Neuroprotection by selective allosteric potentiators of the EP2 prostaglandin receptor.

Activation of the Galphas-coupled EP2 receptor for prostaglandin E2 (PGE(2)) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 microM (i) potentiated the cAMP response to a low concentration of PGE(2) by > 50%; (ii) had no effect on EP4 or beta2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE(2) on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE(2) on EP2 receptors 4- to 5-fold at 10 to 20 microM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE(2) receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE(2) released in a cell-injury setting is neuroprotective.

Asymmetric synthesis of host-directed inhibitors of myxoviruses.

High-throughput screening (HTS) previously identified benzimidazole 1 (JMN3-003) as a compound with broad antiviral activity against different influenza viruses and paramyxovirus strains. In pursuit of a lead compound from this series for development, we sought to increase both the potency and the aqueous solubility of 1. Lead optimization has achieved compounds with potent antiviral activity against a panel of myxovirus family members (EC(50) values in the low nanomolar range) and much improved aqueous solubilities relative to that of 1. Additionally, we have devised a robust synthetic strategy for preparing 1 and congeners in an enantio-enriched fashion, which has allowed us to demonstrate that the (S)-enantiomers are generally 7- to 110-fold more potent than the corresponding (R)-isomers.

Systematic approaches towards the development of host-directed antiviral therapeutics.

Since the onset of antiviral therapy, viral resistance has compromised the clinical value of small-molecule drugs targeting pathogen components. As intracellular parasites, viruses complete their life cycle by hijacking a multitude of host-factors. Aiming at the latter rather than the pathogen directly, host-directed antiviral therapy has emerged as a concept to counteract evolution of viral resistance and develop broad-spectrum drug classes. This approach is propelled by bioinformatics analysis of genome-wide screens that greatly enhance insights into the complex network of host-pathogen interactions and generate a shortlist of potential gene targets from a multitude of candidates, thus setting the stage for a new era of rational identification of drug targets for host-directed antiviral therapies. With particular emphasis on human immunodeficiency virus and influenza virus, two major human pathogens, we review screens employed to elucidate host-pathogen interactions and discuss the state of database ontology approaches applicable to defining a therapeutic endpoint. The value of this strategy for drug discovery is evaluated, and perspectives for bioinformatics-driven hit identification are outlined.

Synthesis and SAR study of N-(4-hydroxy-3-(2-hydroxynaphthalene-1-yl)phenyl)-arylsulfonamides: heat shock protein 90 (Hsp90) inhibitors with submicromolar activity in an in vitro assay.

Heat shock protein 90 is emerging as an important target in cancer chemotherapy. In a program directed toward identifying novel chemical probes for Hsp90, we found N-(4-hydroxy-3-(2-hydroxynaphthalene-1-yl)phenyl)benzene sulfonamide as an Hsp90 inhibitor with very weak activity. In this report, we present a new and general method for the synthesis of a variety of analogs around this scaffold, and discuss their structure-activity relationships.

Discovery of aminoquinolines as a new class of potent inhibitors of heat shock protein 90 (Hsp90): Synthesis, biology, and molecular modeling.

The molecular chaperone Hsp90 plays important roles in maintaining malignant phenotypes. Recent studies suggest that Hsp90 exerts high-affinity interactions with multiple oncoproteins, which are essential for the growth of tumor cells. As a result, research has focused on finding Hsp90 probes as potential and selective anticancer agents. In a high-throughput screening exercise, we identified quinoline 7 as a moderate inhibitor of Hsp90. Further hit identification, SAR studies, and biological investigation revealed several synthetic analogs in this series with micromolar activities in both fluorescent polarization (FP) assay and a cell-based Western blot (WB) assay. These compounds represent a new class of Hsp90 inhibitors with simple chemical structures.

Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation.

NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47phox and p67phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486.

Synthesis and Metabolic Studies of Host Directed Inhibitors for Anti Viral Therapy.

