Activity and structure of human acetyl-CoA carboxylase targeted by a specific inhibitor.

We have studied a series of human acetyl-CoA carboxylase (ACC) 1 and ACC2 proteins with deletions and/or Ser to Ala substitutions of the known phosphorylation sites. In vitro dephosphorylation/phosphorylation experiments reveal a substantial level of phosphorylation of human ACCs produced in insect cells. Our results are consistent with AMPK phosphorylation of Ser29 , Ser80 , Ser1,201 , and Ser1,216 . Phosphorylation of the N-terminal regulatory domain decreases ACC1 activity, while phosphorylation of residues in the ACC central domain has no effect. Inhibition of the activity by phosphorylation is significantly more profound at citrate concentrations below 2 mm. Furthermore, deletion of the N-terminal domain facilitates structural changes induced by citrate, including conversion of ACC dimers to linear polymers. We have also identified ACC2 amino acid mutations affecting specific inhibition of the isozyme by compound CD-017-0191. They form two clusters separated by 60-90 Å: one located in the vicinity of the BC active site and the other one in the vicinity of the ACC1 phosphorylation sites in the central domain, suggesting a contribution of the interface of two ACC dimers in the polymer to the inhibitor binding site.

Discovery and optimization of novel constrained pyrrolopyridone BET family inhibitors.

Novel conformationally constrained BET bromodomain inhibitors have been developed. These inhibitors were optimized in two similar, yet distinct chemical series, the 6-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (A) and the 1-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (B). Each series demonstrated excellent activity in binding and cellular assays, and lead compounds from each series demonstrated significant efficacy in in vivo tumor xenograft models.

Integrated Platform for Expedited Synthesis-Purification-Testing of Small Molecule Libraries.

The productivity of medicinal chemistry programs can be significantly increased through the introduction of automation, leading to shortened discovery cycle times. Herein, we describe a platform that consolidates synthesis, purification, quantitation, dissolution, and testing of small molecule libraries. The system was validated through the synthesis and testing of two libraries of binders of polycomb protein EED, and excellent correlation of obtained data with results generated through conventional approaches was observed. The fully automated and integrated platform enables batch-supported compound synthesis based on a broad array of chemical transformations with testing in a variety of biochemical assay formats. A library turnaround time of between 24 and 36 h was achieved, and notably, each library synthesis produces sufficient amounts of compounds for further evaluation in secondary assays thereby contributing significantly to the shortening of medicinal chemistry discovery cycles.

Development of inverse electron demand Diels-Alder ligation and TR-FRET assays for the determination of ligand-protein target occupancy in live cells.

Determination of target engagement following drug administration under physiological conditions is essential for understanding clinical outcomes of therapeutic candidates. While the list of potential techniques that enable studies of target engagement is continuously expanding, identification of the best method to evaluate interactions between a ligand and its cellular binding partner(s) remains far from straightforward. We developed and compared the applicability of two label-based techniques; inverse electron demand Diels-Alder (IED-DA) ligation-based pull-down and TR-FRET assays for in-cell determination of target occupancy of c-Src kinase and p38-α kinase by the reversible inhibitor Dasatinib. Significantly, none of the assays required engineering proteins-of-interest. Moreover, cellular TR-FRET assay emerged as a very promising platform for the determination of target occupancy of specific protein in a high-throughput manner. Our studies suggest that both IED-DA assay and TR-FRET assay should be considered as methods of choice for the determination of target engagement of small molecule protein binders in live cells.

2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification.

Photoaffinity labels are powerful tools for dissecting ligand-protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C-H/X-H bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. Herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (ACT), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding affinity and specificity of the parent drug. Compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies ACT showed higher photo-cross-linking yields toward their protein targets in vitro based on mass spectrometry analysis. In the in situ target identification studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine. We expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery.

Design strategies to address kinetics of drug binding and residence time.

The kinetics of drug binding and drug residence time are recognized to be important in the clinical effectiveness of drug candidates. In most cases a long residence time of the drug-target complex results in an extended duration of pharmacodynamic activity, even when systemic concentrations of drug have been notably reduced through elimination routes. Hence, if selective for target, long residence times can increase the duration of drug efficacy in vivo and can significantly diminish the potential for off-target-mediated toxicities. Furthermore, a compound with a slower dissociation rate may allow a reduced dosing schedule relative to a compound with a rapid dissociation rate. Factors contributing to long residence time that could be useful to medicinal chemists in the prospective design of compounds with long residence times will be discussed in this perspective. Particular emphasis will be on case studies highlighting how kinetics can be measured, modulated based on supporting structure kinetic relationships and whether these effects are translatable into man.

Resistance to herbicides caused by single amino acid mutations in acetyl-CoA carboxylase in resistant populations of grassy weeds.

Eleven spontaneous mutations of acetyl-CoA carboxylase have been identified in many herbicide-resistant populations of 42 species of grassy weeds, hampering application of aryloxyphenoxypropionate, cyclohexadione and phenylpyrazoline herbicides in agriculture. IC(50) shifts (resistance indices) caused by herbicide-resistant mutations were determined using a recombinant yeast system that allows comparison of the effects of single amino acid mutations in the same biochemical background, avoiding the complexity inherent in the in planta experiments. The effect of six mutations on the sensitivity of acetyl-CoA carboxylase to nine herbicides representing the three chemical classes was studied. A combination of partially overlapping binding sites of the three classes of herbicides and the structure of their variable parts explains cross-resistance among and between the three classes of inhibitors, as well as differences in their specificity. Some degree of resistance was detected for 51 of 54 herbicide/mutation combinations. Introduction of new herbicides targeting acetyl-CoA carboxylase will depend on their ability to overcome the high degree of cross-resistance already existing in weed populations.

Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity.

Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In "proof of concept" experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 muM IC(50) and having no effect on human ACC1 at 100 muM.