Treatment of NIH-3T3 cells with cyclopamine but not IPI-926 confers hyper-responsiveness to Shh stimulation.

<p>Cells were pre-treated with either cyclopamine or IPI-926 for 24 h followed by extensive washing and subsequent treatment with either media alone or media containing 100 nM rShh. Gli1 mRNA levels were assessed by qRT-PCR 21 h post stimulation. Hyper-responsiveness to rShh stimulation is observed in NIH-3T3 cells treated with high doses of cyclopamine (A) but not IPI-926 (B). Note different Y-axis scales between the 2 graphs. Identical experiments carried out in HEPM cells treated with either cyclopamine (C) or IPI-926 (D) does not result in Shh hyper-responsiveness. No Shh hyper-responsiveness was observed in C3H10T1/2 cells (E), pancreatic xenograft tumor stroma (F) or SW-872 cells (G) when pre-treated with 5 µM cyclopamine or 500 nM IPI-926.</p

IPI-926 is significantly more potent than cyclopamine in cell based assays.

<p>IPI-926 inhibits Shh-induced Gli-Luciferase activity with an EC₅₀ value of 9 nM in NIH-3T3 cells (A) and 2 nM in HEPM cells (B) compared to cyclopamine which had an EC₅₀ value of 315 nM in NIH-3T3 cells (A) and 59 nM in HEPM cells (B) (N = 3). (C) IPI-926 inhibits alkaline phosphatase production resulting from Shh-induced C3H10T1/2 cell differentiation with an EC₅₀ value of 12 nM compared to 399 nM for cyclopamine (N = 2). (D) IPI-926 inhibits the binding of BODIPY-labeled cyclopamine to C3H10T1/2 cells overexpressing wild-type human Smo with an EC₅₀ value of 1 nM compared to an EC₅₀ value of 114 nM for unlabeled cyclopamine.</p

Treatment of NIH-3T3 cells with cyclopamine but not IPI-926 results in increased ciliary Smo accumulation.

<p>In the absence of Shh, NIH-3T3 cells were treated for 24 h with either cyclopamine (A) or IPI-926 (B) and the mean fluorescence intensity of ciliary Smo accumulation was calculated. A positive correlation was observed between increasing doses of cyclopamine and mean ciliary Smo fluorescence intensity. Dose responsive Smo accumulation is not observed with IPI-926. Ten µM cyclopamine yielded the maximal Smo fluorescence intensity (63%) and all other data points were normalized to this max value. Dose responsive Smo accumulation is not observed in HEPM cells treated for 24 h with either cyclopamine (C) or IPI-926 (D).</p

Hyper-responsiveness to paracrine Hh signaling was not observed in IPI-926 treated pancreatic xenografts.

<p>(A) In the MiaPaCa pancreatic xenograft model, 50% tumor growth inhibition was observed with daily oral administration of IPI-926 at 40 mg/kg (N = 12 mice per group). (B) Plasma levels of IPI-926 correlate with the degree and duration of stromal mGli1 inhibition after a single dose of IPI-926 at 40 mg/kg. * indicates a p-value <0.003. Modulation of tumor-derived hGli1 was not observed (data not shown).</p

Smo antagonists differentially localize endogenous Smo protein (red) to the PC (green).

<p>In both NIH-3T3 (A) and HEPM cells (C), 24 h treatment with either cyclopamine (CYC, 5 µM) or IPI-926 (500 nM) in the absence of Shh, results in Smo accumulation to the PC while treatment with GDC-0449 (500 nM) does not. Shh treatment results in Smo accumulation to the PC in both NIH-3T3 cells (B) and HEPM cells (D), which can be blocked by GDC-0449 but not with cyclopamine or IPI-926.</p

Orally Active Fumagillin Analogues: Transformations of a Reactive Warhead in the Gastric Environment

Semisynthetic analogues of fumagillin, <b>1</b>, inhibit methionine aminopeptidase-2 (MetAP2) and have entered the clinic for the treatment of cancer. An optimized fumagillin analogue, <b>3</b> (PPI-2458), was found to be orally active, despite containing a spiroepoxide function that formed a covalent linkage to the target protein. In aqueous acid, <b>3</b> underwent ring-opening addition of water and HCl, leading to four products, <b>4–7</b>, which were characterized in detail. The chlorohydrin, but not the diol, products inhibited MetAP2 under weakly basic conditions, suggesting reversion to epoxide as a step in the mechanism. In agreement, chlorohydrin <b>6</b> was shown to revert rapidly to <b>3</b> in rat plasma. In an ex vivo assay, rats treated with purified acid degradants demonstrated inhibition of MetAP2 that correlated with the biochemical activity of the compounds. Taken together, the results indicate that degradation of the parent compound was compensated by the formation of active equivalents leading to a pharmacologically useful level of MetAP2 inhibition