Discovery of potent inhibitors of soluble epoxide hydrolase by combinatorial library design and structure-based virtual screening.

Structure-based virtual screening was applied to design combinatorial libraries to discover novel and potent soluble epoxide hydrolase (sEH) inhibitors. X-ray crystal structures revealed unique interactions for a benzoxazole template in addition to the conserved hydrogen bonds with the catalytic machinery of sEH. By exploitation of the favorable binding elements, two iterations of library design based on amide coupling were employed, guided principally by the docking results of the enumerated virtual products. Biological screening of the libraries demonstrated as high as 90% hit rate, of which over two dozen compounds were single digit nanomolar sEH inhibitors by IC(50) determination. In total the library design and synthesis produced more than 300 submicromolar sEH inhibitors. In cellular systems consistent activities were demonstrated with biochemical measurements. The SAR understanding of the benzoxazole template provides valuable insights into discovery of novel sEH inhibitors as therapeutic agents.

Evaluation of the rubidium efflux assay for preclinical identification of HERG blockade.

Inhibition of the delayed-rectifier potassium channel current, human ether-a-go-go (hERG), by pharmaceutical agents can lead to acquired long QT syndrome and the generation of potentially lethal arrhythmias and sudden death. There remains an unmet need for higher-throughput assays to screen compounds in preclinical development for the potential to block hERG and cause QT prolongation. We evaluated the rubidium efflux assay for its ability to determine block of the hERG potassium channel. hERG-transfected human embryonic kidney-293 cells were cultured on 96-well assay plates and loaded with rubidium ion by incubating in media in which potassium was replaced by 5.4 mM Rb+. Cells were exposed to test compounds and then depolarized with a K+ channel opening buffer containing 50 mM K+. The supernatant was removed, and cells were lysed using 0.1% Triton X-100. Concentration-response curves were generated for test agents by determining the Rb+ efflux using a flame atomic absorption spectrometer. Multiple trials with cisapride yielded 50% inhibitory concentration values between 308.1 +/- 11 nM to 456.3 +/- 24 nM for inhibition of Rb+ efflux and a Z factor of 0.80 +/- 0.07 (n = 5 plates, 12 wells per plate). The values for inhibition of the hERG channel exhibited a rightward shift in potency as compared to those measured using electrophysiological techniques. In addition, we evaluated 19 blinded compounds at 10 microM in the Rb+ efflux assay, and compared results to those using patch clamp electrophysiology and the dofetilide displacement binding assay. The dofetilide displacement binding assay yielded a good correlation with electrophysiological measurements of hERG block. The rubidium efflux assay lacked sensitivity to consistently identify significant channel blockade. In conclusion, the rubidium efflux assay provides a higher-throughput means to identify potent hERG channel blocking agents, but lacks the sensitivity required to accurately determine the potency of blockade.

Inhibition of cyclooxygenase-2 by celecoxib reverses tumor-induced wasting.

There have been a number of reports suggesting inhibition of prostaglandin production may impact tumor-mediated wasting and levels of associated humoral factors such as hypercalcemia. These reductions were achieved using traditional nonsteroidal anti-inflammatory drugs (NSAIDs), which are often contraindicated in cancer patients. This is especially true during chemotherapeutic regimens due to concerns of bleeding from gastrointestinal and hematopoietic toxicities associated with inhibition of the housekeeping cyclooxygenase enzyme COX-1. Here, we report that celecoxib, one of the new class of selective COX-2 inhibitors, has the potential to reverse tumor-mediated wasting and associated humoral factors such as interleukin (IL)-6 and hypercalcemia in preclinical models of cachexia. Tumor bearing mice in late stage cachexia regained weight within days of the start of celecoxib treatment. Two models were tested. The first was the Colon 26 (Col26) syngeneic murine model that induces high levels of circulating IL-6 and hypercalcemia. The second was the human head and neck 1483 HNSCC xenograft model, which is less inflammatory and produces less prostaglandin than Col26. Despite the observation that no significant impact on tumor growth was observed between vehicle and celecoxib-treated animals over the course of the studies, celecoxib rapidly reversed weight loss in both cachectic models. With the added safety of celecoxib over traditional NSAIDs, these results suggest a possible therapeutic use for celecoxib for treating tumor-mediated wasting.

Synergy between celecoxib and radiotherapy results from inhibition of cyclooxygenase-2-derived prostaglandin E2, a survival factor for tumor and associated vasculature.

Previous work has demonstrated that selective cyclooxygenase-2 (COX-2) inhibitors can act synergistically with radiotherapy to improve tumor debulking and control in preclinical models. The underlying mechanism of this remarkable activity has not yet been determined. Here, we report that radiation can elevate intratumoral levels of COX-2 protein and its products, particularly prostaglandin E(2) (PGE(2)). Furthermore, inhibition of COX-2 activity or neutralization of PGE(2) activity enhances radiotherapy even in tumors where COX-2 expression is restricted to the tumor neovasculature. Direct assessment of vascular function by direct contrast enhancement-magnetic resonance imaging showed that the combination of radiation and celecoxib lead to enhanced vascular permeability. These observations suggest that an important mechanism of celecoxib-induced radiosensitization involves inhibition of COX-2-derived PGE(2), thus removing a survival factor for the tumor and its vasculature.