StructureâActivity Relationship for Sulfonamide Inhibition of

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Structure-activity relationship for sulfonamide inhibition of Helicobacter pylori #-carbonic anhydrase. Joyanta K. Modak, Yu-Chih Liu, Claudiu T Supuran, and Anna Roujeinikova J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01333 • Publication Date (Web): 21 Nov 2016 Downloaded from http://pubs.acs.org on November 24, 2016

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Journal of Medicinal Chemistry

Structure-activity relationship for sulfonamide inhibition of Helicobacter pylori α-carbonic anhydrase.

Joyanta K. Modak1,2, Yu C. Liu1, Claudiu T. Supuran3,4 and Anna Roujeinikova1,2,5*

1

Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia

2

Infection and Immunity Program, Monash Biomedicine Discovery Institute; Monash University, Clayton, Victoria 3800, Australia

3

Laboratorio di Chimica Bioinorganica, Polo Scientifico, Università degli Studi di Firenze, Via della Lastruccia 3, Sesto Fiorentino (Florence) Italy

4

Neurofarba Department, Sezione di Scienze Farmaceutiche, Università degli Studi di Firenze, Via U. Schiff 6, Sesto Fiorentino (Florence), Italy

5

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia

*

Corresponding Author

ACS Paragon Plus Environment


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Abstract

α-carbonic anhydrase of Helicobacter pylori (HpαCA) plays an important role in the acclimation of this oncobacterium to the acidic pH of the stomach. Sulfonamide inhibitors of HpαCA possess anti-H. pylori activity. The crystal structures of complexes of HpαCA with a family of acetazolamide-related sulfonamides have been determined. Analysis of the structures revealed that the mode of sulfonamide binding correlates well with their inhibitory activities. In addition, comparisons with the corresponding inhibitor complexes of human carbonic anhydrase II (HCAII) indicated that HpαCA possesses an additional, alternative binding site for sulfonamides that is not present in HCAII. Furthermore, the hydrophobic pocket in HCAII that stabilizes the apolar moiety of sulfonamide inhibitors, is replaced with a more open, hydrophilic pocket in HpαCA. Thus, our analysis identified major structural features can be exploited in the design of selective and more potent inhibitors of HpαCA that may lead to novel antimicrobials.


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Introduction Helicobacter pylori is a Gram negative, neutralophilic bacterium that colonizes human gastric mucosa.1 Around half of the total world’s population is infected with this bacteria, with the prevalence reaching 80% of the population in some developing countries.2,3 H. pylori infections are associated with diseases of the upper gastrointestinal tract such as chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer.4-8 The current standard therapy for anti-H. pylori treatment consists of two broad-spectrum antibiotics (clarithromycin and either metronidazole or amoxicillin) and a proton pump inhibitor. However, the success rate of this regimen has declined over time, falling below 80% globally, mainly due to the spread of resistance to clarithromycin and metronidazole.9-11 Therefore, there is a clear need for identification of novel targets that can be used in the development of alternative treatment strategies for H. pylori infections. Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze a physiologically important process of reversible hydration of CO2 to bicarbonate and protons.12,13 Bacterial CAs provide substrates for many metabolic pathways including pH regulation, CO2 fixation and cyanate degradation, and play an important role in the survival of intracellular pathogens within the host.14-17 H. pylori possesses two different CAs, periplasmic α-carbonic anhydrase (HpαCA) and cytoplasmic β-carbonic anhydrase (HpβCA). Through the activities of HpαCA, HpβCA and urease, H. pylori buffers its periplasm at pH close to 6.1 as a means of acclimation to the harsh acidic environment of the stomach.14,18 Due to their crucial role in H. pylori survival in the host, HpαCA and HpβCA have now gained interest as potential drug targets. Their activities are inhibited by sulfonamides (RSO2NH2), including the clinically used drugs acetazolamide (AAZ), ethoxzolamide (EZA), methazolamide (MZA), topiramate and sulpiride.19,20 Importantly, AAZ



