Non-Zinc Mediated Inhibition of Carbonic Anhydrases: Coumarins Are a New Class of Suicide Inhibitors

Abstract

The X-ray crystal structure of the adduct between the zinc metalloenzyme carbonic anhydrase II (CA, EC 4.2.1.1) with the recently discovered natural product coumarin derivative 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one showed the coumarin hydrolysis product, a cis-2-hydroxycinnamic acid derivative, and not the parent coumarin, bound within the enzyme active site. The bound inhibitor exhibits an extended, two-arm conformation that effectively plugs the entrance to the enzyme active site with no interactions with the catalytically crucial zinc ion. The inhibitor is sandwiched between Phe131, with which it makes an edge-to-face stacking, and Asn67/Glu238sym, with which it makes several polar and hydrogen bonding interactions. This unusual binding mode, with no interactions between the inhibitor molecule and the active site metal ion is previously unobserved for this enzyme class and presents a new opportunity for future drug design campaigns to target a mode of inhibition that differs substantially from classical inhibitors such as the clinically used sulfonamides and sulfamates. Several structurally simple coumarin scaffolds were also shown to inhibit all 13 catalytically active mammalian CA isoforms, with inhibition constants ranging from nanomolar to millimolar. The inhibition is time dependent, with maximum inhibition being observed after 6 h.

Interfering with pH regulation in tumours as a therapeutic strategy

“…Similarly, systematic chemical proteomics studies 107,[111][112][113] have recently shown that another component of the extracellular matrix, periostin, exists in several splice isoforms that are abundantly found in most types of cancers [111][112][113][114] . 42,43 or using nanoparticles to design CAIs 44 . The first study linking the inhibition of CA9 with tumour shrinkage used the fluorescent sulphonamide (compound 1, n=1) and the cationic compound 2 (REF 28).…”

“…Alternative CAIs. Coumarins and thiocoumarins were only recently discovered to have carbonic anhydraseinhibiting properties, and their mechanism of inhibition has subsequently been deciphered 42,43 . The nat ural product 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one (compound 10; FIG.…”

“…2), which represent the de facto enzyme inhibitor. This new class of CAIs binds to carbonic anhydrase in its hydrolysed form at the entrance of the active site, and does not interact with the metal ion, thus constituting a class of CAIs with a new mechanism of action 42,43 . As well as the natural coumarin compound (compound 10), compounds 12 and 13 (GC-205) (FIG.…”

Glycosyl Coumarin Carbonic Anhydrase IX and XII Inhibitors Strongly Attenuate the Growth of Primary Breast Tumors

“…1−7 As shown by kinetic and X-ray crystallographic studies, coumarins are mechanism-based inhibitors that undergo hydrolysis under the influence of the zinc hydroxide, nucleophilically active species of the enzyme, with generation of substituted 2-hydroxycinnamic acids. 1,2 For example, the natural product coumarin A or the simple nonsubstituted derivative B (but also many of their congeners possessing various substitution patterns at the coumarin ring) 2 acts as an effective CAI against many of the mammalian isoforms CA I−CA XV known to date, and the real enzyme inhibitor was demonstrated to be the hydrolyzed coumarins, A1 and B1, formed from the original derivatives A and B, respectively (Scheme 1). 1,2 The adducts of CA II with coumarins A and B have been characterized by X-ray crystallography.…”

“…1,2 For example, the natural product coumarin A or the simple nonsubstituted derivative B (but also many of their congeners possessing various substitution patterns at the coumarin ring) 2 acts as an effective CAI against many of the mammalian isoforms CA I−CA XV known to date, and the real enzyme inhibitor was demonstrated to be the hydrolyzed coumarins, A1 and B1, formed from the original derivatives A and B, respectively (Scheme 1). 1,2 The adducts of CA II with coumarins A and B have been characterized by X-ray crystallography. 1,2 These studies showed the 2-hydroxycinnamic acids A1 and B1 to be bound to the enzyme in an unprecedented way.…”