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1.
Acta Crystallogr D Struct Biol ; 80(Pt 2): 123-136, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38289714

RESUMO

To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Domínio Catalítico , Proteases 3C de Coronavírus , Cristalografia por Raios X
2.
PLoS One ; 13(6): e0199639, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29949628

RESUMO

The use of the tellurium-centered Anderson-Evans polyoxotungstate [TeW6O24]6- (TEW) as a crystallization additive has been described. Here, we present the use of TEW as an additive in the crystallization screening of the nucleotide binding domain (NBD) of HSP70. Crystallization screening of the HSP70 NBD in the absence of TEW using a standard commercial screen resulted in a single crystal form. An identical crystallization screen of the HSP70 NBD in the presence of TEW resulted in both the "TEW free" crystal form and an additional crystal form with a different crystal packing. TEW binding was observed in both crystal forms, either as a well-defined molecule or in overlapping alternate positions suggesting translational disorder. The structures were solved by both molecular replacement and single wavelength anomalous diffraction (SAD) using the anomalous signal of a single bound molecule of TEW. This study adds one more example of TEW binding to a protein and influencing its crystallization behavior.


Assuntos
Proteínas de Choque Térmico HSP70/química , Sítios de Ligação , Cristalização , Escherichia coli , Fluorometria , Estrutura Molecular , Estabilidade Proteica , Desdobramento de Proteína , Temperatura
3.
J Gen Virol ; 95(Pt 7): 1539-1543, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24728713

RESUMO

Osteosarcoma cells U2OS are partially susceptible to adeno-associated virus (AAV)-2 infection, allowing efficient synthesis of Rep proteins and, in a low percentage of cells, capsid production. It is not clear if this partial susceptibility to infection is due to the bone-cell-like nature of these cells or is a result of their transformed properties. Here, we grew osteosarcoma cells in a biomimetic three-dimensional bone-like matrix composed of calcium phosphate and chitosan, and tested whether this would increase or reduce their permissiveness to virus. The osteosarcoma cells grew in the matrix and began to express the alkaline phosphatase bone cell differentiation marker. This was accompanied by a block to their infection by AAV, as indicated by Rep and capsid production. Infection of cells growing in three-dimensional tissue-like matrices could be, in a wider context, a practical way to mimic in vivo conditions.


Assuntos
Dependovirus/crescimento & desenvolvimento , Osteócitos/virologia , Biomimética , Linhagem Celular Tumoral , Humanos , Técnicas de Cultura de Tecidos
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