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and MZA showed anti-H. pylori activity in in vitro assays.21 Thus, the inhibition of HpCAs could be a novel alternative approach for the treatment of H. pylori infections. Since the sulfonamides in current clinical use were designed to target human CA isoforms, their activity against bacterial CAs, including HpαCA and HpβCA, is lower than against human CAs.21 Understanding the structural basis of inhibition and the differences between the inhibitor-binding sites in human CAs and their H. pylori orthologues would be crucial for the design of inhibitors specific to H. pylori. Crystallographic studies of HpCAs in complex with different inhibitors would be an important step towards this goal. The periplasmic location of HpαCA makes it an easier target, as its inhibitors do not need to cross the cytoplasmic membrane. We have previously reported the crystal structures of HpαCA in complex with AAZ and MZA.22 We showed that HpαCA forms a dimer in solution, which is very similar to other bacterial αCAs, but distinct from the mammalian monomeric form of αCA. Despite the difference in the oligomeric state, the HpαCA overall fold is very similar to that of human carbonic anhydrase II (HCAII), which shares a 28% sequence identity with HpαCA.22 The differences between the structures of the two enzymes are mainly in the length of the surface loops. The active site of HpαCA is located in the cone-shaped cavity and contains a catalytic zinc ion that is tetrahedrally coordinated by three histidine residues and a water molecule/hydroxide ion. Notably, the entrance into the HpαCA active site cavity is wider than in HCAII due to a shorter surface loop. However, in both HpαCA and HCAII, residues that are important for the recognition and correct orientation of the substrate molecule (CO2) are conserved and, hence, HpαCA is likely to follow the same reaction mechanism as HCAII, where the substrate is converted into HCO3- via a nucleophilic attack of the reactive zinc-bound H2O/OH- on CO2.22 Our structural analysis revealed that the two sulfonamide oxygens of the


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inhibitors occupy positions similar to those of the oxygen atoms of the CO2 substrate, whereas its Zn-coordinating sulfonamide nitrogen atom is bound at the site for the catalytic H2O molecule. Sulfonamides AAZ (KI, 21 nM) and MZA (KI, 225 nM) therefore act as site-directed inhibitors of HpαCA by mimicking the catalytic transition state of the CO2 hydration reaction. In this study, we present the analysis of the structure-activity relationship for a family of AAZ- and MZA-related sulfonamide inhibitors of HpαCA, whose structures, IUPAC names and inhibitory constants (KI) are presented in Figure 1 and Supporting Information Table S1. We identify the features that define their inhibitory activity against HpαCA and pinpoint the differences with HCAII that can be exploited in the design of specific and more potent inhibitors of HpαCA.

Results and discussion In order to investigate the structure-activity relationship for sulfonamide inhibition of HpαCA, we determined the crystal structures of HpαCA in complex with four different sulfonamide inhibitors representing two different classes of sulfonamides, modeled the complexes with three other sulfonamides from this family and integrated this data with the previously determined structures of the complexes with acetazolamide (AAZ) and methazolamide (MZA).22 The inhibitors discussed in this study include clinical drugs AAZ, MZA, EZA, dorzolamide (DZA) and brinzolamide (BRZ), and related compounds 1, 2, 3 and benzolamide (BZA) (Figure 1). The chemical structure of all inhibitors has a five-membered ring linked to a sulfonamide moiety and either decorated with R1 and N-R2 substituents at positions 4 and 5 (class 1), respectively, or fused with a 6-membered ring (class 2) as illustrated in Figure 1. The crystals of the HpαCA



