ABSTRACT
Selective inhibitors of DYRK1A are of interest for the treatment of cancer, Type 2 diabetes and neurological disorders. Optimization of imidazo [1,2-b]pyridazine fragment 1 through structure-activity relationship exploration and in silico drug design efforts led to the discovery of compound 17 as a potent cellular inhibitor of DYRK1A with selectivity over much of the kinome. The binding mode of compound 17 was elucidated with X-ray crystallography, facilitating the rational design of compound 29, an imidazo [1,2-b]pyridazine with improved kinase selectivity with respect to closely related CLK kinases.
Subject(s)
Diabetes Mellitus, Type 2 , Iohexol/analogs & derivatives , Pyridazines , Humans , Dyrk Kinases , Diabetes Mellitus, Type 2/drug therapy , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Pyridazines/chemistryABSTRACT
Herein, we describe a systematic SAR- and SPR-investigation of the peptidomimetic hydroxy-proline based VHL-ligand VH032, from which most to-date published VHL-targeting PROTACs have been derived. This study provides for the first time a consistent data set which allows for direct comparison of structural variations including those which were so far hidden in patent literature. The gained knowledge about improved VHL binders was used to design a small library of highly potent BRD4-degraders comprising different VHL exit vectors. Newly designed degraders showed favorable molecular properties and significantly improved degradation potency compared to MZ1.
Subject(s)
Nuclear Proteins , Von Hippel-Lindau Tumor Suppressor Protein , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Ligands , Nuclear Proteins/metabolism , Proteolysis , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1-4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing four amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.
Subject(s)
Hypertension , Ubiquitin-Protein Ligases , Adaptor Proteins, Signal Transducing/genetics , Humans , Microfilament Proteins/genetics , Phosphorylation , Proline , Protein Serine-Threonine Kinases/genetics , UbiquitinABSTRACT
SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.
Subject(s)
Aniline Compounds/pharmacology , Maleimides/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Aniline Compounds/chemistry , Aniline Compounds/metabolism , Binding Sites , Cell Line, Tumor , Cell Movement/drug effects , HEK293 Cells , Humans , Maleimides/chemistry , Maleimides/metabolism , Microfilament Proteins/metabolism , Molecular Docking Simulation , Molecular Structure , Phosphorylation/drug effects , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Structure-Activity RelationshipABSTRACT
Polymorphisms in the region of the calmodulin-dependent kinase isoform D (CaMK1D) gene are associated with increased incidence of diabetes, with the most common polymorphism resulting in increased recognition by transcription factors and increased protein expression. While reducing CaMK1D expression has a potentially beneficial effect on glucose processing in human hepatocytes, there are no known selective inhibitors of CaMK1 kinases that can be used to validate or translate these findings. Here we describe the development of a series of potent, selective, and drug-like CaMK1 inhibitors that are able to provide significant free target cover in mouse models and are therefore useful as in vivo tool compounds. Our results show that a lead compound from this series improves insulin sensitivity and glucose control in the diet-induced obesity mouse model after both acute and chronic administration, providing the first in vivo validation of CaMK1D as a target for diabetes therapeutics.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 1/antagonists & inhibitors , Diet/adverse effects , Drug Discovery , Insulin Resistance , Obesity/drug therapy , Obesity/metabolism , Protein Kinase Inhibitors/pharmacology , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 1/chemistry , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Models, Molecular , Obesity/chemically induced , Protein Conformation , Protein Kinase Inhibitors/therapeutic useABSTRACT
There are currently no approved drugs for the treatment of emerging viral infections, such as dengue and Ebola. Adaptor-associated kinase 1 (AAK1) is a cellular serine-threonine protein kinase that functions as a key regulator of the clathrin-associated host adaptor proteins and regulates the intracellular trafficking of multiple unrelated RNA viruses. Moreover, AAK1 is overexpressed specifically in dengue virus-infected but not bystander cells. Because AAK1 is a promising antiviral drug target, we have embarked on an optimization campaign of a previously identified 7-azaindole analogue, yielding novel pyrrolo[2,3- b]pyridines with high AAK1 affinity. The optimized compounds demonstrate improved activity against dengue virus both in vitro and in human primary dendritic cells and the unrelated Ebola virus. These findings demonstrate that targeting cellular AAK1 may represent a promising broad-spectrum antiviral strategy.
