{"doi":"10.3389/fphar.2022.1040039","title":"Application of temperature-responsive HIS-tag fluorophores to differential scanning fluorimetry screening of small molecule libraries","abstract":"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 histidine-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 HIS-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 T m values with an average coefficient of variation of 0.51% and median coefficient of variation of compound T m 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.","journal":"Frontiers in Pharmacology","year":2022,"id":286981,"datarank":0.24141568686511508,"base_score":1.6094379124341003,"endowment":1.6094379124341003,"self_citation_contribution":0.24141568686511508,"citation_network_contribution":0.0,"self_endowment_contribution":0.24141568686511508,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9587,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":228430,"name":"Bolormaa Baljinnyam","orcid":"0000-0001-9548-4965","position":1,"is_corresponding":false},{"id":268258,"name":"Zina Itkin","orcid":"0000-0003-1686-1326","position":2,"is_corresponding":false},{"id":636400,"name":"Sankalp Jain","orcid":"0000-0002-9370-9611","position":3,"is_corresponding":false},{"id":268294,"name":"Ganesha Rai","orcid":"0000-0001-9763-9641","position":4,"is_corresponding":false},{"id":12541,"name":"Alexey Zakharov","orcid":"0000-0003-2466-1711","position":5,"is_corresponding":false},{"id":245047,"name":"Utpal Pal","orcid":"0000-0002-7504-4628","position":6,"is_corresponding":false},{"id":228423,"name":"Anton Simeonov","orcid":"0000-0002-4523-9977","position":7,"is_corresponding":false},{"id":228400,"name":"Michael Ronzetti","orcid":"0000-0001-5034-7692","position":0,"is_corresponding":true}],"reference_count":45,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T00:29:56.781756Z","pmid":"36506591","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}