Tetralogy of fallot with persistent fifth aortic arch: echocardiographic diagnosis.

Although persistent, fifth aortic arch (P5A) is an uncommon anomaly. Its incidence is unknown due largely to failure of recognition. Fundamental to its diagnosis is an adequate understanding of the various settings in which P5A may manifest. One of the more frequently reported manifestations of this unusual anomaly is a systemic-to-pulmonary connection in the presence of pulmonary atresia, with or without a ventriculoseptal defect. This report describes a new case of P5A in an infant with tetralogy of Fallot but not pulmonary atresia, which was diagnosed echocardiographically. The echo-Doppler characteristics of P5A in the presence of right ventricular outflow obstruction are described, and the implications regarding surgery for the accompanying defects are discussed. To the authors' best knowledge, the described case is the first reported instance of P5A in this specific setting.

Silencing of HSP90 cochaperone AHA1 expression decreases client protein activation and increases cellular sensitivity to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin.

AHA1 (activator of HSP90 ATPase) is a cochaperone of the ATP-dependent molecular chaperone, HSP90, which is involved in the maturation, stabilization/degradation, and function of oncogenic proteins. HSP90 operates in a multimeric complex driven by the binding and hydrolysis of ATP. Treatment of cells with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) results in the degradation of client proteins via the ubiquitin-proteasome pathway. As AHA1 increases the ATPase activity of HSP90, we hypothesized that modulation of AHA1 expression could influence the activity of client proteins and/or the cellular response to 17-AAG. We show that the basal expression of AHA1 is different across a panel of human cancer cell lines, and that treatment with 17-AAG resulted in sustained AHA1 up-regulation. Increasing the expression of AHA1 did not affect the sensitivity to 17-AAG, but did increase C-RAF activity and the levels of phosphorylated MEK1/2 and ERK1/2 without affecting total levels of these proteins or of client proteins C-RAF, ERBB2, or CDK4. Conversely, small interfering RNA-selective knockdown of >80% of AHA1 expression decreased C-RAF activity and reduced the levels of MEK1/2 and ERK1/2 phosphorylation. Moreover, the AHA1 knockdown resulted in a significant (P < 0.05) increase in sensitivity to 17-AAG, due in part to a 2- to 3-fold increase in apoptosis. These results show that the reduction of AHA1 levels could decrease the phosphorylation of key signal transduction proteins, and for the first time, separate the activation and stabilization functions of HSP90. Furthermore, AHA1 knockdown could sensitize cancer cells to 17-AAG. We conclude that modulation of AHA1 might be a potential therapeutic strategy to increase sensitivity to HSP90 inhibitors.

Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues.

Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors.