Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F.

Deregulation of cap-dependent translation is associated with cancer initiation and progression. The rate-limiting step of protein synthesis is the loading of ribosomes onto mRNA templates stimulated by the heterotrimeric complex, eukaryotic initiation factor (eIF)4F. This step represents an attractive target for anticancer drug discovery because it resides at the nexus of the TOR signaling pathway. We have undertaken an ultra-high-throughput screen to identify inhibitors that prevent assembly of the eIF4F complex. One of the identified compounds blocks interaction between two subunits of eIF4F. As a consequence, cap-dependent translation is inhibited. This compound can reverse tumor chemoresistance in a genetically engineered lymphoma mouse model by sensitizing cells to the proapoptotic action of DNA damage. Molecular modeling experiments provide insight into the mechanism of action of this small molecule inhibitor. Our experiments validate targeting the eIF4F complex as a strategy for cancer therapy to modulate chemosensitivity.

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.

Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.

A set of time-resolved fluorescence resonance energy transfer assays for the discovery of inhibitors of estrogen receptor-coactivator binding.

Therapeutic block of estrogen action is typically achieved with conventional antagonists (CAs), compounds that displace estradiol from the estrogen receptor (ER) and induce formation of an ER conformation that cannot bind to coactivator proteins, such as the steroid receptor coactivators (SRCs). As an alternative mode for blocking estrogen action, the authors seek small molecules that act as coactivator binding inhibitors (CBIs)-that is, they compete directly with SRC3 for interaction with estradiol-bound ER. CBIs would be interesting mechanistic probes of estrogen action and might also provide an alternative, more durable endocrine therapy for hormone-responsive breast cancer, where cellular adaptations lead to resistance to CAs. The authors have designed and optimized a set of time-resolved fluorescence resonance energy transfer (TR-FRET) assays to monitor the interaction of ER with SRC3 and ligands, and they have used them in high-throughput screens to discover small-molecule CBIs that are able to disrupt this interaction. These assays also distinguish CBIs from CAs. These robust and sensitive "mix-and-measure" assays use low concentrations of ER labeled with a europium chelate as FRET donor and a Cy5-labeled SRC as acceptor. This multiplexed protocol produces excellent signal-to-noise ratios (>100) and Z' values (>0.8).

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.

An assessment of the performance of the Baxter elastomeric (LV10) Infusor™ pump under hyperbaric conditions.

INTRODUCTION: There are limited data on the use of elastomeric infusion pumps during hyperbaric oxygen treatment. AIM: This study evaluated the flow rate of the Baxter elastomeric LV10 Infusor™ pump under normobaric (101.3 kPa) and three hyperbaric conditions of 203 kPa, 243 kPa and 284 kPa. METHODS: Elastomeric pumps were secured to participants in the same manner as for a typical patient, except that a container collected the delivered antibiotic solution. Pumps and tubing were weighed before and after the test period to determine volume delivered and to calculate flow rates at sea level and the three commonly used hyperbaric treatment pressures at two different time periods, 0-2 hours (h) and 19-21 h into the infusion. RESULTS: The mean flow rates in ml·h⁻¹ (SD) were: 9.5 (0.4), 10.3 (0.6), 10.4 (0.6), 10.4 (0.5) at 0-2 h and 10.5 (1.0), 12.2 (0.6), 9.4 (0.5), 10.3 (0.9) at 19-21 h for the normobaric, 203 kPa, 243 kPa and 284 kPa conditions respectively. There was no significant association between flow rate and time period (P = 0.166) but the 203 kPa flow rates were significantly faster than the other flow rates (P = 0.008). In retrospect, the 203 kPa experiments had all been conducted with the same antibiotic solution (ceftazidime 6 g). Repeating that experimental arm using flucloxacillin 8 g produced flow rates of 10.4 (0.8) ml·h⁻¹, with no significant associations between flow rate and time period (P = 0.652) or pressure (P = 0.705). CONCLUSION: In this study, the flow rate of the Baxter LV10 Infusor™ device was not significantly affected by increases in ambient pressure across the pressure range of 101.3 kPa to 284 kPa, and flow rates were generally within a clinically acceptable range of 9-12 ml·h⁻¹. However, there was evidence that the specific antibiotic solution might affect flow rates and this requires further study.

Performance of the Baxter Infusor LV10 under hyperbaric conditions.

INTRODUCTION: Elastomeric drug delivery devices are a simple way to provide long-term IV therapy to patients in the outpatient setting. Patients receiving hyperbaric oxygen therapy occasionally need these devices. This study compared the performance of the Baxter infusor LV10 elastomeric device in repetitive conditions at pressures of 101.3 kPa and 243 kPa. METHODS: Ten Baxter infusor LV10 elastomeric devices were pressurised in a hyperbaric chamber to 243 kPa over a two-hour period consistent with a standard medical treatment run. This process was repeated 10 times for each device giving a total of 20 hours under pressure. The fluid delivered by each device was measured and the device weighed at the end of each pressurisation. Ten control devices containing identical drugs were tested in the same manner at 101.3 kPa over the same time period. RESULTS: No significant differences in output of the devices were observed between hyperbaric and control conditions. The flow rates measured in both study groups were 35% lower than the manufacturer's stated flow rate, possibly due to lower test environment temperature and outdated devices used in the tests. CONCLUSION: Despite lower than expected flow rates, this study demonstrated no significant difference in the delivery rate of the Baxter infusor LV10 under 243 kPa hyperbaric conditions compared with room pressure.

Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits.

NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits.

A dual-readout F2 assay that combines fluorescence resonance energy transfer and fluorescence polarization for monitoring bimolecular interactions.

Förster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed "Dual-Readout F(2) assay" with F(2) for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F(2) assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.

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.