Synthesis of polyfluoro ketones for selective inhibition of human phospholipase A2 enzymes.

The development of selective inhibitors for individual PLA(2) enzymes is necessary in order to target PLA(2)-specific signaling pathways, but it is challenging due to the observed promiscuity of known PLA(2) inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro, and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA(2), a very important enzyme for which specific, potent, reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the alpha' position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA(2) inhibitors.

Structure-activity relationships of natural and non-natural amino acid-based amide and 2-oxoamide inhibitors of human phospholipase A(2) enzymes.

A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthesized. Their activity on two human intracellular phospholipases (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase (GV sPLA(2)) was evaluated. We show that an amide based on (R)-gamma-norleucine is a highly selective inhibitor of GV sPLA(2).

Rhodanine derivatives as selective protease inhibitors against bacterial toxins.

In this study, we analyzed a series of rhodanine derivatives, as potential inhibitors of bacterial toxins, namely the proteases anthrax lethal factor and the botulinum neurotoxin type A. Conducting an extensive structure-activity relationship study on rhodanine derivatives, we profiled their selectivity against the two bacterial toxins and two related human metalloproteases using in vitro assays. In addition, we examined initial in vitro ADME-Tox properties of selected compounds and their ability to protect lethal factor-induced cell death of macrophages. These data allowed the selection of one additional drug candidate for which preliminary in vivo efficacy studies against anthrax spores were conducted. Integration of these results with our structure-activity relationship studies provides a framework for the development of potential drug candidates against anthrax and botulinum.

Structure-based discovery of a new class of Bcl-xL antagonists.

Apoptosis, or programmed cell death, plays a key role in normal tissue homeostasis ensuring a proper balance between cell production and cell loss. Anti-apoptotic Bcl-2-family proteins are central regulators of the apoptotic pathway and due to their ability to confer tumor resistance to chemotherapy or radiation, have been recently validated as targets for cancer drug discovery. Since the crucial interaction between pro- and anti-apoptotic members occurs via a conserved region located on the surface of the protein, a viable way to inhibit the anti-death activity of Bcl-2 proteins is to design small molecule inhibitors that occupy this cavity. Here, we describe a structure-based approach that led to the identification of four small molecule inhibitors directed at the hydrophobic groove on the surface of the Bcl-2 family protein Bcl-xL. The compounds were characterized in a number of assays including in vitro binding using 15N-labeled protein, a displacement DELFIA assay, and a cell-based viability assay with human cancer cells.

Accelerating the discovery of biologically active small molecules using a high-throughput yeast halo assay.

The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation, resulting in the identification of crambescidin 800 as a potent antifungal agent.

Oxidative inhibition of human soluble catechol-O-methyltransferase.

A common polymorphism in the human gene for catechol-O-methyltransferase results in replacement of Val-108 by Met in the soluble form of the protein (s-COMT) and has been linked to breast cancer and neuropsychiatric disorders. The 108M and 108V variants are reported to differ in their thermal stability, with 108M COMT losing catalytic activity more rapidly. Because human s-COMT contains seven cysteine residues and includes CXXC and CXXS motifs that are associated with thiol-disulfide redox reactions, we examined the effects of reducing and oxidizing conditions on the enzyme. In the absence of a reductant 108M s-COMT lost activity more rapidly than 108V, whereas in the presence of 4 mm dithiothreitol (DTT) we found no significant differences in the stability of the two variants at 37 degrees C. DTT also restored most of the activity that was lost upon incubation at 37 degrees C in the absence of DTT. Mass spectrometry showed that cysteines 188 and 191 formed an intramolecular disulfide bond when s-COMT was incubated with oxidized glutathione, whereas cysteines 69, 95, 157, and 173 formed protein-glutathione adducts. Replacing Cys-95 by serine protected 108M s-COMT against inactivation in the absence of a reductant; C33S and Cys-188 mutations had little effect, and C69S was destabilizing. The sequences surrounding the reactive cysteine residues of human s-COMT and other proteins that form glutathione adducts at identified sites all include Pro and/or Gly and most include a hydrogen-bonding residue, suggesting that glutathiolation at conserved sites plays a physiologically important role.