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/chemical synthesis , Pyridines/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Cell Line , Chemistry Techniques, Synthetic , Humans , Molecular Docking Simulation , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Pyridines/chemistry , Pyridines/metabolism , Structure-Activity RelationshipABSTRACT
The group A p21-activated kinases (PAKs) exist in an auto-inhibited form until activated by GTPase binding and auto-phosphorylation. In the auto-inhibited form, a regulatory domain binds to the kinase domain (KD) blocking the binding of substrates, and CDC42 or Rac binding to the regulatory domain relieves this auto-inhibition allowing auto-phosphorylation on the KD activation loop. We have determined the crystal structure of the PAK3 catalytic domain and by small angle X-ray scattering, the solution-phase structures of full-length inactive PAK1 and PAK3. The structures reveal a compact but elongated molecular shape that demonstrates that, together with multiple independent biophysical measurements and in contrast with previous assumptions, group A PAKs are monomeric both before and after activation, consistent with an activation mechanism of cis-auto-inhibition and initial cis-auto-phosphorylation, followed by transient dimerisation to allow trans-auto-phosphorylation for full activation, yielding a monomeric active PAK protein.
Subject(s)
p21-Activated Kinases/chemistry , Binding Sites , Biophysical Phenomena , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation , Humans , Kinetics , Models, Molecular , Phosphorylation , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Scattering, Small Angle , Solutions , X-Ray Diffraction , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolismABSTRACT
ß-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.
Subject(s)
Clathrin/metabolism , Endocytosis/physiology , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Protein Serine-Threonine Kinases/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway/physiology , Animals , Feedback, Physiological , HEK293 Cells , Humans , Male , Mice , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5 : 5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB-BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.
Subject(s)
Cullin Proteins/chemistry , Cullin Proteins/metabolism , Potassium Channels, Voltage-Gated/chemistry , Potassium Channels, Voltage-Gated/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Crystallography, X-Ray/methods , Cullin Proteins/genetics , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Potassium Channels, Voltage-Gated/genetics , Protein Binding/physiology , Protein Structure, Secondary , Protein Structure, Tertiary , Ubiquitin-Protein Ligases/geneticsABSTRACT
The highly diverse Numb-associated kinase (NAK) family has been linked to broad cellular functions including receptor-mediated endocytosis, Notch pathway modulation, osteoblast differentiation, and dendrite morphogenesis. Consequently, NAK kinases play a key role in a diverse range of diseases from Parkinson's and prostate cancer to HIV. Due to the plasticity of this kinase family, NAK kinases are often inhibited by approved or investigational drugs and have been associated with side effects, but they are also potential drug targets. The presence of cysteine residues in some NAK family members provides the possibility for selective targeting via covalent inhibition. Here we report the first high-resolution structures of kinases AAK1 and BIKE in complex with two drug candidates. The presented data allow a comprehensive structural characterization of the NAK kinase family and provide the basis for rational design of selective NAK inhibitors.
Subject(s)
Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Binding Sites , Conserved Sequence , Cysteine/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Models, Molecular , Protein Binding , Protein Kinase Inhibitors/metabolism , Transcription Factors/metabolismABSTRACT
Keap1 is a highly redox-sensitive member of the BTB-Kelch family that assembles with the Cul3 protein to form a Cullin-RING E3 ligase complex for the degradation of Nrf2. Oxidative stress disables Keap1, allowing Nrf2 protein levels to accumulate for the transactivation of critical stress response genes. Consequently, the Keap1-Nrf2 system is extensively pursued for the development of protein-protein interaction inhibitors that will stabilize Nrf2 for therapeutic effect in conditions of neurodegeneration, inflammation, and cancer. Here we review current progress toward the structure determination of Keap1 and its protein complexes with Cul3, Nrf2 substrate, and small-molecule antagonists. Together the available structures establish a rational three-dimensional model to explain the two-site binding of Nrf2 as well as its efficient ubiquitination.
Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Models, Molecular , NF-E2-Related Factor 2/chemistry , Oxidative Stress/physiology , Signal Transduction/physiology , Animals , Humans , Intracellular Signaling Peptides and Proteins/metabolism , NF-E2-Related Factor 2/metabolismSubject(s)
Benzamides/chemistry , Death-Associated Protein Kinases/antagonists & inhibitors , Death-Associated Protein Kinases/chemistry , Protein Kinase Inhibitors/chemistry , Pyridazines/chemistry , Receptors, N-Methyl-D-Aspartate/chemistry , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Death-Associated Protein Kinases/genetics , Enzyme Assays , Gene Expression Regulation , High-Throughput Screening Assays , Humans , Kinetics , Ligands , Microfluidic Analytical Techniques , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Binding , Receptors, N-Methyl-D-Aspartate/geneticsABSTRACT
WNK1 [with no lysine (K)] and WNK4 regulate blood pressure by controlling the activity of ion co-transporters in the kidney. Groundbreaking work has revealed that the ubiquitylation and hence levels of WNK isoforms are controlled by a Cullin-RING E3 ubiquitin ligase complex (CRL3KLHL3) that utilizes CUL3 (Cullin3) and its substrate adaptor, KLHL3 (Kelch-like protein 3). Loss-of-function mutations in either CUL3 or KLHL3 cause the hereditary high blood pressure disease Gordon's syndrome by stabilizing WNK isoforms. KLHL3 binds to a highly conserved degron motif located within the C-terminal non-catalytic domain of WNK isoforms. This interaction is essential for ubiquitylation by CRL3KLHL3 and disease-causing mutations in WNK4 and KLHL3 exert their effects on blood pressure by disrupting this interaction. In the present study, we report on the crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif. This reveals an intricate web of interactions between conserved residues on the surface of the Kelch domain ß-propeller and the WNK4 degron motif. Importantly, many of the disease-causing mutations inhibit binding by disrupting critical interface contacts. We also present the structure of the WNK4 degron motif in complex with KLHL2 that has also been reported to bind WNK4. This confirms that KLHL2 interacts with WNK kinases in a similar manner to KLHL3, but strikingly different to how another KLHL protein, KEAP1 (Kelch-like enoyl-CoA hydratase-associated protein 1), binds to its substrate NRF2 (nuclear factor-erythroid 2-related factor 2). The present study provides further insights into how Kelch-like adaptor proteins recognize their substrates and provides a structural basis for how mutations in WNK4 and KLHL3 lead to hypertension.
Subject(s)
Blood Pressure/physiology , Carrier Proteins/physiology , Hypertension/genetics , Intracellular Signaling Peptides and Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Adaptor Proteins, Signal Transducing , Amino Acid Motifs , Blood Pressure/genetics , Carrier Proteins/genetics , Crystallization , Crystallography, X-Ray , Cullin Proteins/genetics , Cullin Proteins/physiology , Humans , Microfilament Proteins/physiology , Minor Histocompatibility Antigens , Nerve Tissue Proteins/physiology , Ubiquitination , WNK Lysine-Deficient Protein Kinase 1ABSTRACT
A series of highly potent indole-3-glyoxylamide based antiprion agents was previously characterized, focusing on optimization of structure-activity relationship (SAR) at positions 1-3 of the indole system. New libraries interrogating the SAR at indole C-4 to C-7 now demonstrate that introducing electron-withdrawing substituents at C-6 may improve biological activity by up to an order of magnitude, and additionally confer higher metabolic stability. For the present screening libraries, both the degree of potency and trends in SAR were consistent across two cell line models of prion disease, and the large majority of compounds showed no evidence of toxic effects in zebrafish. The foregoing observations thus make the indole-3-glyoxylamides an attractive lead series for continuing development as potential therapeutic agents against prion disease.
