5.6 ENZYME INHIBITOR EXAMPLES FOR THE TREATMENT OF BREAST CANCER
5.6.6.3 Mechanism of Action for STS and STS Inhibitors
All potent irreversible STS inhibitors reported to date share a common pharmaco- phore, that is, a phenol sulfamate ester with substituents that exploit favorable hydrophobic interactions with the enzyme active site. Although nonsulfamoylated phenolic compounds such as 5.162and5.163were potent reversible inhibitors of STS, the sulfamate derivative 5.164was an irreversible inhibitor with a potency superior to its phenolic counterpart (see steroidal inhibitors in Section 5.6.6.1).
TheN-monoalkyl (e.g., 5.183) and N,N-dialkyl derivatives of EMATE (e.g., 5.184) were weak reversible inhibitors of STS, although N-acetyl-EMATE (5.185), but not the benzoyl derivative, inhibited the enzyme irreversibly, albeit less potently than EMATE. Analogs of EMATE in which the 3-O-atom was replaced by other heteroatoms (S, 5.186and N, 5.187) were only weak reversible inhibitors of STS.
All these findings have demonstrated that a free sulfamate group (i.e., H2NSO2O–) with no substitutions at the N-atom is the prerequisite for highly potent irreversible STS inhibition.
Despite the fact that STS is bound to the membrane of the endoplasmic reticulum, it was successfully isolated and purified. The crystal structure of human STS has recently been reported in the literature. The overall shape of the protein is mushroom- like, with the crown protruding toward the lumen side of, and the stalk traversing through the lipid bilayer of, the endoplasmic reticulum. STS is similar to its closely related soluble enzymes arylsulfatase A (ASA) and arylsulfatase B (ASB), whose crystal structures were published a few years ago, and shares with them a similar catalytic site topology and a unique characteristic universal to all sulfatases, i.e., posttranslational modification of a conserved cysteine residue to a formylglycine (FGly, ∑-CHO) residue. As observed for ASB, the resting state of human STS at the catalytic site consists of a sulfated gem-diol form of FGly [i.e., ~CH(OH)OSO3–], which is coordinated to a bivalent Ca2+cation. The steroid scaffold recognition site of STS is composed of mainly hydrophobic residues that interact favorably with
E1S via hydrophobic contacts. It has been demonstrated that [~CH(OH)OSO3–] is crucial to the hydrolysis of sulfate ester by ASB. The putative mechanism of STS is depicted in Figure 5.9. The first step involves the regeneration of the gem-diol form of FGly via the attack of a molecule of water on the FGly intermediate. One of the hydroxyl groups of the gem-diol form of FGly then attacks the sulfur atom of E1S, releasing E1 and regenerating the sulfated gem-diol form of FGly.
Because the sulfamate group is designed to mimic the sulfate group, it is rea- sonable to expect that the mechanism of action of sulfamate-based STS inhibitors such as EMATE would also involve the [~CH(OH)OSO3–] residue. One of the proposed mechanisms of STS inhibition by EMATE is shown in Figure 5.10. It is not clear if structure I is a dead-end product or will undergo further modifications to yield a species that irreversibly inactivates the enzyme.
However, it is also possible that irreversibly inhibiting sulfamate esters (e.g., EMATE) could inhibit STS in a more random manner by a specific or nonspecific sulfamoylation of amino acid residues in the active site. Such proposed mechanisms are shown in Figure 5.11. Path A involves an attack by a nucleophilic amino acid residue in the active site. Path B involves the generation of a sulfonylamine species via an E1cBprocess, possibly initiated by an enzyme-catalyzed N-proton abstraction and stimulated by hydrogen bonding to the bridging O-atom. The N-proton of a sulfamate group is fairly acidic, considering that EMATE has a pKa value of 9.5 FIGURE 5.9 Proposed reaction scheme for cleavage of estrone sulfate by steroid sulfatase.
(Adapted from Woo, L.W.L., Purohit, A., Malini, B. et al. (2000). Chemistry & Biology, 7, 773–791.)
FIGURE 5.10 Proposed mechanism of steroid sulfatase inhibition by EMATE via a nucleo- philic attack on the sulfamoyl group by the gem-diol form of formylglycine residue in the enzyme active site. Structure Iis proposed to be a dead-end product. (Adapted from Woo, L.W.L., Purohit, A., Malini, B. et al. (2000). Chemistry & Biology, 7, 773–791.)
measured in 70% aqueous methanol. This value is expected to be lower, perhaps by one or two units, when EMATE is in an aqueous environment. With what is known about the topology of the active site of STS, the attacking nucleophile in Path A and the N-proton abstracting amino acid residue (:B) in Path B could well be either one of the conserved lysine or histidine residues lining the active site of STS. The end product of both paths is a sulfamoylated essential amino acid residue that would have led to irreversible inactivation of the enzyme.
5.6.7 FUTURE DIRECTIONS
It has clearly been established that inhibition of the aromatase enzyme is highly effective in the management of HDBC. Although letrozole and anastrazole were originally licensed as the second-line treatment after the failure of tamoxifen in postmenopausal women with advanced breast cancer, evidence has emerged from many recent trials to suggest that these agents are indeed superior to tamoxifen as the first-line treatment for HDBC. With these NSAIs showing better toxicity profiles than tamoxifen, it is envisaged that letrozole or anastrazole would eventually chal- lenge the established role of tamoxifen in the treatment of breast cancer. Despite the reports of so many fourth-generation NSAIs, it is not known if any of these FIGURE 5.11 Proposed random specific or nonspecific sulfamoylation by EMATE of an essential amino acid residue in the steroid sulfatase active site. Path A: via an attack by a nucleophilic amino acid residue in the active site other than the gem-diol residue (:Nu-H).
Path B: via the generation of a sulfonylamine species. No regeneration of the enzyme active form from the sulfamoylated intermediate is expected. :B,a proton-abstracting amino acid residue; X, a hydrogen-bond-donating amino acid residue or a coordinating metal ion. Dashed line: hydrogen-bonding. (Adapted from Woo, L.W.L., Purohit, A., Malini, B. et al. (2000).
Chemical Biology, 7, 773–791.)
agents would successfully progress to the clinical development stage. Unless some of these newer agents are shown to possess a therapeutic profile superior to existing agents, it is anticipated that letrozole and anastrazole would remain the gold standard of NSAIs for many years to come.
The recent entry of the nonsteroidal STS inhibitor 667COUMATE into Phase I trial is a promising advance. The results of a follow-on Phase II trial would allow the role of STS inhibition in the management of HDBC to be assessed. Once a clinical proof-of-concept has been established, it is anticipated that a few more leading steroidal and nonsteroidal STS inhibitors will be scheduled for clinical development.
There is strong evidence to suggest that more effective estrogen deprivation in patients with breast cancer that is sensitive to estrogenic stimulation can only be achieved if both aromatase and STS are inhibited at the same time. Although administering an aromatase inhibitor in conjunction with an STS inhibitor as two separate agents might be an obvious choice in a combined endocrine therapy, an attractive alternative approach is to design a dual aromatase and sulfatase inhibitor (DASI) that will inhibit both enzymes as a single agent. This concept has recently been validated by the report on compound A (see Structure A) that showed dual inhibition of both enzymes. It is anticipated that DASIs such as compound A should allow the therapeutic potential of dual inhibition of estrogen formation in breast tumors to be evaluated.
ACKNOWLEDGMENT
The author thanks Barry V.L. Potter for comments on the manuscript.
FURTHER READING
Reviews on Aromatase Inhibitors
1. Recanatini, M., Cavalli, A., and Valenti, P. (2002). Nonsteroidal aromatase inhibitors:
recent advances. Medicinal Research Reviews, 22, 282–304.
2. Brodie, A. (2002). Aromatase inhibitors in breast cancer. Trends in Endocrinology and Metabolism, 13, 61–65.
Aromatase Mechanisms
1. Cole, P.A. and Robinson, C.H. (1990). Mechanism and inhibition of cytochrome P450 aromatase.Journal of Medicinal Chemistry, 33, 2933–2942.
2. Akhtar, M., Njar, V.C., and Wright, J.N. (1993). Mechanistic studies on aromatase and related C–C bond cleavage P450 enzymes. Journal of Steroid Biochemistry and Molecular Biology, 44, 375–387.
3. Kao, Y.C., Korzekwa, K.R., Laughton, C.A. et al. (2001). Evaluation of the mecha- nism of aromatase cytochrome P450. A site-directed mutagenesis study. European Journal of Biochemistry, 268, 243–251.
Reviews on Steroid Sulfatase Inhibitors
1. Reed, M.J., Purohit, A., Woo, L.W.L., and Potter, B.V.L. (1996). The development of steroid sulfatase inhibitors. Endocrine-Related Cancer, 3, 9–23.
2. Poirier, D., Ciobanu, L.C. and Maltais, R. (1999). Steroid sulfatase inhibitors. Expert Opinion on Therapeutic Patents, 9, 1083–1099.
3. Nussbaumer, P. and Billich, A. (2003). Steroid sulfatase inhibitors. Expert Opinion on Therapeutic Patents, 13, 605–625.
4. Smith, H.J., Nichols, P.J., Simons, C., and Le Lain, R. (2001). Inhibitors of steroido- genesis as agents for the treatment of hormone-dependent cancers. Expert Opinion on Therapeutic Patents, 11, 789–824.
Crystal Structure of STS
1. Hernandez-Guzman, F.G., Higashiyama, T., Pangborn, W. et al. (2003). Structure of human estrone sulfatase suggests functional roles of membrane association. Journal of Biological Chemistry, 278, 22989–22997.
Proposed Mechanisms of Irreversible Steroid Sulfatase Inhibitors
1. Woo, L.W.L., Purohit, A., Malini, B. et al. (2000). Potent active site-directed inhibition of steroid sulfatase by tricyclic coumarin-based sulfamates. Chemistry & Biology, 7, 773–791.
Dual Aromatase and Sulfatase Inhibitors
1. Woo, L.W.L., Sutcliffe, O.B., Bubert, C. et al. (2003). First dual aromatase-steroid sulfatase inhibitors. Journal of Medicinal Chemistry, 46, 3193–3196.
5.7 ENZYME INHIBITOR EXAMPLES FOR THE