Targeting host cell factors required for virus replication provides an alternative to targeting pathogen components and represents a promising approach to develop broad-spectrum antiviral therapeutics. High-throughput screening (HTS) identified two classes of inhibitors (2 and 3) with broad-spectrum antiviral activity against ortho- and paramyxoviruses including influenza A virus (IAV), measles virus (MeV), respiratory syncytial virus (RSV), and human parainfluenza virus type 3 (HPIV3). Hit-to-lead optimization delivered inhibitor, 28a, with EC50 values of 0.88 and 0.81 µM against IAV strain WSN and MeV strain Edmonston, respectively. It was also found that compound 28a delivers good stability in human liver S9 fractions with a half-life of 165 minutes. These data establish 28a as a promising lead for antiviral therapy through a host-directed mechanism.

Host-directed Inhibitors of Myxoviruses: Synthesis and in vitro Biochemical Evaluation.

Drugs targeted to viral proteins are highly vulnerable to the development of viral resistance. One little explored approach to the treatment of viral diseases is the development of agents that target host factors required for virus replication. Myxoviruses are predominantly associated with acute disease and, thus, ideally suited for this approach since the necessary treatment time is anticipated to be limited. High-throughput screening previously identified benzimidazole 22407448 with broad anti-viral activity against different influenza virus and paramyxovirus strains. Hit to lead chemistry has generated 6p (JMN3-003) with potent antiviral activity against a panel of myxovirus family members exhibiting EC(50) values in the low nanomolar range.

Cancer and virus leads by HTS, chemical design and SEA data mining.

A variety of medicinal chemistry approaches can be used for the identification of hits, generation of leads and to accelerate the development of drug candidates. The Emory Chemical and Biology Discovery Center (ECBDC) has been an active participant in the NIH's high-throughput screening (HTS) endeavor to identify potent small molecule probes for poorly studied proteins. Several of Emory's projects relate to cancer or virus infection. We have chosen three successful examples including discovery of potent measles virus RNA-dependent RNA polymerase inhibitors, development of Heat Shock Protein 90 (Hsp90) blockers and identification of angiogenesis inhibitors using transgenic Zebrafish as a HTS model. In parallel with HTS, a unique component of the Emory virtual screening (VS) effort, namely, substructure enrichment analysis (SEA) program has been utilized in several cases.

Relationship among ligand conformations in solution, in the solid state, and at the Hsp90 binding site: geldanamycin and radicicol.

The unknown effects of a receptor's environment on a ligand's conformation presents a difficult challenge in predicting feasible bioactive conformations, particularly if the receptor is ill-defined. The primary hypothesis of this work is that a structure's conformational ensemble in solution presents viable candidates for protein binding. The experimental solution profile can be achieved with the NAMFIS (NMR analysis of molecular flexibility in solution) method, which deconvolutes the average NMR spectrum of small flexible molecules into individual contributing conformations with varying populations. Geldanamycin and radicicol are structurally different macrocycles determined by X-ray crystallography to bind to a common site on the cellular chaperone heat shock protein 90 (Hsp90). Without benefit of a receptor structure, NAMFIS has identified the bioactive conformers of geldanamycin and radicicol in CDCl3 solution with populations of 4% and 21%, respectively. Conversely, docking the set of NAMFIS conformers into the unliganded proteins with GLIDE followed by MM-GBSA scoring reproduces the experimental crystallographic binding poses.

Conformations of laulimalide in DMSO-d6.

Laulimalide is one of the newest naturally occurring macrolides known to act as a microtubule stabilizing agent with properties similar to Taxol. It also stands as being one of the most flexible with 18 rotatable bonds. This large number of rotatable bonds allows for approximately 3(18) potential conformers. To examine the conformational energy surface of laulimalide, we have performed an NAMFIS deconvolution analysis for laulimalide in DMSO-d6. The latter has been supplemented with a post-NAMFIS energy analysis at the Becke3LYP/6-31G level that examines the opposing effects of internal hydrogen bonding and syn-pentane interactions. In this way, we have identified 15 laulimalide conformations that can be classified into 5 different families: Supine, Convex, Cobra, Stretch, and Concave motifs.