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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.
Hepatitis Delta virus (HDV) is a satellite of Hepatitis B virus with a single-stranded circular RNA genome. HDV RNA genome synthesis is carried out in infected cells by cellular RNA polymerases with the assistance of the small hepatitis delta antigen (S-HDAg). Here we show that S-HDAg binds the bromodomain (BRD) adjacent to zinc finger domain 2B (BAZ2B) protein, a regulatory subunit of BAZ2B-associated remodeling factor (BRF) ISWI chromatin remodeling complexes. shRNA-mediated silencing of BAZ2B or its inactivation with the BAZ2B BRD inhibitor GSK2801 impairs HDV replication in HDV-infected human hepatocytes. S-HDAg contains a short linear interacting motif (SLiM) KacXXR, similar to the one recognized by BAZ2B BRD in histone H3. We found that the integrity of the S-HDAg SLiM sequence is required for S-HDAg interaction with BAZ2B BRD and for HDV RNA replication. Our results suggest that S-HDAg uses a histone mimicry strategy to co-activate the RNA polymerase II-dependent synthesis of HDV RNA and sustain HDV replication.
Since hyperactivity of the protein kinase DYRK1A is linked to several neurodegenerative disorders, DYRK1A inhibitors have been suggested as potential therapeutics for Down syndrome and Alzheimer’s disease. Most published inhibitors to date suffer from low selectivity against related kinases or from unfavorable physicochemical properties. In order to identify DYRK1A inhibitors with improved properties, a series of new chemicals based on [b]-annulated halogenated indoles were designed, synthesized, and evaluated for biological activity. Analysis of crystal structures revealed a typical type-I binding mode of the new inhibitor 4-chlorocyclohepta[b]indol-10(5H)-one in DYRK1A, exploiting mainly shape complementarity for tight binding. Conversion of the DYRK1A inhibitor 8-chloro-1,2,3,9-tetrahydro-4H-carbazol-4-one into a corresponding Mannich base hydrochloride improved the aqueous solubility but abrogated kinase inhibitory activity.
Serine/arginine-protein kinase 1 (SRPK1) regulates alternative splicing of VEGF-A to pro-angiogenic isoforms and SRPK1 inhibition can restore the balance of pro/antiangiogenic isoforms to normal physiological levels. The lack of potency and selectivity of available compounds has limited development of SRPK1 inhibitors, with the control of alternative splicing by splicing factor-specific kinases yet to be translated. We present here compounds that occupy a binding pocket created by the unique helical insert of SRPK1, and trigger a backbone flip in the hinge region, that results in potent (<10 nM) and selective inhibition of SRPK1 kinase activity. Treatment with these inhibitors inhibited SRPK1 activity and phosphorylation of serine/arginine splicing factor 1 (SRSF1), resulting in alternative splicing of VEGF-A from pro-angiogenic to antiangiogenic isoforms. This property resulted in potent inhibition of blood vessel growth in models of choroidal angiogenesis in vivo. This work identifies tool compounds for splice isoform selective targeting of pro-angiogenic VEGF, which may lead to new therapeutic strategies for a diversity of diseases where dysfunctional splicing drives disease development.
The function of the p53 transcription factor family is dependent on several folded domains. In addition to a DNA-binding domain, members of this family contain an oligomerization domain. p63 and p73 also contain a C-terminal Sterile α-motif domain. Inhibition of most transcription factors is difficult as most of them lack deep pockets that can be targeted by small organic molecules. Genetic knock-out procedures are powerful in identifying the overall function of a protein, but they do not easily allow one to investigate roles of individual domains. Here we describe the characterization of Designed Ankyrin Repeat Proteins (DARPins) that were selected as tight binders against all folded domains of p63. We determine binding affinities as well as specificities within the p53 protein family and show that DARPins can be used as intracellular inhibitors for the modulation of transcriptional activity. By selectively inhibiting DNA binding of the ΔNp63α isoform that competes with p53 for the same promoter sites, we show that p53 can be reactivated. We further show that inhibiting the DNA binding activity stabilizes p63, thus providing evidence for a transcriptionally regulated negative feedback loop. Furthermore, the ability of DARPins to bind to the DNA-binding domain and the Sterile α-motif domain within the dimeric-only and DNA-binding incompetent conformation of TAp63α suggests a high structural plasticity within this special conformation. In addition, the developed DARPins can also be used to specifically detect p63 in cell culture and in primary tissue and thus constitute a very versatile research tool for studying the function of p63.