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complexes with BZA, EZA, compound 1 and compound 3 belonged to the P21 space group with eight protein subunits in the asymmetric unit. The structures of the protein subunits from the four complexes could be superposed with an average pairwise Cα root mean square deviation (r.m.s.d.) value of 0.5 Å, showing no significant differences. The analysis and discussion therefore focuses on the similarities and differences in the relative positions of the respective inhibitors in the enzyme’s active site. Two different binding modes of compound 1 and comparison with AAZ The chemical structure of compound 1 (R1 = R2 = H) is a truncated version of that of AAZ (Figure 1). Difference Fourier maps calculated for the HpαCA/compound 1 complex, based on the molecular replacement (MR) solution obtained with the coordinates of the free enzyme, clearly showed the presence of one inhibitor molecule bound in the active site of each protein subunit. Interestingly, structural alignment of the eight protein subunits in the asymmetric unit revealed that compound 1 binds to HpαCA in two different modes (A and B) (Figure 2 and Supporting Information Figure S1), in both of which the N atom of the sulfonamide moiety of the inhibitor coordinates the catalytic zinc ion in the active site. Binding mode A of compound 1, observed in four out of eight subunits, is very close to that of the AAZ molecule previously observed in the crystal structure of the HpαCA/AAZ complex (Supporting Information Figure S1A).22 In this mode, the N atom of sulfonamide moiety forms a hydrogen bond with the Oγ atom of Thr191 (2.9 Å), whilst one of the two sulfonamide oxygen atoms is within hydrogen bonding distance from the backbone NH of Thr191 (2.9 Å) (Figure 2 and Supporting Information Figure S1A). The sulfonamide group is further stabilized by van der Waals contacts with the side chains of Val141, Thr191 and Trp201. The thiadiazole ring is located in the hydrophobic side of the active site and makes van der Waals contacts with Val131, Leu190 and


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Ala192. The 5-amino group of the inhibitor points out of the pocket and is exposed to the solvent. In binding mode B, the 5-amino-thiadiazole ring of compound 1 is bound in a small, partially hydrophobic cavity formed by residues Trp23, Thr83, His84, Thr86, His110 and Ala192, and the inhibitor is oriented at an approximate angle of 60° to binding mode A (Figure 2 and Supporting Information Figure S1B). In common with mode A, the N atom of the sulfonamide group forms a hydrogen bond with the Oγ atom of Thr191 (2.6 Å), and the O1 atom of the sulfonamide group forms a hydrogen bond with the backbone NH moiety of Thr191 (3.1 Å). An additional hydrogen bond that is present in mode B, but absent in mode A, is that between the O1 atom of the sulfonamide group and the backbone NH group of Ala192 (2.9 Å). The thiadiazole ring in binding mode B forms van der Waals interactions with Trp23, His110 and Ala192. The 5-amino group, which is completely exposed to the solvent in mode A, is stabilized by van der Waals contacts with the carbonyl oxygen atoms of Thr83 and His84 and the side chain of Thr86 in mode B. Structural superposition revealed that the pocket accommodating the 5-amino-thiadiazole ring bound in mode B is occupied by a molecule of cryo-protectant glycerol in the HpαCA/AAZ complex. Modeling the AAZ molecule based on the coordinates of compound 1 bound to HpαCA in mode B resulted in a clash between the acetamido group of AAZ and protein residues Thr83, His84 and Thr86, which explains why binding mode B was not observed for AAZ.22 Compound 1 (KI = 323 nM) is a less potent inhibitor of HpαCA than AAZ (KI = 21 nM). Superposition of the structure of the HpαCA complex with AAZ with that of the corresponding complex with compound 1 in binding mode A shows that the acetamido moiety of AAZ contributes additional favorable interactions between the inhibitor molecule and the enzyme in



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the form of a hydrogen bond between the acetamido group and the Nδ atom of Asn108, and a van der Waals contact with the side chain of Lys88 (Supporting Information Figure S1). Comparison of compound 1 binding sites in HpαCA and HCAII Superposition of the structures of HpαCA and its human orthologue HCAII revealed that 206 of 258 Cα atoms could be overlapped with an r.m.s.d. of 1.6 Å, showing the overall similarity of the two enzymes despite only 28% amino-acid sequence identity between them.22 Inspection of their respective superposed complexes with compound 1 (PDB ID: 2HNC)23 revealed that the binding mode of the inhibitor in HCAII is remarkably close to mode A in HpαCA (Supporting Information Figure S2) with a large number of conserved residues involved in stabilizing the inhibitor in HpαCA and HCAII (in parentheses) as follows: Val131 (Val121), Val141 (Val143), Leu190 (Leu198), Thr191 (Thr199) and Trp201 (Trp209). Modeling binding mode B for compound 1 in the active site of HCAII based on this superposition resulted in a steric clash between the thiadiazole ring and the Cγ atom of Thr200 (Ala192 in HpαCA) (Supporting Information Figure S2), consistent with the HCAII’s preference for mode A. Furthermore, the presence of a hydroxyl group on Thr200 in HCAII, as compared to Ala192 in HpαCA, allows formation of an additional hydrogen bond with the nitrogen atom of the compound 1’s heterocyclic ring, resulting in a tighter binding to the human orthologue (KI (HpαCA) = 323 nM, KI (HCAII) = 60 nM, Supporting Information Table S1). Evidence of positional disorder of benzolamide (BZA) bound to HpαCA Electron density maps calculated for the HpαCA crystals soaked in a solution containing BZA showed the location of BZA molecules in each subunit of HpαCA, except chain F, where the occupancy of the binding site was very low. The 2-sulfonamide moiety of BZA forms


