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Hepatocyte nuclear factor 4α (HNF4α) is a ligand-sensing transcription factor and presents as a potential drug target in metabolic diseases and cancer. In humans, mutations in the HNF4α gene cause maturity-onset diabetes of the young (MODY), and the elevated activity of this protein has been associated with gastrointestinal cancers. Despite the high therapeutic potential, available ligands and structure–activity relationship knowledge for this nuclear receptor are scarce. Here, we disclose a chemically diverse collection of orthogonally validated fragment-like activators as well as inverse agonists, which modulate HNF4α activity in a low micromolar range. These compounds demonstrate the druggability of HNF4α and thus provide a starting point for medicinal chemistry as well as an early tool for chemogenomics.
The FUBP1-FUSE complex is an essential component of a transcription molecular machinery that is necessary for tight regulation of expression of many key genes including c-Myc and p21. FUBP1 utilizes its four articulated KH modules, which function cooperatively, for FUSE nucleotide binding. To understand molecular mechanisms fundamental to the intermolecular interaction, we present a set of crystal structures, as well ssDNA-binding characterization of FUBP1 KH domains. All KH1-4 motifs were highly topologically conserved, and were able to interact with FUSE individually and independently. Nevertheless, differences in nucleotide binding properties among the four KH domains were evident, including higher nucleotide-binding potency for KH3 as well as diverse nucleotide sequence preferences. Variations in amino acid compositions at one side of the binding cleft responsible for nucleobase resulted in diverse shapes and electrostatic charge interaction, which might feasibly be a contributing factor for different nucleotide-binding propensities among KH1-4. Nonetheless, conservation of structure and nucleotide-binding property in all four KH motifs is essential for the cooperativity of multi KH modules present in FUBP1 towards nanomolar affinity for FUSE interaction. Comprehensive structural comparison and ssDNA binding characteristics of all four KH domains presented here provide molecular insights at a fundamental level that might be beneficial for elucidating the mechanisms of the FUBP1-FUSE interaction.
Nuclear receptor related 1 (Nurr1) is an orphan ligand-activated transcription factor and considered as neuroprotective transcriptional regulator with great potential as therapeutic target for neurodegenerative diseases. However, the collection of available Nurr1 modulators and mechanistic understanding of Nurr1 are limited. Here, we report the discovery of several structurally diverse non-steroidal anti-inflammatory drugs as inverse Nurr1 agonists demonstrating that Nurr1 activity can be regulated bidirectionally. As chemical tools, these ligands enable unraveling the co-regulatory network of Nurr1 and the mode of action distinguishing agonists from inverse agonists. In addition to its ability to dimerize, we observe an ability of Nurr1 to recruit several canonical nuclear receptor co-regulators in a ligand-dependent fashion. Distinct dimerization states and co-regulator interaction patterns arise as discriminating factors of Nurr1 agonists and inverse agonists. Our results contribute a valuable collection of Nurr1 modulators and relevant mechanistic insights for future Nurr1 target validation and drug discovery.
The retinoid X receptor (RXR) is a ligand-sensing transcription factor acting mainly as a universal heterodimer partner for other nuclear receptors. Despite presenting as a potential therapeutic target for cancer and neurodegeneration, adverse effects typically observed for RXR agonists, likely due to the lack of isoform selectivity, limit chemotherapeutic application of currently available RXR ligands. The three human RXR isoforms exhibit different expression patterns; however, they share high sequence similarity, presenting a major obstacle toward the development of subtype-selective ligands. Here, we report the discovery of the saturated fatty acid, palmitic acid, as an RXR ligand and disclose a uniform set of crystal structures of all three RXR isoforms in an active conformation induced by palmitic acid. A structural comparison revealed subtle differences among the RXR subtypes. We also observed an ability of palmitic acid as well as myristic acid and stearic acid to induce recruitment of steroid receptor co-activator 1 to the RXR ligand-binding domain with low micromolar potencies. With the high, millimolar endogenous concentrations of these highly abundant lipids, our results suggest their potential involvement in RXR signaling.
YEATS-domain-containing MLLT1 is an acetyl/acyl-lysine reader domain, which is structurally distinct from well-studied bromodomains and has been strongly associated in development of cancer. Here, we characterized piperazine-urea derivatives as an acetyl/acyl-lysine mimetic moiety for MLLT1. Crystal structures revealed distinct interaction mechanisms of this chemotype compared to the recently described benzimidazole-amide based inhibitors, exploiting different binding pockets within the protein. Thus, the piperazine-urea scaffold offers an alternative strategy for targeting the YEATS domain family.
Dysfunction of YEATS-domain-containing MLLT1, an acetyl/acyl-lysine dependent epigenetic reader domain, has been implicated in the development of aggressive cancers. Mutations in the YEATS domain have been recently reported as a cause of MLLT1 aberrant reader function. However, structural basis for the reported alterations in affinity for acetyled/acylated histone has remained elusive. Here, we report the crystal structures of both insertion and substitution present in cancer, revealing significant conformational changes of the YEATS-domain loop 8. Structural comparison demonstrates that such alteration not only altered the binding interface for acetylated/acylated histones, but the sequence alterations in the T1 loop may enable dimeric assembly consistent inducing self-association behavior. Nevertheless, we show that also the MLLT1 mutants can be targeted by developed acetyllysine mimetic inhibitors with affinities similarly to wild type. Our report provides a structural basis for the altered behaviors and potential strategy for targeting oncogenic MLLT1 mutants.
The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus, targeting this protein domain may offer a therapeutic strategy to combat the current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally-occurring nucleotides, small molecules as well as nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and it provides chemical starting points for future inhibitor development.
