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Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli.

ABSTRACT: Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is often not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay in E. coli to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ mol^-1 more stable than the wild-type. In one case, the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ mol^-1 more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.

SUBMITTER: Hamborg L

PROVIDER: S-EPMC8373930 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli.

Hamborg Louise L Granata Daniele D Olsen Johan G JG Roche Jennifer Virginia JV Pedersen Lasse Ebdrup LE Nielsen Alex Toftgaard AT Lindorff-Larsen Kresten K Teilum Kaare K

Communications biology 20210818 1

Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is often not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay in E. coli to select variants of the barley chymotrypsin inhibitor 2 wit ...[more]

PMID: 34408246

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Project description:BackgroundMelanoma is the most aggressive and deadly form of skin cancer with increasing case numbers worldwide. The development of inhibitors targeting mutated BRAF (found in around 60% of melanoma patients) has markedly improved overall survival of patients with late-stage tumors, even more so when combined with MEK inhibitors targeting the same signaling pathway. However, invariably patients become resistant to this targeted therapy resulting in rapid progression with treatment-refractory disease. The purpose of this study was the identification of new kinase inhibitors that do not lead to the development of resistance in combination with BRAF inhibitors (BRAFi), or that could be of clinical benefit as a 2nd line treatment for late-stage melanoma patients that have already developed resistance.MethodsWe have screened a 274-compound kinase inhibitor library in 3 BRAF mutant melanoma cell lines (each one sensitive or made resistant to 2 distinct BRAFi). The screening results were validated by dose-response studies and confirmed the killing efficacies of many kinase inhibitors. Two different tools were applied to investigate and quantify potential synergistic effects of drug combinations: the Chou-Talalay method and the Synergyfinder application. In order to exclude that resistance to the new treatments might occur at later time points, synergistic combinations were administered to fluorescently labelled parental and resistant cells over a period of > 10 weeks.ResultsEight inhibitors targeting Wee1, Checkpoint kinase 1/2, Aurora kinase, MEK, Polo-like kinase, PI3K and Focal adhesion kinase killed melanoma cells synergistically when combined with a BRAFi. Additionally, combination of a Wee1 and Chk inhibitor showed synergistic killing effects not only on sensitive cell lines, but also on intrinsically BRAFi- and treatment induced-resistant melanoma cells. First in vivo studies confirmed these observations. Interestingly, continuous treatment with several of these drugs, alone or in combination, did not lead to emergence of resistance.ConclusionsHere, we have identified new, previously unexplored (in the framework of BRAFi resistance) inhibitors that have an effect not only on sensitive but also on BRAFi-resistant cells. These promising combinations together with the new immunotherapies could be an important step towards improved 1st and 2nd line treatments for late-stage melanoma patients.

Dataset Information

Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli.

Publications

Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli.

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