Active Site Ring‐Opening of a Thiirane Moiety and Picomolar Inhibition of Gelatinases

Abstract

Abstract(±)-2-[(4-Phenoxyphenylsulfonyl)methyl]thiirane 1 is a potent and selective mechanism-based inhibitor of the gelatinase sub-class of the zinc-dependent matrix metalloproteinase (MMP) family. Inhibitor 1 has excellent activity in in vivo models of gelatinase-dependent disease. We demonstrate that the mechanism of inhibition is a rate-limiting gelatinase-catalyzed thiolate generation via deprotonation adjacent to the thiirane, with concomitant thiirane opening. A corollary to this mechanism is the prediction that thiol-containing structures, related to thiiraneopened 1, will possess potent MMP inhibitory activity. This prediction was validated by the synthesis of the product of this enzyme-catalyzed reaction on 1, which exhibited a remarkable K i of 530 pM against MMP-2. Thiirane 1 acts as a caged thiol, unmasked selectively in the active sites of gelatinases. This mechanism is unprecedented in the substantial literature on inhibition of zinc-dependent hydrolases. Keywords thiirane; latent thiolate; zinc protease; tight-binding inhibitionThe central position of the epoxide ring in organic synthesis derives from the ease of its synthesis, and the ability of Brønsted or Lewis acids to control its opening by nucleophiles. Thiiranes, three-membered rings containing a sulfur atom, are typically less reactive than epoxides. Due to their latent reactivity toward nucleophiles, the aziridines, epoxides and thiiranes all have been used as irreversible enzyme inhibitors (1-3). In the pioneering work of Kim et al., epoxybutanoic acid covalently modified the carboxylate of the glutamate in the active site of the zinc protease, carboxypeptidase A (4). The catalytic zinc ion of this protease, which activates the scissile amide bond of the substrate during normal turnover, here functions as a Lewis acid for epoxide O-alkylation of this glutamate (5). The conceptual extension of this principle to the creation of efficacious matrix metalloproteinase inhibitors, zinc proteases involved in the pathophysiology of inter alia human inflammation and tumor metastasis, is an objective of this laboratory (6). We previously reported the discovery, the core SAR, and initial computational and experimental mechanistic studies with the thiirane-containing structure 1 (7-12). Thiirane 1 (also known as SB-3CT) exerts * Corresponding author: Shahriar Mobashery, mobashery@nd.edu. Supporting InformationAdditional Supporting Information may be found in the on-line version of this article: enzymatic assay methods, computational methods, and synthetic procedures. NIH Public Access Author ManuscriptChem Biol Drug Des. Author manuscript; available in PMC 2010 December 1. (25), and T-cell lymphoma metastasis to the liver (26). In contrast, the lack of efficacy of 1 in cell culture models of ovarian cancer cell metastasis implicates the activity of other MMPs during collagen degradation in this cancer (27). The favorable biological outcome in MMP-dependent disease models using 1 as a gelatinase MMP subclass inhibitor stimulated this further mechani...

Sulfonate-Containing Thiiranes as Selective Gelatinase Inhibitors

“…As reported earlier in our mechanistic studies of the thiirane-type gelatinase inhibitors, slow-binding behavior is a hallmark of the mechanism of action of this type of inhibitors. 12,19,20 We showed that inhibitor 1 exhibited slow-binding inhibition of gelatinases, which we proposed to be at the root of selectivity. 12 As proposed earlier, gelatinases deprotonate R to the aromatic sulfone moiety, which leads to the opening of the thiirane ring and the release of the thiolate.…”

“…12,19,20 We showed that inhibitor 1 exhibited slow-binding inhibition of gelatinases, which we proposed to be at the root of selectivity. 12 As proposed earlier, gelatinases deprotonate R to the aromatic sulfone moiety, which leads to the opening of the thiirane ring and the release of the thiolate. Hence, the thiirane behaves as a caged thiolate, which becomes available within the active site of gelatinases.…”

“…We have demonstrated that the mechanism of inhibition is the second one and that compound 1 is a slow-binding inhibitor. 12 The thiirane moiety in these inhibitors serves as a caged thiolate, which is unleashed in the active site of gelatinases (closely related MMP-2 and MMP-9) to result in potent inhibition of these enzymes. 12 The deprotonation reaction that leads to opening of the thiirane moiety is the rate-limiting step, which leads to slow-binding and, at times, tight-binding inhibition of gelatinases.…”

“…12 The thiirane moiety in these inhibitors serves as a caged thiolate, which is unleashed in the active site of gelatinases (closely related MMP-2 and MMP-9) to result in potent inhibition of these enzymes. 12 The deprotonation reaction that leads to opening of the thiirane moiety is the rate-limiting step, which leads to slow-binding and, at times, tight-binding inhibition of gelatinases. [13][14][15] A major route of metabolism of these compounds is hydroxylation at the para position in the terminal phenyl group.…”

Selective Gelatinase Inhibitor Neuroprotective Agents Cross the Blood-Brain Barrier

“…6 The compounds of the thiirane class have a unique mechanism of action, involving ring-opening of the thiirane ring at the active site of gelatinases and generation of the thiolate, which is a picomolar and tight-binding inhibitor. 7 The blood-brain barrier (BBB) forms a physical separation between circulating blood and the brain parenchyma and consists of tight junctions around endothelial cells surrounded by basement membrane. The BBB prevents pathogens and large hydrophilic molecules from entering the brain, while allowing transport of substances needed by the central nervous system (CNS) such as glucose, fatty acids, and vitamins.…”