Characterization of Specific N-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 (3a and 4a) 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. Cellular target engagement experiments further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding mode observed in the cocrystal structures.

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 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.

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. Indazole-based inhibitors are up to 60-fold more potent with two substrates (gastrin, minigastrin) relative to the others. Thus there is a component of uncompetitive inhibition because a specific phosphoacceptor enhances potency but is not strictly required. This substrate-specific inhibitory potency enhancement correlates with phosphoacceptor active site saturation and is not observed with other related kinases. The effect is confined to a specific catalytic domain conformation because autophosphorylation eliminates the potency enhancement as does the addition of the juxtamembrane domain (20 amino acids). Indazole inhibitor structure-activity analysis reveals that the magnitude of potency enhancement correlates with the size of the substituent that binds in a regulatory region of the active site. VEGFR drugs profiled with VEGFR2-CD using minigastrin have potency well-correlated with inhibition of full-length, cellular VEGFR2 autophosphorylation, an indication that the minigastrin-induced conformation is biologically relevant. These findings raise the possibility that inhibitors directed toward a common target can have different biological effects based on the kinase-substrate complexes present in different cellular contexts.

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.

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. However, because many organic compounds absorb in the ultraviolet region, they can interfere with coumarin-based fluorescence assays. Red-shifted fluorescent dyes such as peptidyl rhodamine derivatives are useful because there is generally less interference from organic compounds outside the ultraviolet wavelengths. In this report, rhodamine-based fluorogenic substrates, such as bis-(Leu)(2)-Rhod110 and bis-(Ala-Pro)-Rhod110, were developed for leucine aminopeptidase and dipeptidyl aminopeptidase. Novel, tandem rhodamine substrates such as Ala-Pro-Rhod110-Leu were designed with 2 protease cleavage sites and used to assay 2 proteases in a multiplex format. General endpoint high-throughput screening (HTS) assays were also developed for leucine aminopeptidase, dipeptidyl aminopeptidase, and trypsin that incorporated both amidomethylcoumarin and rhodamine-based fluorogenic substrates into a single screening format. These dual-substrate assays allowed for the successful screening of the LOPAC trade mark collection and natural product extracts despite high levels of fluorescence interference.