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interactions with the active-site Zn2+ ion and the main chain of Thr191 (Figure 3A) that are very similar to those observed in the crystal complexes with compound 1, AAZ and MZA.22 The thiadiazole ring forms van der Waals contacts with His110, Val131, Val141, Leu190, Thr191, Ala192, and Pro193. The O1 and O2 oxygen atoms of the 5-sulfonamido group are within hydrogen bonding distance from the side chains of Asn108 and Lys88, respectively. The apolar phenyl ring lies in an energetically unfavorable environment, with one side of it exposed to the solvent and the other side packing against the protein surface area that contains both polar (Asp107, Asn108, Lys133) and apolar (Val131 and Leu139) residues (Figure 3A). Superposition of the structures of all subunits in the asymmetric unit revealed a significant degree of positional disorder of this inhibitor in the crystal (Figure 3B). The BZA molecule has been trapped in several slightly different orientations in different protein subunits, which is a manifestation of the fact that, in solution, the BZA molecule bound to HpαCA has some degree of thermal disorder and performs ~8 degree pivotal movement about the sulfur atom of the 2-sulfonamide moiety and ~25 degree rotation about the S-C-N-S torsion angle, scanning through different possible binding modes. Furthermore, the electron density for the inhibitor in subunit E indicates the presence of two distinctly different binding modes, in one of which the phenylsulfonamido moiety flips by ~180° (Figure 3C) and binds in the cavity lined with polar residues Thr83, His84, Thr86 and His110, likely at high energetic cost. The positional disorder of BZA observed in the crystal is likely to be the consequence of multiple unfavorable interactions between the phenyl ring of the inhibitor and the protein atoms, consistent with a low inhibitory potency of BZA towards HpαCA (KI = 315 nM).



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Comparison of BZA binding sites in HpαCA and HCAII Superposition of the structure of the HpαCA/BZA complex with the corresponding complex of HCAII (PDB ID: 3D8W)24 (also produced by crystal soaking in the inhibitor solution) revealed that the mode of BZA binding is similar (Figure 3D), which is in agreement with the fact that a large number of residues involved in BZA binding in HpαCA are strongly conserved in its human orthologue (in parentheses) Asn108 (Gln92), His110 (His94), Val131 (Val121), Leu139 (Leu141), Val141 (Val143), Leu190 (Leu198), Thr191 (Thr199) and Pro193 (Pro201). The most significant differences between the BZA binding sites of the two enzymes are the presence of a short helix 130-136 in HCAII that is absent in the H. pylori enzyme and an Ala→Thr substitution in HCAII at a position corresponding to Ala192 in HpαCA. These differences result in additional interactions that stabilize BZA bound to HCAII: hydrophobic interactions between the apolar side chains of the helix residues Phe131 and Val135 and the phenyl moiety of BZA, and a hydrogen bond between the side chain of Thr200 and a nitrogen atom of the 5-membered heterocyclic ring of BZA. These additional stabilizing interactions are consistent with a significantly higher potency of BZA towards HCAII (KI = 9 nM) than HpαCA (KI = 315 nM). Tert-butyl substituent on phenyl ring of compound 3 forms stabilizing interactions with HpαCA Despite the lower resolution (2.9 Å) of the data for the crystal complex of HpαCA with compound 3, difference Fourier maps unambiguously indicated the position of the inhibitor molecule (Figure 4). The electron density around this molecule was somewhat better in subunit A. Therefore, the detailed analysis of compound 3 interactions with HpαCA is discussed with reference to that subunit. Compared to the BZA structure, compound 3 contains a CH3


