Characterization of Specific -α-Acetyltransferase 50 (Naa50) Inhibitors Identified Using a DNA Encoded Library.

Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds ( and ) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate.

Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A.

S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.

Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors.

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate.

Substrate-specific conformational regulation of the receptor tyrosine kinase VEGFR2 catalytic domain.

The contributions of the phosphoacceptor and the catalytic domain context to protein kinase biology and inhibitor potency are routinely overlooked, which can lead to mischaracterization of inhibitor and receptor functions. The receptor tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR2) is studied as a model system using a series of phosphoacceptor substrates (k(cat)/K(m) 684-116,000 M(-1) s(-1)) to assess effects on catalysis and inhibitor binding. ATP-competitive inhibitor potency toward the VEGFR2 catalytic domain (VEGFR2-CD) varies with different phosphoacceptor substrates, which is unexpected because the phosphoacceptors do not affect K(m,ATP) values.

Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays.

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains.

Kinase inhibition that hinges on halogen bonds.

A major challenge for the discovery of protein kinase inhibitors is to identify potent, selective, and novel pharmacophores. In this issue, Fedorov et al. (2011) describes KH-CB19, an ATP-competitive inhibitor of cdc2-like kinase that interacts with the ATP hinge region through a halogen-bonding motif.

Identification of novel pyrrolopyrazoles as protein kinase C β II inhibitors.

A novel series of pyrrolopyrazole-based protein kinase C β II inhibitors has been identified from high-throughput screening. Herein, we report our initial structure-activity relationship studies with a focus on optimizing compound ligand efficiency and physicochemical properties, which has led to potent inhibitors with good cell permeability.

Discovery of a novel class of targeted kinase inhibitors that blocks protein kinase C signaling and ameliorates retinal vascular leakage in a diabetic rat model.

Protein kinase C (PKC) family members such as PKCbetaII may become activated in the hyperglycemic state associated with diabetes. Preclinical and clinical data implicate aberrant PKC activity in the development of diabetic microvasculature abnormalities. Based on this potential etiological role for PKC in diabetic complications, several therapeutic PKC inhibitors have been investigated in clinical trials for the treatment of diabetic patients.

Characterization of the CHK1 allosteric inhibitor binding site.

Checkpoint kinase 1 (CHK1) is a key element in the DNA damage response pathway and plays a crucial role in the S-G(2)-phase checkpoint. Inhibiting CHK1 is a therapeutic strategy involving abrogation of the G2/M mitotic checkpoint defense of tumor cells toward lethal damage induced by DNA-directed chemotherapeutic agents. To date, most CHK1 inhibition approaches have involved targeting the ATP site of this kinase.

Breaching the DNA damage checkpoint via PF-00477736, a novel small-molecule inhibitor of checkpoint kinase 1.

Checkpoints are present in all phases of the cell cycle and are regarded as the gatekeepers maintaining the integrity of the genome. Many conventional agents used to treat cancer impart damage to the genome and activate cell cycle checkpoints. Many tumors are defective in the tumor suppressor p53 and therefore lack a functional G(1) checkpoint.

Structure-based design and synthesis of (5-arylamino-2H-pyrazol-3-yl)-biphenyl-2',4'-diols as novel and potent human CHK1 inhibitors.

The cocrystal structure of a library hit was used to design a novel series of CHK1 inhibitors. The new series retained the critical hydrogen-bonding groups of the resorcinol moiety for binding but lacked the phenolic anilide moiety. The newly designed compounds exhibited similar enzymatic activity, while demonstrating increased cellular potency.

Structure of the catalytic domain of human protein kinase C beta II complexed with a bisindolylmaleimide inhibitor.

The conventional protein kinase C isoform, PKCII, is a signaling kinase activated during the hyperglycemic state and has been associated with the development of microvascular abnormalities associated with diabetes. PKCII, therefore, has been identified as a therapeutic target where inhibitors of its kinase activity are being pursued for treatment of microvascular-related diabetic complications. In this report, we describe the crystal structure of the catalytic domain of PKCbetaII complexed with an inhibitor at 2.

Bicyclic amidine inhibitors of nitric oxide synthase: discovery of perhydro-iminopyrindine and perhydro-iminoquinoline as potent, orally active inhibitors of inducible nitric oxide synthase.

Syntheses and nitric oxide synthase inhibitory activity of cyclic amidines containing 5,6- 6,6- and 7,6-fused systems are described. X-ray structure determination facilitated the assignment of the stereochemistry of the most active compounds perhydro-2-iminoisoquinoline (8a) and perhydro-2-iminopyrindine (10a). Both 8a and 10a are very potent inhibitors of iNOS, with excellent selectivity over eNOS and they are orally active in rats with long duration suitable for once or twice a day dosing.

Synthesis of analogs of (1,4)-3- and 5-imino oxazepane, thiazepane, and diazepane as inhibitors of nitric oxide synthases.

A series of 3- and 5-imino analogs from oxazepane, thiazepane, and diazepane was prepared and evaluated as inhibitors of human nitric oxide synthesis (NOS). The most potent iNOS inhibitor was the thiazepane analog 25 (IC(50) = 0.19 microM).

Evaluation of pyrrolidin-2-imines and 1,3-thiazolidin-2-imines as inhibitors of nitric oxide synthase.

Syntheses and evaluation of pyrrolidin-2-imines and 1,3-thiazolidin-2-imines as inhibitors of nitric oxide synthase (NOS) are discussed. An extensive SAR was established for pyrrolidin-2-imines class of compounds. The amidines came out as the most potent inhibitors in addition to displaying selectivity.

Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates.

Components within synthetic chemical and natural product extract libraries often interfere with fluorescence-based assays. Fluorescence interference can result when the intrinsic spectral properties of colored compounds overlap with the fluorescent probes. Typically, fluorescence-based protease assays use peptide amidomethylcoumarin derivatives as substrates.