Biology subjects

Ronzetti, M. H.

Publications and source records attributed to Ronzetti, M. H..

3 recordsLinked to original sources

bioRxiv2022

Application of Temperature-Responsive HIS-Tag Fluorophores to Differential Scanning Fluorimetry Screening of Small Molecule Libraries

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in 6xHIS-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this histidine-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.

biochemistry↗

bioRxiv2022

Protein Refolding Guided by High-Throughput Differential Scanning Fluorimetry: a case study of an HtrA-Family Bacterial Protease

The high temperature requirement A (HtrA) serine protease family presents an attractive target class for antibacterial therapeutics development. These proteins possess dual protease and chaperone functions and contain numerous binding sites and regulatory loops, displaying diverse oligomerization patterns dependent on substrate type and occupancy. HtrA proteins that are natively purified coelute with contaminating peptides and activating species, shifting oligomerization and protein structure to differently activated populations. Here, a redesigned HtrA production results in cleaner preparations with high yields by overexpressing and purifying target protein from inclusion bodies under denaturing conditions, followed by a high-throughput screen for optimal refolding buffer composition using function-agnostic biophysical techniques that do not rely on target-specific measurements. We use the Borrelia burgdorferi HtrA to demonstrate the effectiveness of our function-agnostic approach, while characterization with both new and established biophysical methods shows the retention of proteolytic and chaperone activity of the refolded protein. This systematic workflow and toolset will translate to the production of HtrA-family proteins in higher quantities of pure and monodisperse composition than the current literature standard, with applicability to a broad array of protein purification strategies. StatementThe production of a therapeutically-relevant protein family sensitive to coeluting contaminants is greatly improved by optimized expression and refolding workflow. A miniaturized, high-throughput system supported by a function-agnostic biophysics assay and modified data analysis scripts results in a refolded protein that is highly pure, monodisperse, and retains proteolytic and chaperone activity. This approach has broad applicability towards hard-to-express proteins and proteins sensitive to coeluting species. Additionally, novel methods are presented to characterize protein chaperone activity.

biochemistry↗

bioRxiv2022

A real-time cellular thermal shift assay (RT-CETSA) to monitor target engagement

Determining a molecules mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a proteins unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered that withstood unfolding at temperatures greater than 90 degrees Celsius and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. The RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule. SignificanceValidating target engagement is a critical step when characterizing a small molecule modulator. The cellular thermal shift assay (CETSA) is a common approach to examine target engagement, as alterations in the thermal stability of a protein can be conferred by ligand binding. An advantage of CETSA is that it does not require modification of the protein target or small molecule. Major limitations are the throughput and ease-of-use, as the traditional detection method uses western blots, which limits the number of samples that can be processed. Higher-throughput CETSA methods have been developed but are performed at a single temperature and require target-specific optimization. We developed a high-throughput real-time CETSA to circumvent these challenges, providing a rapid and cost-effective strategy to assess on-target activity of a small molecule in living cells.

pharmacology and toxicology↗