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substituent at position 4 of the thiadiazole ring and a tert-butyl substituent at position 4 of the phenyl ring (Figure 1). Similar to BZA and other sulfonamide inhibitors of HpαCA described above, the 2-sulfonamide N atom of compound 3 coordinates directly to the Zn2+ ion, while the 2-sulfonamide moiety and thiadiazole ring form interactions with His110, Val131, Val141, Leu190, Thr191 Ala192, Pro193 and Trp201 (Figure 4). In common with BZA, the oxygen atoms of the 5-sulfonamido group of compound 3 form hydrogen bonds with Lys88 and Asn108. The CH3 substituent at the thiadiazole ring (absent in BZA) forms a van der Waals contact with Pro194. The tert-butyl phenyl ring of compound 3 binds in the same pocket as the phenyl group of BZA (Figure 4). The aliphatic tert-butyl substituent forms stabilising hydrophobic interactions with the side chain of Leu139 and the aliphatic part of the side chain of Lys133, which compensates for an unfavorable contact with the polar side chain of Asp107 and accounts for the tighter binding of compound 3 to HpαCA (KI = 8.2 nM) in comparison to BZA. Analysis of ethoxzolamide binding mode and comparison to AAZ The structure of the ethoxzolamide (EZA) complex of HpαCA was determined at 2.2 Å resolution. The Fourier maps in the active site showed unambiguous electron density for the inhibitor (Figure 5A) in all chains except chain D. Comparisons with the crystal structures of the different HpαCA/sulfonamide inhibitor complexes discussed above showed that the 2sulfonamide moiety of EZA overlaps well with that of other sulfonamides, and the thiazole ring forms similar hydrophobic interactions with Val131, Leu190 and Ala192. Inspection of the superposition of the structures of the HpαCA complexes with EZA and AAZ (Figure 5B) showed that the bicyclic ring of the EZA molecule and the thidiazole ring of AAZ partially overlap. In comparison to AAZ, the EZA molecule is pivoted about the sulfur atom of the 2sulfonamide moiety in the plane of the ring, towards the loop 189-199. The benzene ring fused to



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the thiazole moiety of EZA forms van der Waals contacts with Pro193 and Pro194 (Figure 5). In comparison to AAZ, the proximity of the benzene ring of the EZA molecule to the partially negatively charged carbonyl oxygen of Pro193 is energetically unfavorable. Furthermore, EZA forms one less hydrogen bond with the protein than AAZ, as it lacks the acetamido group which, in the crystal complex with AAZ, is hydrogen-bonded to the side chain of Asn108 (Figure 5B). In addition, the apolar ethyl tail of the EZA molecule is mostly solvent exposed. Cumulatively, these energetically unfavorable factors result in a higher inhibitory constant value of EZA for HpαCA (KI = 193 nM) in comparison to AAZ (KI = 21 nM).

Comparison of EZA binding sites in HpαCA and HCAII Superposition of the crystal structures of the respective EZA complexes of HpαCA and HCAII (PDB ID: 3CAJ)25 revealed a remarkably similar binding mode of EZA in the two enzymes (Figure 5C). The most noticeable differences in the EZA/protein interactions concern the α-helix 130-136 in HCAII (absent in HpαCA), and residues Thr200 and Gln92 in HCAII that occupy positions corresponding to Ala191 and Asn108 in HpαCA, respectively. The 130-136 α-helix of HCAII forms a wall of the hydrophobic pocket accommodating the 6-ethoxy tail of EZA, with the apolar side chain of Phe131 forming stabilizing van der Walls interactions with the inhibitor molecule. The side chain of Thr200 forms a hydrogen bond with the nitrogen atom of the inhibitor’s thiazole ring, whilst the side chain of Gln92 is within the van der Waals contact distance from the benzene ring of EZA. These additional multiple stabilizing interactions present in the HCAII/EZA complex are consistent with a much higher potency (KI = 8 nM) of EZA towards the human CAII as compared to HpαCA (KI = 193 nM).


