Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Abstract

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein’s physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a ‘Gold Standard’ for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes

“…The observed ΔT m in the absence and presence of MgCl 2 (1 mM) was 9.1 °C, which is similar to the ΔT m observed in the protein-probe assay (10.9 °C). 26 We 26 and others 34,35 previously demonstrated that protein concentration can significantly impact not only protein detectability but also the observed T m values. Thus, we determined T m values of HRAS and NRAS at concentrations of 100, 250, and 500 nM in the one-step peptide-probe protocol; T m values were slightly different and protein concentration-dependent (Figure S30).…”

“…The observed T m values and ΔT m (2.9 °C) were slightly higher than those in the 96-well plate protocol, possibly due to differences in assay buffers, plates, volumes, proteins, and AZA concentrations (Table S1). 15,26,34,35 Next, we selected RAS as a target protein to further study the one-step peptide-probe thermal protocol. We first mimicked the PLI-type stabilization of KRAS (500 nM) in both the absence and presence of MgCl 2 (1 mM) (Figure 3A).…”

“…This finding contrasts with our original protein-probe assay, where KRAS showed higher detectability compared to other RAS proteins. 26 To gain insight into the difference in detectability among the RAS proteins, even with the same Eu-probe, we conducted assays using two shorter versions of KRAS that lacked the high variable region (HVR). 26 These truncated KRAS proteins showed higher detectability than full-length KRAS, and their T m values (iMet-KRAS (2−169): 48.4 ± 1.5 °C; Ac-KRAS (2−169): 46.6 ± 1.0 °C) were consistent with expectations (Figure S31).…”

“…26 To gain insight into the difference in detectability among the RAS proteins, even with the same Eu-probe, we conducted assays using two shorter versions of KRAS that lacked the high variable region (HVR). 26 These truncated KRAS proteins showed higher detectability than full-length KRAS, and their T m values (iMet-KRAS (2−169): 48.4 ± 1.5 °C; Ac-KRAS (2−169): 46.6 ± 1.0 °C) were consistent with expectations (Figure S31). These results indicate that the high KRAS detectability with the original protein-probe prefers the positively charged HVR on full-length KRAS, not present in HRAS and NRAS.…”

Advances in Molecular Research of Oncogenes

“…With regards to the methodological articles, Kopra et al [ 3 ] described a new methodology to evaluate and characterize newly designed drug candidates. The authors designed a protocol improving previously established methodologies, of which the sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level.…”