Using yeast surface display to engineer a soluble and crystallizable construct of hematopoietic progenitor kinase 1 (HPK1).

Hematopoietic progenitor kinase 1 (HPK1) is an intracellular kinase that plays an important role in modulating tumor immune response and thus is an attractive target for drug discovery. Crystallization of the wild-type HPK1 kinase domain has been hampered by poor expression in recombinant systems and poor solubility. In this study, yeast surface display was applied to a library of HPK1 kinase-domain variants in order to select variants with an improved expression level and solubility.

Optimization of Nicotinamides as Potent and Selective IRAK4 Inhibitors with Efficacy in a Murine Model of Psoriasis.

IRAK4 is an attractive therapeutic target for the treatment of inflammatory conditions. Structure guided optimization of a nicotinamide series of inhibitors has been expanded to explore the IRAK4 front pocket. This has resulted in the identification of compounds such as with improved potency and selectivity.

Discovery of Pyridazinone and Pyrazolo[1,5-]pyridine Inhibitors of C-Terminal Src Kinase.

C-terminal Src kinase (CSK) functions as a negative regulator of T cell activation through inhibitory phosphorylation of LCK, so inhibitors of CSK are of interest as potential immuno-oncology agents. Screening of an internal kinase inhibitor collection identified pyridazinone lead , and a series of modifications led to optimized compound . Compound showed potent activity in biochemical and cellular assays and demonstrated the ability to increase T cell proliferation induced by T cell receptor signaling.

Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165.

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain.

Identification of -Methyl Nicotinamide and -Methyl Pyridazine-3-Carboxamide Pseudokinase Domain Ligands as Highly Selective Allosteric Inhibitors of Tyrosine Kinase 2 (TYK2).

As a member of the Janus (JAK) family of nonreceptor tyrosine kinases, TYK2 plays an important role in mediating the signaling of pro-inflammatory cytokines including IL-12, IL-23, and type 1 interferons. The nicotinamide , identified by a SPA-based high-throughput screen targeting the TYK2 pseudokinase domain, potently inhibits IL-23 and IFNα signaling in cellular assays. The described work details the optimization of this poorly selective hit () to potent and selective molecules such as and .

Identification of Imidazo[1,2-]pyridazine Derivatives as Potent, Selective, and Orally Active Tyk2 JH2 Inhibitors.

In sharp contrast to a previously reported series of 6-anilino imidazopyridazine based Tyk2 JH2 ligands, 6-((2-oxo-1-substituted-1,2-dihydropyridin-3-yl)amino)imidazo[1,2-]pyridazine analogs were found to display dramatically improved metabolic stability. The N1-substituent on 2-oxo-1,2-dihydropyridine ring can be a variety of alkyl, aryl, and heteroaryl groups, but among them, 2-pyridyl provided much enhanced Caco-2 permeability, attributed to its ability to form intramolecular hydrogen bonds. Further structure-activity relationship studies at the C3 position led to the identification of highly potent and selective Tyk2 JH2 inhibitor , which proved to be highly effective in inhibiting IFNγ production in a rat pharmacodynamics model and fully efficacious in a rat adjuvant arthritis model.

An improved protocol for amino acid type-selective isotope labeling in insect cells.

An improved expression protocol is proposed for amino acid type-specific [C], [N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449-456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media.

Crystal structures of apo and inhibitor-bound TGFβR2 kinase domain: insights into TGFβR isoform selectivity.

The cytokine TGF-β modulates a number of cellular activities and plays a critical role in development, hemostasis and physiology, as well as in diseases including cancer and fibrosis. TGF-β signals through two transmembrane serine/threonine kinase receptors: TGFβR1 and TGFβR2. Multiple structures of the TGFβR1 kinase domain are known, but the structure of TGFβR2 remains unreported.

Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders.

Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases.

Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase Domain.

Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach.

The influence of adnectin binding on the extracellular domain of epidermal growth factor receptor.

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies.

The structure of human tau-tubulin kinase 1 both in the apo form and in complex with an inhibitor.

Tau-tubulin kinase 1 (TTBK1) is a dual-specificity (serine/threonine and tyrosine) kinase belonging to the casein kinase 1 superfamily. TTBK1 is a neuron-specific kinase that regulates tau phosphorylation. Hyperphosphorylation of tau is implicated in the pathogenesis of Alzheimer's disease.

Development of novel, 384-well high-throughput assay panels for human drug transporters: drug interaction and safety assessment in support of discovery research.

Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.

X-ray crystal structure of bone marrow kinase in the x chromosome: a Tec family kinase.

Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton's tyrosine kinase and IL-2-inducible T-cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X-ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.

Structural basis for CARM1 inhibition by indole and pyrazole inhibitors.

CARM1 (co-activator-associated arginine methyltransferase 1) is a PRMT (protein arginine N-methyltransferase) family member that catalyses the transfer of methyl groups from SAM (S-adenosylmethionine) to the side chain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors such as p53.

Discovery of 6-(aminomethyl)-5-(2,4-dichlorophenyl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamides as potent, selective dipeptidyl peptidase-4 (DPP4) inhibitors.

Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species.

Discovery of pyrrolopyridine-pyridone based inhibitors of Met kinase: synthesis, X-ray crystallographic analysis, and biological activities.

Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4.

Structural basis for the high-affinity binding of pyrrolotriazine inhibitors of p38 MAP kinase.

The crystal structure of unphosphorylated p38alpha MAP kinase complexed with a representative pyrrolotriazine-based inhibitor led to the elucidation of the high-affinity binding mode of this class of compounds at the ATP-binding site. The ligand binds in an extended conformation, with one end interacting with the adenine-pocket hinge region, including a hydrogen bond from the carboxyl O atom of Met109. The other end of the ligand interacts with the hydrophobic pocket of the binding site and with the backbone N atom of Asp168 in the DFG activation loop.

Time-resolved limited proteolysis of mitogen-activated protein kinase-activated protein kinase-2 determined by LC/MS only.

Mass spectrometry has gained prominence in limited proteolysis studies largely due to its unparalleled precision in determining protein molecular mass. However, proteolytic fragments usually cannot be identified through direct mass measurement, since multiple subsequences of a protein can frequently be matched to observed masses of proteolytic fragments. Therefore, additional information from N-terminal sequencing is often needed.

Involvement of DPP-IV catalytic residues in enzyme-saxagliptin complex formation.

The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.

The structure of Dasatinib (BMS-354825) bound to activated ABL kinase domain elucidates its inhibitory activity against imatinib-resistant ABL mutants.

Chronic myeloid leukemia (CML) is caused by the constitutively activated tyrosine kinase breakpoint cluster (BCR)-ABL. Current frontline therapy for CML is imatinib, an inhibitor of BCR-ABL. Although imatinib has a high rate of clinical success in early phase CML, treatment resistance is problematic, particularly in later stages of the disease, and is frequently mediated by mutations in BCR-ABL.

Structural and functional characterization of CFE88: evidence that a conserved and essential bacterial protein is a methyltransferase.

CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase.

Correlation of high-throughput pregnane X receptor (PXR) transactivation and binding assays.

Pregnane X receptor (PXR) transactivation and binding assays have been developed into high-throughput assays, which are robust and reproducible (Z' > 0.5). For most compounds, there was a good correlation between the results of the transactivation and binding assays.