Although overexpression and hyperactivity of protein kinases are causative for a wide range of human cancers, protein kinase inhibitors currently approved as cancer drugs address only a limited number of these enzymes. To identify new chemotypes addressing alternative protein kinases, the basic structure of a known PLK1/VEGF-R2 inhibitor class was formally dissected and reassembled. The resulting 7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones were synthesized and proved to be dual inhibitors of Aurora A kinase and VEGF receptor kinases. Crystal structures of two representatives of the new chemotype in complex with Aurora A showed the ligand orientation in the ATP binding pocket and provided the basis for rational structural modifications. Congeners with attached sulfamide substituents retained Aurora A inhibitory activity. In vitro screening of two members of the new kinase inhibitor family against the cancer cell line panel of the National Cancer Institute (NCI) showed antiproliferative activity in the single-digit micromolar concentration range in the majority of the cell lines.
The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure-activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.
We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.
Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.
The p300/CBP‐associated factor (PCAF) and related GCN5 bromodomain‐containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine‐based L‐45 (dubbed L‐Moses) as the first potent, selective, and cell‐active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)‐(−)‐norephedrine furnished L‐45 in enantiopure form. L‐45 was shown to disrupt PCAF‐Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co‐crystal structure of L‐45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L‐45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell‐permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.
Phenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still pose a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies.
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.
Cdc2-like kinases (CLKs) represent a family of serine-threonine kinases involved in the regulation of splicing by phosphorylation of SR-proteins and other splicing factors. Although compounds acting against CLKs have been described, only a few show selectivity against dual-specificity tyrosine phosphorylation regulated-kinases (DYRKs). We here report a novel CLK inhibitor family based on a 6,7-dihydropyrrolo[3,4-g]indol-8(1H)-one core scaffold. Within the series, 3-(3-chlorophenyl)-6,7-dihydropyrrolo[3,4-g]indol-8(1H)-one (KuWal151) was identified as inhibitor of CLK1, CLK2 and CLK4 with a high selectivity margin towards DYRK kinases. The compound displayed a potent antiproliferative activity in an array of cultured cancer cell lines. The X-ray structure analyses of three members of the new compound class co-crystallized with CLK proteins corroborated a molecular binding mode predicted by docking studies.
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 P300/CBP-associated factor plays a central role in retroviral infection and cancer development, and the C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening approaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors.
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.
Publicly available compound and bioactivity databases provide an essential basis for data-driven applications in life-science research and drug design. By analyzing several bioactivity repositories, we discovered differences in compound and target coverage advocating the combined use of data from multiple sources. Using data from ChEMBL, PubChem, IUPHAR/BPS, BindingDB, and Probes & Drugs, we assembled a consensus dataset focusing on small molecules with bioactivity on human macromolecular targets. This allowed an improved coverage of compound space and targets, and an automated comparison and curation of structural and bioactivity data to reveal potentially erroneous entries and increase confidence. The consensus dataset comprised of more than 1.1 million compounds with over 10.9 million bioactivity data points with annotations on assay type and bioactivity confidence, providing a useful ensemble for computational applications in drug design and chemogenomics.
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.
Selectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, noncanonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK, and CLK. By use of the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a noncanonical binding mode in CLK1, providing a rationale for selectivity over the closely related CLK3 which harbors a smaller DFG-1 alanine. Our data suggest that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.
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.
MKK7 (MEK7) is a key regulator of the JNK stress signaling pathway and targeting MKK7 has been proposed as a chemotherapeutic strategy. Detailed understanding of the MKK7 structure and factors that impact its activity is therefore of critical importance. Here, we present a comprehensive set of MKK7 crystal structures revealing insights into catalytic domain plasticity and the role of the N-terminal regulatory helix, conserved in all MAP2Ks, mediating kinase activation. Crystal structures harboring this regulatory helix revealed typical structural features of active kinase, providing exclusively a first model of the MAP2K active state. A small molecule screening campaign yielded multiple scaffolds, including type-II irreversible inhibitors a binding mode that has not been reported previously. We also observed an unprecedented allosteric pocket located in the N-terminal lobe for the approved drug ibrutinib. Collectively, our structural and functional data expand and provide alternative targeting strategies for this important MAP2K kinase.
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.
Selectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, non-canonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK and CLK. Using the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a non-canonical binding mode in CLK1, providing a rational for selectivity over the closely-related CLK3 which harbors a smaller DFG-1 alanine. Our data suggests that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.
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.