Minimum Significant Ratio of Selectivity Ratios (MSRSR) and Confidence in Ratio of Selectivity Ratios (CRSR): Quantitative Measures for Selectivity Ratios Obtained by Screening Assays

Abstract

Abbreviations: CRSR, confidence in ratio of selectivity ratios; JAK, Janus kinase; MSR, minimum significant ratio; MSR 95 , minimum significant ratio at 95% confidence; MSSR, minimum significant selectivity ratio; MSRSR, minimum significant ratio of selectivity ratios; MSRSR 95 : minimum significant ratio of selectivity ratios at 95% confidence; pSTAT3, phosphorylation of STAT3, pSTAT5, phosphorylation of STAT5; RA, rheumatoid arthritis; SAR, structure-activity relationship; SE, standard error of the mean; SR, selectivity ratio. AbstractDevelopment of inhibitor compounds selective against undesirable targets is critical in drug discovery. Selectivity ratios for candidate compounds are evaluated by dividing potencies from two assays assessing the off-target and target. Because all potency measurements have underlying uncertainty, understanding error propagation is essential to interpreting selectivity data. Assay noise introduces ambiguity in the statistical significance of selectivity ratios, particularly at low replicate numbers when compounds are often prioritized for subsequent testing. The ability to differentiate potency results for any pair of compounds in one assay is evaluated using a metric called minimum significant ratio (MSR). Potency results of one compound tested in a pair of assays can be differentiated by the minimum significant selectivity ratio (MSSR). To differentiate selectivity ratios for any pair of compounds, we extend this concept by proposing two new parameters called the minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR). Importantly, these tools can be used after a single selectivity measurement. We describe these methods and illustrate their usefulness using structure-activity relationship data from a Janus kinase inhibitor project, in which these tools informed a cogent retesting strategy and enabled rapid and objective decision making.

Discovery and Optimization of Pyrrolopyrimidine Inhibitors of Interleukin-1 Receptor Associated Kinase 4 (IRAK4) for the Treatment of Mutant MYD88L265P Diffuse Large B-Cell Lymphoma

“…The thioether (34) offered similar potency and rat hepatocyte metabolism but at higher lipophilicity (Δlog D +1.5) and with a consequent reduction in aqueous solubility (5 μM compared with 130 μM for 28 at pH 7.4). Replacement of the pyrrolopyrimidine with a pyrrolopyridine (35) and incorporation of a 3-cyano group (36) did not offer any advantage. On the overall balance of properties, compound 28 was selected for further profiling.…”

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

“…7 and Table 6). To interrogate common ␥-chain signaling, we used an IL-2-stimulated T-blast assay that requires the activity of Jak3 and Jak1 to phosphorylate STAT5 (33). In this system, Compounds 1 and 2 show full inhibition of STAT5 phosphorylation with moderate potency (EC 50 values of 1.3 and 0.5 M, respectively).…”

Cell-Based In Vitro Assay Automation: Balancing Technology and Data Reproducibility/Predictability

“…The minimum significant ratio (MSR) decision tool was used to measure the reproducibility and variability of potencies between assay runs. [11][12][13][14] The MSR is defined as the smallest ratio between the potencies of two compounds that is statistically significant. The mean ratio (MR), the geometric average fold difference in potency between two runs, was also determined.…”