Imidazo[1,2-b]pyridazines as inhibitors of DYRK kinases.

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.

Chemical Properties, Ruminal Fermentation, Gas Production and Digestibility of Silages Composed of Spineless Cactus and Tropical Forage Plants for Sheep Feeding.

The aim was to evaluate the chemical composition, carbohydrates, protein fractionation and in vitro gas production of silages composed of spineless cactus and tropical forages and their effect on sheep performance. Treatments consisted of silages: corn silage (CS), spineless cactus silage (SCS), spineless cactus + gliricidia (SCG), spineless cactus + buffel grass silage (SCBG) and spineless cactus + pornunça (SCP). Silos were opened 60 days after ensiling, and analyses were carried out. The digestibility test lasted for 36 days, with eight animals per treatment. A completely randomized design was adopted. Considering carbohydrate fractionation, CS, SCS and SCBG silages had higher total carbohydrate content (p = 0.001). The SCS silage presented a higher A + B1 fraction (p = 0.001). The SCBG and SCG silages showed a higher B2 fraction (p < 0.0001) compared to the CS and SCS silages. The SCBG and SCP silages presented a higher C fraction (p = 0.001). For protein fractionation, the SCP and SCG silages showed higher crude protein contents (p = 0.001). The CS and SCS silages showed a higher A fraction (p = 0.001). The SCBG silage presented a higher B1 + B2 fraction (p = 0.001). The SCG silage showed a higher B3 fraction (p = 0.006) compared to SCBG silage. The SCS and SCP silages showed a higher C fraction (p = 0.001). Exclusive SCS silage showed higher in vitro dry matter digestibility (p = 0.001), dry matter degradability (p = 0.001) and total gas production (p = 0.001). The use of the SCBG, SCP and SCG silages to feed sheep increased the dry matter intake (p < 0.001). Sheep fed the SCG silage showed greater dry matter and crude protein digestibility compared to the sheep fed the CS, SCS and SCP silages (p = 0.002). There was a higher water intake (p < 0.001) with the use of the SCS and SCG silages to feed the sheep. The SCP and SCG silages provided a greater intake (p < 0.001) and excretion (p < 0.001) of nitrogen by the animals. Although there were no differences between the treatments for daily gains, lambs that received the spineless cactus-based silage associated with tropical forages showed higher gains (160-190 g/day) than lambs that received CS silage (130 g/day). Thus, the use of spineless cactus associated with buffelgrass, pornunça and gliricidia to prepare mixed silages (60:40) to feed sheep has potential use to feed sheep, with positive effects on nutrient degradation and increases in dry matter intake. Under experimental conditions, we recommend the exclusive use of spineless cactus silage associated with buffel grass, pornunça and gliricidia in feeding sheep in semi-arid regions, as it provides nutrients, water and greater daily gains compared to corn silage.

Discovery of pyrazolo[3,4-d]pyrimidines as novel mitogen-activated protein kinase kinase 3 (MKK3) inhibitors.

The dual-specificity protein kinase MKK3 has been implicated in tumor cell proliferation and survival, yet its precise role in cancer remains inconclusive. A critical step in elucidating the kinase's involvement in disease biology is the identification of potent, cell-permeable kinase inhibitors. Presently, MKK3 lacks a dedicated tool compound for these purposes, along with validated methods for the facile screening, identification, and optimization of inhibitors. In this study, we have developed a TR-FRET-based enzymatic assay for the detection of MKK3 activity in vitro and a BRET-based assay to assess ligand binding to this enzyme within intact human cells. These assays were instrumental in identifying hit compounds against MKK3 that share a common chemical scaffold, sourced from a library of bioactive kinase inhibitors. Initial hits were subsequently expanded through the synthesis of novel analogs. The resulting structure-activity relationship (SAR) was rationalized using molecular dynamics simulations against a homology model of MKK3. We expect our findings to expedite the development of novel, potent, selective, and bioactive inhibitors, thus facilitating investigations into MKK3's role in various cancers.

Discovery of novel benzothiophene derivatives as potent and narrow spectrum inhibitors of DYRK1A and DYRK1B.

