Anticancer steroid sulfatase inhibitors: synthesis of a potent fluorinated second-generation agent, in vitro and in vivo activities, molecular modeling, and protein crystallography

Abstract

An improved steroid sulfatase inhibitor was prepared by replacing the N-propyl group of the second-generation steroid-like inhibitor (2) with a N-3,3,3-trifluoropropyl group to give (10). This compound is 5-fold more potent in vitro, completely inhibits rat liver steroid sulfatase activity after a single oral dose of 0.5 mg/kg, and exhibits a significantly longer duration of inhibition over (2). These biological properties are attributed to the increased lipophilicity and metabolic stability of (10) rendered by its trifluoropropyl group and also the potential H-bonding between its fluorine atom(s) and Arg 98 in the active site of human steroid sulfatase. Like other sulfamates, (10) is expected to be sequestered, and transported by, erythrocytes in vivo because it inhibits human carbonic anhydrase II (hCAII) potently (IC 50 , 3 nmol/L). A congener (4), which possesses a N-(pyridin-3-ylmethyl) substituent, is even more active (IC 50 , 0.1 nmol/L). To rationalize this, the hCAII-(4) adduct, obtained by cocrystallization, reveals not only the sulfamate group and the backbone of (4) interacting with the catalytic site and the associated hydrophobic pocket, respectively, but also the potential H-bonding between the N-(pyridin-3-ylmethyl) group and NE 2 of Gln 136 . Like (2), both (10) and its phenolic precursor (9) are non-estrogenic using a uterine weight gain assay. In summary, a highly potent, long-acting, and nonestrogenic steroid sulfatase inhibitor was designed with hCAII inhibitory properties that should positively influence in vivo behavior. Compound (10) and other related inhibitors of this structural class further expand the armory of steroid sulfatase inhibitors against hormone-dependent breast cancer. [Mol Cancer Ther 2008;7(8):2435 -44]

Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations

“…Crystal structure of hCA II in complex with compound 3 was determined at 1.80 Å resolution, revealing a clear electron density for the inhibitor molecule in the enzyme active site (Figure 2 [49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65] , compound 1 interacts directly with the zinc ion of the active site, with its sulphamate nitrogen atom N1 (for atom numbering see Figure 1) displacing the water molecule/hydroxide ion, which in the not-inhibited enzyme occupies the fourth coordination position. Additional hydrogen bonds between the sulphamate moiety and residues within the enzyme active site contribute to stabilise the binding.…”

“…To understand if the different position assumed by N2 and O3 atoms in the enzyme active site was associated to a peculiarity of the two complexes under investigation, or to a more general behaviour of sulphamate and sulphamide derivatives, a comparative analysis of all hCA II/sulphamate and hCA II/sulphamide structures available in the PDB was undertaken 25,26,[49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][67][68][69][70][71] . Surprisingly, the analysis of all these structures revealed that, independently of the nature of the moiety attached to the ZBG, the distance between the Thr200OG1 atom and the sulphamide nitrogen N2 in hCA II/sulphamide complexes was generally shorter than the corresponding distance between the sulphamate oxygen O3 and the same enzyme atom in hCA II/sulphamate complexes (see Tables 3 and 4).…”

Geometry optimization of steroid sulfatase inhibitors - the influence on the free binding energy with STS

“…The STS inhibitors presented in Table 1 (optimized at above describe three levels of theory: (i) MM+, (ii) PM7 and (iii) DFT B3LYP 6-31++G*) were analysed in order to select compounds structurally similar to 667-COUMATE that was thoroughly studied according to mode of inhibitor binding to STS [34]. Hierarchical cluster analysis (HCA) with Euclidian distances and Ward's method of linkage [34] was applied.…”

“…Hierarchical cluster analysis (HCA) with Euclidian distances and Ward's method of linkage [34] was applied. Selected inhibitors were saved as PDB file format for input to PatchDock software [35].…”

“…To compare the influence of the inhibitors' geometries on their free binding energy with the steroid sulfatase we have selected inhibitors structurally similar to 667-COUMATE that was proven to bind with the binding pocket of the protein [34]. The selection was performed with the application of HCA with Euclidian distances and Ward's method of linkage [43].…”