Serine protease inhibitor AEBSF reduces dengue virus infection via decreased cholesterol synthesis

Sreelatha, Liji; Malakar, Shilu; Songprakhon, Pucharee; Morchang, Atthapan; Srisawat, Chatchawan; Noisakran, Sansanee; Yenchitosomanus, Pa-thai; Limjindaporn, Thawornchai

doi:10.1016/j.virusres.2019.197672

Cited by 10 publications

(6 citation statements)

References 42 publications

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“…However, the overall culture productivity was significantly reduced. (Data not shown) Inhibitor Approach: The protease inhibitor 4‐(2‐aminoethyl) benzenesulfonyl fluoride (AEBSF) was added to the bioreactor during the cell culture process to inhibit proteolytic activity 17,18 . Daily additions of AEBSF were spiked into the culture starting on day 4 and continued through day 14, initial additions were 100 μM AEBSF and then gradually increased to 500 μM by day 14.…”

Section: Resultsmentioning

confidence: 99%

Analytical characterization of broadly neutralizing antibody CAP256LS heavy chain clipping during manufacturing development

Gollapudi¹,

Rosales‐Zavala²,

Ivleva³

et al. 2022

Biotechnology Progress

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Broadly neutralizing antibody (bNAb) CAP256-VRC26.25 (abbreviated CAP256LS), a human IgGI monoclonal antibody targeting the V1V2 site of the HIV-1 envelope, has demonstrated high therapeutic potential as a broadly neutralizing monoclonal antibody against HIV-1. During the process development, a heavy chain fragmentation (clipping) was observed, that led to a relative potency reduction. In this report, we highlighted a series of process and product mitigation strategies deployed to advance this product. We have detailed how analytical characterization tools, especially the microchip reduced capillary gel electrophoresis (CGE-SDS), played a pivotal role in identifying the development issues and in providing measurements to guide implementation of mitigation strategies.

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Section: Resultsmentioning

confidence: 99%

Analytical characterization of broadly neutralizing antibody CAP256LS heavy chain clipping during manufacturing development

Gollapudi¹,

Rosales‐Zavala²,

Ivleva³

et al. 2022

Biotechnology Progress

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“…In mutating Cys34 to serine, Prp was essentially changed from a cysteine protease to a serine protease. As with most other serine proteases, the high pI of the hydroxyl group within serine requires a proton-accepting group to form the seroxide nucleophilic group that will attack the substrate [ 56 ]. Cysteine, however, can form its nucleophilic group through deprotonation by either a proton acceptor or solvent at physiological pH [ 55 ].…”

Section: Resultsmentioning

confidence: 99%

Phage-Related Ribosomal Proteases (Prps): Discovery, Bioinformatics, and Structural Analysis

2022

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Many new antimicrobials are analogs of existing drugs, sharing the same targets and mechanisms of action. New antibiotic targets are critically needed to combat the growing threat of antimicrobial-resistant bacteria. Phage-related ribosomal proteases (Prps) are a recently structurally characterized antibiotic target found in pathogens such as Staphylococcus aureus, Clostridioides difficile, and Streptococcus pneumoniae. These bacteria encode an N-terminal extension on their ribosomal protein L27 that is not present in other bacteria. The cleavage of this N-terminal extension from L27 by Prp is necessary to create a functional ribosome. Thus, Prp inhibition may serve as an alternative to direct binding and inhibition of the ribosome. This bioinformatic and structural analysis covers the discovery, function, and structural characteristics of known Prps. This information will be helpful in future endeavors to design selective therapeutics targeting the Prps of important pathogens.

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“…Multiple process strategies were evaluated to minimize the proteolytic cleavage observed during cell culture 6 , 7 . These included (1) a fill-draw approach that resembled a repeated fed batch, where 75% of the bioreactor was harvested on day 10 and refilled with fresh media every 3 days (2) inclusion of protease inhibitors for serine proteases 8 , 9 in the cell culture to effectively inhibit proteolytic activity and (3) alternating tangential flow (ATF)-Perfusion cell culture 10 , 11 , allowing continuous antibody harvest. All three approaches minimized CAP256 cleavage below 5%; however, due to risks associated with scaling and transferring these processes, product stability risks, and long-term development risks, it was determined that engineering out the proteolytic cleavage site was better suited for long term clinical product development.…”

Section: Introductionmentioning

confidence: 99%

Engineering of HIV-1 neutralizing antibody CAP256V2LS for manufacturability and improved half life

Zhang

Gollapudi

Gorman

et al. 2022

Sci Rep

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The broadly neutralizing antibody (bNAb) CAP256-VRC26.25 has exceptional potency against HIV-1 and has been considered for clinical use. During the characterization and production of this bNAb, we observed several unusual features. First, the antibody appeared to adhere to pipette tips, requiring tips to be changed during serial dilution to accurately measure potency. Second, during production scale-up, proteolytic cleavage was discovered to target an extended heavy chain loop, which was attributed to a protease in spent medium from 2-week culture. To enable large scale production, we altered the site of cleavage via a single amino acid change, K100mA. The resultant antibody retained potency and breadth while avoiding protease cleavage. We also added the half-life extending mutation LS, which improved the in vivo persistence in animal models, but did not impact neutralization activity; we observed the same preservation of neutralization for bNAbs VRC01, N6, and PGDM1400 with LS on a 208-virus panel. The final engineered antibody, CAP256V2LS, retained the extraordinary neutralization potency of the parental antibody, had a favorable pharmacokinetic profile in animal models, and was negative in in vitro assessment of autoreactivity. CAP256V2LS has the requisite potency, developability and suitability for scale-up, allowing its advancement as a clinical candidate.

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Serine protease inhibitor AEBSF reduces dengue virus infection via decreased cholesterol synthesis

Cited by 10 publications

References 42 publications

Analytical characterization of broadly neutralizing antibody CAP256LS heavy chain clipping during manufacturing development

Analytical characterization of broadly neutralizing antibody CAP256LS heavy chain clipping during manufacturing development

Phage-Related Ribosomal Proteases (Prps): Discovery, Bioinformatics, and Structural Analysis

Engineering of HIV-1 neutralizing antibody CAP256V2LS for manufacturability and improved half life

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