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Docking studies of compound 2, brinzolamide (BRZ) and dorzolamide (DZA): To further investigate structure-activity relationships in sulfonamides, three related sulfonamide derivatives (compound 2, BRZ and DZA) were modeled into the HpαCA active site. The chemical structure of compound 2 is very similar to that of compound 1, with the main difference being an additional R1 methyl group at position 4 of the thiadiazole ring (Figure 1). The crystal structure of the HpαCA/compound 1 complex was therefore used for modeling the binding mode of compound 2 by placing its sulfonamide and thiadiazole moiety in the same position as those of compound 1 in its respective crystal complex. Modeling compound 2 in binding mode A observed for compound 1 (Figure 6) positioned its apolar 4-methyl group within energetically unfavorable distance from the partially negatively charged main-chain carbonyl oxygens of Ala192 and Pro193, similar to the situation observed for the structurally similar MZA in its respective crystal complex with HpαCA.22 Modeling compound 2 in binding mode B (Figure 6) positioned its methyl group close to the partially negatively charged main-chain carbonyl oxygen of His84. This structural analysis explains the reduced inhibitory potency (KI = 549 nM) of compound 2 compared to compound 1 (KI = 310 nM). BRZ and DZA were modeled based on the crystal structure of the complex with the structurally similar EZA, the 6-ethoxy-1,3-benzo group of which was replaced with the substituted thiazine ring for BRZ and substituted thiopyran ring for DZA (Figure 1). The sulfonamide and thiophene moieties of BRZ and DZA were placed in the same position as the respective sulfonamide and thiazole moieties of EZA, and their heterocyclic six-membered rings



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were overlapped with the benzene ring of EZA (Figure 7). The modeling placed the two oxygen atoms of the sulfodioxide moiety of both BRZ and DZA within a hydrogen bonding distance from the side-chain amines of Lys88 and Lys133. In addition, the N atom of the ethylamine group of BRZ and DZA was within a hydrogen bonding distance from the main-chain carbonyl oxygen of Pro193. Furthermore, the ethyl moiety of BRZ and DZA was within van der Waals contact distance from Trp23, His84 and Ala192 (Figure 7). However, superposition of the modeled BRZ and DZA complexes of HpαCA onto the crystal structures of the respective complexes of HCAII26 did not produce a good overlap of the inhibitor molecules, despite a significant degree of conservation between the active sites of the two enzymes (Figure 8). Thus, based on our modelling study, we cannot exclude the possibility that the actual binding modes of BRZ and DZA in HpαCA may be somewhat different to that of EZA. Detailed analysis of these superpositions revealed that some of the key stabilizing interactions between the protein and BRZ/DZA in HCAII cannot be formed in the H. pylori enzyme. One of the differences concerns the 130-136 helix in HCAII that has no structural equivalent in HpαCA. This helix harbours apolar residues Phe131 and Val135 that contribute to the hydrophobic environment for the apolar methoxypropyl/methyl substituent of the six-membered ring of BRZ/DZA (Figure 8). In addition, the polar side chain of Thr196 in HpαCA is substituted with leucyl (Leu204) in human CAII, which enhances the hydrophobicity of the binding pocket for the methoxypropyl tail. These structural differences between HpαCA and HCAII are consistent with the respective differences in potency of BRZ/DZA (KI = 210 nM/ 4360 nM for HpαCA compared to 3 nM/9 nM for HCAII).


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Overview of the pockets in the HpαCA active site that accommodate different classes of sulfonamide inhibitors In this study, we presented analysis of the complexes between HpαCA and a series of sulfonamide inhibitors containing a common structural core consisting of a five-membered ring linked to a sulfonamide moiety. Based on this analysis, we classified HpαCA residues forming interactions with different classes of sulfonamide inhibitors discussed in this study, according to the binding pocket they belong to. Residues Val131, Val141, Leu190, Thr191, Ala192, Trp201 (colored green in Figure 9A) interact with the common structural core and therefore contribute to binding of all inhibitors. Residues Trp23, Thr83, His84, Thr86 and His110 (colored red) form a hydrophilic pocket that accommodates the alternative binding mode (mode B) of compound 1 and BZA. Residues Pro193 and Pro194 (orange) form a surface that interacts with the R1 substituent (methyl group) of compound 3 and MZA, and with the fused bicyclic ring of EZA (Figure 9A). The R2 substituents of AAZ, BZA in the binding mode A and compound 3 are bound within the pocket lined with mostly polar residues Lys88, Asp107, Asn108, Lys133, Leu139 (colored cyan in Figure 9A). We noted that the most potent inhibitor in the analyzed series, compound 3 (KI = 8 nM), forms extensive interactions with this pocket via its bulky apolar R2 tert-butyl phenyl moiety. Whilst the hydrophobic nature of this moiety is matched by the apolar side of the pocket formed by the side chain of Leu139 and the aliphatic part of the side chain of Lys133, its proximity to the polar side chains of Lys88, Asp107 and Asn108 is a destabilizing factor. Our structural analysis therefore suggests that sulfonamides with a bulky, polar or amphipathic R2 substituent group may have higher potency towards HpαCA than compound 3.