The discovery of potent and selective inhibitors for understudied kinases can provide relevant pharmacological tools to illuminate their biological functions. DYRK1A and DYRK1B are protein kinases linked to chronic human diseases. Current DYRK1A/DYRK1B inhibitors also antagonize the function of related protein kinases, such as CDC2-like kinases (CLK1, CLK2, CLK4) and DYRK2. Here, we reveal narrow spectrum dual inhibitors of DYRK1A and DYRK1B based on a benzothiophene scaffold. Compound optimization exploited structural differences in the ATP-binding sites of the DYRK1 kinases and resulted in the discovery of 3n, a potent and cell-permeable DYRK1A/DYRK1B inhibitor. This compound has a different scaffold and a narrower off-target profile compared to current DYRK1A/DYRK1B inhibitors. We expect the benzothiophene derivatives described here to aid establishing DYRK1A/DYRK1B cellular functions and their role in human pathologies.

Discovery and Characterization of Selective and Ligand-Efficient DYRK Inhibitors.

Dual-specificity tyrosine-regulated kinase 1A (DYRK1A) regulates the proliferation and differentiation of neuronal progenitor cells during brain development. Consequently, DYRK1A has attracted interest as a target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Down's syndrome. Recently, the inhibition of DYRK1A has been investigated as a potential treatment for diabetes, while DYRK1A's role as a mediator in the cell cycle has garnered interest in oncologic indications. Structure-activity relationship (SAR) analysis in combination with high-resolution X-ray crystallography leads to a series of pyrazolo[1,5-b]pyridazine inhibitors with excellent ligand efficiencies, good physicochemical properties, and a high degree of selectivity over the kinome. Compound 11 exhibited good permeability and cellular activity without P-glycoprotein liability, extending the utility of 11 in an in vivo setting. These pyrazolo[1,5-b]pyridazines are a viable lead series in the discovery of new therapies for the treatment of diseases linked to DYRK1A function.

Water intake and ingestive behavior of sheep fed diets based on silages of cactus pear and tropical forages.

The objective was to evaluate the water intake and ingestive behavior of sheep fed diets containing silages of cactus pear combined with tropical forages. Forty sheep without defined breed, intact, with initial average weight of 22.65 ± 1.01 kg were distributed in a completely randomized design with 5 treatments and 8 replications. The experimental diets consisted of cactus pear silage (CPS), cactus pear + buffel grass silage (CPBS), cactus pear + gliricidia silage (CPGS), cactus pear + pornunça silage (CPPS), and corn silage (CS). CPGS provided higher water intake via food, total water intake, metabolic water, and excretion via feces and urine (P < 0.05). Animals that received diets containing CS showed higher water intake via drinking fountain, less efficient feeding and rumination of dry matter, less efficient rumination of neutral detergent fiber, grams of dry matter per cud, grams of neutral detergent fiber per cud, and the shortest average time spent in chewing per cud (P < 0.05). CPGS, CPPS, and CS provided longer times for rumination and numbers of cuds per day (P < 0.05). CPS showed animals spending more time in idleness, lower quantity of cuds per minute, higher concentration of crystals in urine, with a higher frequency of ammonia-magnesium phosphate and calcium oxalate. Silages based on cactus pear are an alternative to the supply of water via food for sheep in semi-arid.

Binding and structural analyses of potent inhibitors of the human Ca2+/calmodulin dependent protein kinase kinase 2 (CAMKK2) identified from a collection of commercially-available kinase inhibitors.

Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.

Validation of the protein kinase PfCLK3 as a multistage cross-species malarial drug target.

The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the Plasmodium falciparum protein kinase PfCLK3, which we used in combination with chemogenetics to validate PfCLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for PfCLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of PfCLK3 mediated rapid killing of asexual liver- and blood-stage P. falciparum and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against P. berghei and P. knowlesi Hence, our data establish PfCLK3 as a target for drugs, with the potential to offer a cure-to be prophylactic and transmission blocking in malaria.

SGC-GAK-1: A Chemical Probe for Cyclin G Associated Kinase (GAK).

We describe SGC-GAK-1 (11), a potent, selective, and cell-active inhibitor of cyclin G-associated kinase (GAK), together with a structurally related negative control SGC-GAK-1N (14). 11 was highly selective in an in vitro kinome-wide screen, but cellular engagement assays defined RIPK2 as a collateral target. We identified 18 as a potent RIPK2 inhibitor lacking GAK activity. Together, this chemical probe set can be used to interrogate GAK cellular biology.

WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop.

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.

Structural Analysis of Inhibitor Binding to CAMKK1 Identifies Features Necessary for Design of Specific Inhibitors.

The calcium/calmodulin-dependent protein kinases (CAMKKs) are upstream activators of CAMK1 and CAMK4 signalling and have important functions in neural development, maintenance and signalling, as well as in other aspects of biology such as Ca2+ signalling in the cardiovascular system. To support the development of specific inhibitors of CAMKKs we have determined the crystal structure of CAMKK1 with two ATP-competitive inhibitors. The structures reveal small but exploitable differences between CAMKK1 and CAMKK2, despite the high sequence identity, which could be used in the generation of specific inhibitors. Screening of a kinase inhibitor library revealed molecules that bind potently to CAMKK1. Isothermal titration calorimetry revealed that the most potent inhibitors had binding energies largely dependent on favourable enthalpy. Together, the data provide a foundation for future inhibitor development activities.

1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors.

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.

Identification and Optimization of 4-Anilinoquinolines as Inhibitors of Cyclin†G Associated Kinase.

4-Anilinoquinolines were identified as potent and narrow-spectrum inhibitors of the cyclin G associated kinase (GAK), an important regulator of viral and bacterial entry into host cells. Optimization of the 4-anilino group and the 6,7-quinoline substituents produced GAK inhibitors with nanomolar activity, over 50 000-fold selectivity relative to other members of the numb-associated kinase (NAK) subfamily, and a compound (6,7-dimethoxy-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine; 49) with a narrow-spectrum kinome profile. These compounds may be useful tools to explore the therapeutic potential of GAK in prevention of a broad range of infectious and systemic diseases.

Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations.

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors.

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.

High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1.

Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome.

PURPOSE: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. METHODS: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c.3277C>T, a nonsense mutation, and c.3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. RESULTS: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. CONCLUSIONS: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors.

Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3-dependent manner. A 9-amino-acid peptide derived from full-length TR3 with polyarginine tail (TR3-r8) recapitulates TR3's binding specificity, displaying high affinity for Bcl-B. TR3-r8 peptide was used to screen for small molecule Bcl-B inhibitors. A fluorescence polarization assay (FPA) employing fluorescein isothiocyanate (FITC)-labeled TR3-r8 peptide (FITC-TR3-r8) and Bcl-B protein was optimized, with nonfluorescent TR3-r8 serving to demonstrate reversible, competitive binding. Approximately 50,000 compounds were screened at 3.75 mg/L, yielding 145 reproducible hits with > or =50% FITC-TR3-r8 displacement (a confirmed hit rate of 0.29%). After dose-response analyses and counterscreening with an unrelated FITC-based FPA, 6 candidate compounds remained. Nuclear magnetic resonance (NMR) showed that 2 of these compounds bound Bcl-B, but not glutathione S-transferase (GST) control protein. One Bcl-B-binding compound was unable to displace FITClabeled BH3 peptides from Bcl-B, confirming a unique binding mechanism compared with traditional antagonists of antiapoptotic Bcl-2-family proteins. This compound bound Bcl-B with Kd 1.94 +/- 0.38 microM, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrated that this compound induced Bcl-B-dependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins.

Structure of the thiazole biosynthetic enzyme THI1 from Arabidopsis thaliana.

Thiamin pyrophosphate is an essential coenzyme in all organisms that depend on fermentation, respiration or photosynthesis to produce ATP. It is synthesized through two independent biosynthetic routes: one for the synthesis of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (pyrimidine moiety) and another for the synthesis of 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate (thiazole moiety). Herein, we will describe the three-dimensional structure of THI1 protein from Arabidopsis thaliana determined by single wavelength anomalous diffraction to 1.6A resolution. The protein was produced using heterologous expression in bacteria, unexpectedly bound to 2-carboxylate-4-methyl-5-beta-(ethyl adenosine 5-diphosphate) thiazole, a potential intermediate of the thiazole biosynthesis in Eukaryotes. THI1 has a topology similar to dinucleotide binding domains and although details concerning its function are unknown, this work provides new clues about the thiazole biosynthesis in Eukaryotes.