{"doi":"10.1016/j.jbc.2022.102683","title":"Gambogic acid and juglone inhibit RNase P through distinct mechanisms","abstract":"The first step in transfer RNA (tRNA) maturation is the cleavage of the 5′ end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 μM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multisubunit protein enzyme and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex, while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor complex and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships. The first step in transfer RNA (tRNA) maturation is the cleavage of the 5′ end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 μM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multisubunit protein enzyme and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex, while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor complex and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships. Transfer RNAs are transcribed as precursor transcripts (pre-tRNA) with extra nucleotides at both the 5′ and 3′ ends and undergo several modifications before they are functional for use in protein synthesis. The first step in tRNA maturation across all domains of life is the removal of the 5′ leader through phosphodiester bond hydrolysis catalyzed by ribonuclease P (RNase P) in the presence of Mg2+ (1Perreault J.P. Altman S. Pathway of activation by magnesium ions of substrates for the catalytic subunit of RNase P from Escherichia coli.J. Mol. Biol. 1993; 230: 750-756Crossref PubMed Scopus (51) Google Scholar, 2Beebe J.A. Kurz J.C. Fierke C.A. Magnesium ions are required by Bacillus","journal":"Journal of Biological Chemistry","year":2022,"id":274621,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9619,"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":419253,"name":"Agnes Karasik","orcid":null,"position":1,"is_corresponding":false},{"id":476587,"name":"Kipchumba Kaitany","orcid":"0000-0003-4085-5399","position":2,"is_corresponding":false},{"id":63441,"name":"Carol A. Fierke","orcid":"0000-0002-1481-0579","position":3,"is_corresponding":false},{"id":418435,"name":"Markos Koutmos","orcid":"0000-0003-0933-6312","position":4,"is_corresponding":false},{"id":943687,"name":"Nancy Wu Meyers","orcid":null,"position":0,"is_corresponding":true}],"reference_count":53,"raw_metadata":null,"created_at":"2026-07-19T00:28:12.324621Z","pmid":"36370850","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":[]}