Slow self-activation enhances the potency of viridin prodrugs

Abstract

When the viridin wortmannin (Wm) is modified by reaction with certain nucleophiles at the C20 position, the compounds obtained exhibit an improved antiproliferative activity even though a covalent reaction between C20 and a lysine in the active site of PI3 kinase is essential to Wm's ability to inhibit this enzyme. Here we show that this improved potency results from an intramolecular attack by the C6 hydroxyl group that slowly converts these inactive prodrugs to the active species Wm over the 48 h duration of the antiproliferative assay. Our results provide a guide for selecting Wm-like compounds to maximize kinase inhibition with the variety of protocols used to assess the role of PI3 kinase in biological systems, or for achieving optimal therapeutic effects in vivo . In addition, the slow self-activation of WmC20 derivatives provides a mechanism that can be exploited to obtain kinase inhibitors endowed with physical and pharmacokinetic properties far different from man-made kinase inhibitors because they do not bind to kinase active sites.

Figure 1. Synthesis of fluorescent WmC20 derivatives

Wm 1 is modified at C11 by the attachment of NBD and a 6 carbon linker to obtain NBD-Wm, 3. When 3 is modified at C20, it becomes a self-activating fluorescent derivative by virtue of an intramolecular attack of the hydroxyl at C6 which forms Wm as shown in greater detail in Figure 1 of (29). The physical properties of low molecular weight fluorescent derivatives (4a, 4b) can be further altered by reaction with an amine “carrier,” as illustrated by their reaction with amino dextran to form 6a and 6b. Oxygens at C3 and C6 flanking C20 form hydrogen bonds when Wm (or NBD-Wm) has reacted with a lysine in the ATP binding site of PI3 kinase gamma, see discussion regarding Figure 4.

Figure 2. Time course for the formation of wortmannylated cell protein when cells were incubated with NBD-Wm or NBD-WmC20 compounds

Multiple wortmannylated (NBDylated) proteins were seen with the principal bands at 35 kDa (possibly a doublet), 55 kDa and 75 kDa. With all compounds the 55 kDa band appears first. Time course of cell protein labeling (total area of 35, 55 and 75 kDa bands) was further analyzed as shown in Figure 3A.

Figure 3. Time courses for the behavior of fluorescent WmC20 derivatives in cells

(A) Time course for the formation of wortmannylated cell protein with the C20 reactive NBD-Wm 3 or NBD-WmC20 compounds. (B) Time course for the change in cell fluorescence, reflecting uptake of NBD. The relative cell fluorescence (RCF) is shown on a short time scale (0-1 h) and long time scale (0-24 h). The y-axis is the same for both. Lines in (A) or (B) (0-1 h) were obtained by fitting data to the equations given in Methods, with the relative initial rates given in Table 1.

Figure 4. Binding of NBD-Wm 3 into the active site of PI3 kinase gamma

Model is based on the published structure of Wm in the ATP site of PI3 kinase gamma (PDB: 1E7U) with NBD adduct added. (A) Desacyl Wm (Wm lacking the acetyl group at C11) is orange. The NBD adduct (NBD and six carbon linker) is colored cyan, with nitrogen in blue and oxygen in red. The NBD and the six-carbon linker protrude out of the ATP-binding pocket into the solvent while Wm is buried deeply in the ATP-binding pocket. (B). Another view of NBD-Wm showing the relationship between the surface of the enzyme and NBD-Wm. Lysine 833 covalently reacted with the C20 of Wm, is in magenta and extends through a blue, positively, charged surface. The proximity of the surface prevents Wm’s modified at C20 from binding in the ATP-binding pocket.

Figure 5. Fate of NBD-WmC20 derivatives in biological systems

(A) PI3 kinase assay: With a 0.5 h incubation, fast and slow activating WmC20 derivatives partially activate by generating NBD-Wm 3 and have higher IC50’s than non C20 protected forms of Wm like 3. 3 is stable and active under these conditions and has a low IC50. (B) Antiproliferative assay: NBD-Wm in culture media reacts with nucleophiles like amino acids, yielding a mixture of NBD-WmC20, components of which slowly regenerate NBD-Wm, endowing the culture media with a continuing antiproliferative activity though NBD-Wm has disappeared. When a slowly NBD-Wm forming prodrug (4a or 6a) is used, the viridin NBD-Wm forms over the assay period. The slow formation of 4a or 6a an increased potency relative to 3, though the slow formation of NBD-Wm makes these compounds poor PI3 kinase inhibitors. NBD-Wm rapidly enters cells after release.