Carbonic Anhydrase Inhibitors:  Stacking with Phe131 Determines Active Site Binding Region of Inhibitors As Exemplified by the X-ray Crystal Structure of a Membrane-Impermeant Antitumor Sulfonamide Complexed with Isozyme II

Abstract

Structure for the adduct of carbonic anhydrase II with 1-N-(4-sulfamoylphenyl-ethyl)-2,4,6-trimethylpyridinium perchlorate, a membrane-impermeant antitumor sulfonamide, is reported. The phenylethyl moiety fills the active site, making van der Waals interactions with side chains of Gln192, Val121, Phe131, Leu198, Thr200. The 2,4,6-trimethylpyridinium functionality is at van der Waals distance from the aliphatic chain of Ile91 being involved in strong offset face-to-face stacking with Phe131. Analyzing X-ray crystal structures of such adducts, two binding modes were observed: some inhibitors bind with their tail within the hydrophobic half of the active site, defined by residues Phe131, Val135, Leu198, Pro202, Leu204. Other derivatives bind with their tail in a different region, pointing toward the hydrophilic half and making strong parallel stacking with Phe131. This interaction orients the inhibitor toward the hydrophilic part of the active site. Impossibility to participate in it leads to its binding within the hydrophobic half. Such findings are relevant for designing better inhibitors targeting isozymes II, IX, and XII, some of which are overexpressed in hypoxic tumors.

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

“…182 In addition to structural information, NMR also characterizes the internal motion of bound inhibitors [270][271][272][273][274] and of the enzyme active site itself. 272 Except for pentafluorobenzene-sulfonamide (212), which contains magnetically distinct environments for each fluorine atom when bound to HCA II, 271 the aromatic ring of benzenesulfonamides rotates rapidly on the NMR time scale about its C(1)-C(4) axis. 270,273,274 BCA II forms a 1:1 complex with p-methylbenzenesulfon-amide (6 , Table 10) at pH 6.0.…”

“…In a study relevant to the binding of arylsulfonamides to CA II, Krishnamurthy et al have recently reported the binding of fluorinated benzenesulfonamides (207)(208)(209)(210)(211)(212) to BCA II. 182 By constructing a quantitative structure-activity relationship (QSAR), the investigators separated the influence of fluorination of the ring on electrostatic-Lewis basicity of ArSO 2 NH − toward the Zn II cofactor and toward the hydrogen-bond network (interactions that they assumed were dependent on pK a )-and hydrophobic-contacts of the phenyl ring with the enzyme (an interaction that they assumed was dependent on log P, the logarithm of the octanol/aqueous buffer partition coefficient)-interactions (eq 22; Figure 16A).…”

“…As mentioned previously, Krishnamurthy et al constructed a QSAR between the affinity (K d ArSO 2 NH − ) of fluorinated benzenesulfonamides (207)(208)(209)(210)(211)(212) for BCA II and pK a and log P of the ligands (eq 22; Figure 16A). 182 Assuming that the two hydrogen bonds between the enzyme and ligand were equal to one another in energy ( Figure 13B), their analysis allows the partitioning of affinity to the structural interactions of the Zn II -N bond, the hydrogen-bond network, and the hydrophobic contacts of the phenyl ring between the ligand and enzyme (Figure 18; section 10.2.4).…”

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies

“…Compound 2 on the other hand, which is also a very strong CA IX inhibitor (K i of 14 nM) 125,295 , belongs to a class of positively charged, membrane-impermeant compounds previously reported by one of our groups 355 , which are highly attractive for targeting CA IX with its extracellular active site, since such compounds do not inhibit intracellular CAs, and may thus lead to drugs with fewer side effects as compared to the currently available compounds (acetazolamide is the prototypical one 293 ), which indiscriminately inhibit all CAs 293 . The X-ray crystal structure of compound 2 in adduct with CA II (whose active site is very similar to that of CA IX, as shown recently by homology modeling) has been reported recently 356 . It has been observed that the positively charged .…”

“…It should be mentioned that since the X-ray structure of CA IX is not yet available, most studies used the CA II structure for modeling and designing CA IX inhibitors. We stress again that positively charged compounds, of which 2 is a representative, may have the advantage of selectively inhibiting only CA IX in vivo, due to their membrane impermeability 356 . Indisulam 3 is a sulfonamide derivative (originally called E7070) discovered through extensive screening/synthetic studies, which showed powerful anticancer activity in vitro and in vivo 349 .…”