. Airflow heating and inline temperature control by thermistors enabled highaccuracy temperature management in PCR tubes (±0.1 °C) allowing us to perform highresolution thermal shift assays (TSA) from exemplary biological applications.

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Section: Validation and Characterizationsupporting

confidence: 88%

Section: Validation and Characterizationmentioning

confidence: 99%

A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments

et al. 2022

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“…Protease inhibition selectivity : Fluorometric assays of the ZIKV NS2B/NS3 protease were performed as described previously . The assay was carried out in triplicates at 25 °C in assay buffer (50 mM Tris, pH 9.0, 20 % glycerol and 1 mM CHAPS).…”

Section: Methodsmentioning

confidence: 99%

Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A

et al. 2020

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Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with drugresistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active-site cysteine. A broad series of derivatives were synthesized to derive structure-activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were found to have single-digit micromolar K i values and caused up to a 66 % reduction of S. aureus fibrinogen attachment at an effective inhibitor concentration of 10 μM. This new molecule class exhibited minimal cytotoxicity, low bacterial growth inhibition and impaired sortase-mediated adherence of S. aureus cells. a 100 % � 0.5 % 93 % � 5.6 % n.i. 7 a 98 % � 0.1 % 14 % � 8.9 % 18 % � 1.1 % 7 f 60 % � 0.5 % n.i. 12 % � 3.1 % 7 g 18 % � 3.9 % n.i. 17 % � 7.7 % 7 h 69 % � 1.7 % 23 % � 11 % 13 % � 8.0 % 12 a 90 % � 0.5 % n.i. 15 % � 5.7 % n.i. = no inhibition at 20 μM compound concentration. All results include the mean value and standard deviations from triplicate measurements.

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“…Intermolecular protease cleavage can be easily probed by mixing catalytically inactivated enzymes with a wildtype protease. [44] Incubating an excess of purified, catalytically inactive pro-rhodesain C150A with catalytically active, mature, wildtype rhodesain led to a continuous decrease of the high molecular band corresponding to pro-rhodesain in a gel-shift assay (Fig. 6A, B).…”

Section: Resultsmentioning

confidence: 99%

Structure, interdomain dynamics and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes

Johé¹,

Jaenicke²,

Neuweiler³

et al. 2020

Preprint

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Rhodesain is the lysosomal cathepsin L-like cysteine protease of T. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in E. coli and determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH-sensitivity of pro-enzyme cleavage in (trypanosomal) CathL-like proteases.

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Cis autocatalytic cleavage of glycine‐linked Zika virus NS2B‐NS3 protease constructs

Cited by 14 publications

References 24 publications

A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments

A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments

Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A

Structure, interdomain dynamics and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes

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