Subject(s)
Indoles/chemistry , Indoles/pharmacology , Microsomes/drug effects , Prions/drug effects , Animals , Cell Line , Drug Discovery , Indoles/adverse effects , Structure-Activity Relationship , ZebrafishABSTRACT
Structure-activity relationships within the indole-3-glyoxylamide series of antiprion agents have been explored further, resulting in discovery of several new compounds demonstrating excellent activity in a cell line model of prion disease (EC50 <10â nM). After examining a range of substituents at the para-position of the N-phenylglyoxylamide moiety, five-membered heterocycles containing at least two heteroatoms were found to be optimal for the antiprion effect. A number of modifications were made to probe the importance of the glyoxylamide substructure, although none were well tolerated. The most potent compounds did, however, prove largely stable towards microsomal metabolism, and the most active library member cured scrapie-infected cells indefinitely on administration of a single treatment. The present results thereby confirm the indole-3-glyoxylamides as a promising lead series for continuing in vitro and in vivo evaluation against prion disease.
Subject(s)
Indoles , Prion Diseases/drug therapy , Prions , Small Molecule Libraries , Sulfonylurea Compounds , Animals , Cell Line , High-Throughput Screening Assays , Indoles/chemistry , Kinetics , Ligands , Mice , Models, Molecular , Prions/antagonists & inhibitors , Prions/metabolism , Protein Conformation , Quantitative Structure-Activity Relationship , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Sulfonylurea Compounds/chemistryABSTRACT
Amide derivatives of 2,4-diarylthiazole-5-carboxylic acids were synthesised and tested for efficacy in a cell line model of prion disease. A number of compounds demonstrating antiprion activity were thereby identified from the screening libraries, showing improved potency and reproducibility of results relative to amide derivatives of the related 2,4-diphenyl-5-aminothiazole, which have been documented previously. Thus, 'switching' the sense of the amide bond at thiazole C5 revealed a more promising lead series of potential prion disease therapeutics. Furthermore, 3,5-diaryl-1,2,4-thiadiazoles isolated as by-products during library synthesis provided a handful of additional examples possessing an antiprion effect, thereby augmenting the set of newly identified active compounds. Evaluation of binding to cellular prion protein (PrP(C)) showed only weak affinities at best, suggesting that the newly identified antiprion agents do not mediate their biological effect through direct interaction with PrP(C).
Subject(s)
Amides/chemistry , Prions/antagonists & inhibitors , Thiazoles/chemistry , Animals , Cell Line , Humans , Mice , Microsomes, Liver/metabolism , Prions/metabolism , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/toxicityABSTRACT
The anthrax protein protective antigen (PA) is responsible for cell-surface recognition and aids the delivery of the toxic anthrax enzymes into host cells. By targeting PA and preventing it from binding to host cells, it is hoped that the delivery of toxins into the cell will be inhibited. The current assay reported for PA is a low throughput functional assay. Here, the high throughput screening method using differential scanning fluorimetry (DSF) was developed and optimized to screen a number of libraries from various sources including a selection of FDA-approved drugs as well as hits selected by a virtual screening campaign. DSF is a rapid technique that uses fluorescence to monitor the thermal unfolding of proteins using a standard QPCR instrument. A positive shift in the calculated melting temperature (Tm), of the protein in the presence of a compound, relative to the Tm of the unbound protein, indicates that stabilization of the protein by ligand binding may have occurred. Optimization of the melting assay showed SYPRO Orange to be an ideal dye as a marker and lead to the reduction of DMSO concentration to <1% (v/v) in the final assay. The final assay volume was minimized to 25 L with 5 g protein per well of 96-well plate. In addition, a buffer, salt and additive screen lead to the selection of 10 mM HEPES-NaOH pH 7.5, 100 mM NaCl as the assay buffer. This method has been shown here to be useful as a primary method for the detection of small-molecule PA ligands, giving a hit rate of approximately 7%. These ligands can then be studied further using PA functional assays to confirm their biological activities before being selected as lead compounds for the treatment of anthrax.