It is well understood that small molecule drugs are bound by plasma proteins. This plasma protein binding (PPB) varies dramatically from compound to compound and is determined indirectly as a 'PPB adjusted' IC₅₀. Despite the inaccuracies of this PPB adjusted IC₅₀ the alternative gold standard of in vivo measurement of free drug is complex and therefore restricted to late optimization stages of drug development.
Scientists at Prelude Therapeutics and Locanabio have built on the well-described and widely used in-cell western assay (ICW) to develop a direct, sensitive and quantitative measurement of drug potency in the presence of plasma protein using the model of protein kinase inhibitors on target cells. The assay format is suitable for both adherent and non-adherent cells and for drugs which display their maximal response in under 6 hours and where expression/modification of a protein is affected by the drug treatment (acting as the reporter).
The reporter for the protein kinase inhibitor was RNA pol II B1 detected by antibody and fluorescence. The far-red DNA dye DRAQ5 (optimized at 2.5 µM final concentration in blocking buffer with IRdye 800CW-tagged secondary antibody, incubated for 1h at RT) was used to normalise cell number between the plate wells. Plates were scanned using the LICOR Odyssey Imaging system. The whole process is complete in 2 days for suspension cells and 3 days for adherent cells (1 day for adherence).
Importantly, the data for a list of compounds tested show striking discrepancies between the PPB adjusted IC₅₀ and this direct functional cell-based assay, with the latter showing good accord with other reports in the literature for the reference compound used.
This automatable high throughput ICW assay format is amenable to earlier phases of the drug discovery process due to its simplicity, speed. It is both quantitative and sensitive.
Reference:
Zhang, Yang W., et al. "Human Plasma In‐Cell Western Assays—An In vitro Predictor for In vivo Pharmacology in Oncology Drug Discovery." Current Protocols 1.2 (2021): e51.
Related blogposts:
Assaying for Virus Neutralizing Antibodies. Dec. 15, 2020
No comments :
Post a Comment