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Summative analysis of the differences between the sulfonamide binding sites in HpαCA and HCAII. In order to identify the structural differences between HpαCA and its human orthologue CAII that can be exploited in the rational design of H. pylori-specific inhibitors, we have summarized the analysis of the superpositions of the corresponding sulfonamide complexes of HpαCA and HCAII. The results are presented in Figure 9B by mapping these differences on the representative structure of the HpαCA/sulfonamide crystal complex (with compound 3) and the crystal structure of HCAII (PDB ID: 2VVA).27 We note that the residues that interact with the common structural core of all sulfonamides discussed here, and therefore play an important role in the binding of all inhibitors, are strongly conserved in HpαCA and HCAII (residue IDs for HCAII in parentheses): Val131 (Val121), Val141 (Val143), Leu190 (Leu198), Thr191 (Thr199), Ala192 (Thr200), Trp201 (Trp209). This observation is consistent with the fact that the two enzymes showed a remarkably similar mode of binding to compound 1, the structure of which represents the common motif present in all inhibitors in this study. The only difference between the residues surrounding this common structural core in the two enzymes is Thr200 in HCAII in place of Ala192 in HpαCA. Comparisons between the HpαCA complex structures and the crystal structures of sulfonamide complexes of HCAII23-25 showed that this substitution results in one additional hydrogen bond between the side chain of Thr200 and the nitrogen atom of the 5-membered heterocyclic ring of the inhibitor. All of the sulfonamides in this study that contain a nitrogen atom in the respective position of the 5-membered heterocyclic ring showed higher potency towards HCAII than HpαCA (not including compound 3, for which KI for HCAII is not available) (Supporting Information Table S1), which indicates that this additional hydrogen bond between HCAII and


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the common structural core of the inhibitor plays an important role in stabilizing the bound inhibitor in the HCAII active site. Further analysis of this superposition revealed that the small hydrophilic pocket that accommodates the alternative binding mode (mode B) of compound 1 and BZA in HpαCA (highlighted in red in Figure 9A) is obstructed by the Cγ and Oγ atoms of Thr200 in HCAII (Figures 3D and 9B, and Supporting Information Figure S2). Inhibitors targeting this pocket would therefore be selective to HpαCA over HCAII. The most significant structural differences between the two enzymes are in the pocket that accommodates the R2/X group of the sulfonamide inhibitors (highlighted in cyan in Figure 9A). In HCAII, this pocket is flanked on one side by the helix harbouring apolar residues Phe131 and Val135 and by the side chains of Leu204 and Ile91, which render this surface strongly hydrophobic (Figure 9B). In contrast, this helix is absent in the HpαCA structure, the pocket is more open to the solvent and is lined on this side with polar residues Asp107 (corresponding to Ile91 in HCAII), Lys133 and Thr196 (corresponding to Leu204 in HCAII). This suggests that sulfonamides with polar or amphipathic R2/X group are likely to have preference towards HpαCA over HCAII. On the other side, this pocket is lined with mainly polar residues in both HpαCA and its human orthologue (Figure 9B). Although the shape and hydrophilicity of this surface is similar in the two proteins, the residues that form it are not conserved between the two enzymes (Thr86, Thr83, Lys88, Asn108 in HpαCA corresponding to Ala65, Asn62, Asn67, Gln92 in HCAII), and these additional, subtle differences may therefore also be exploited in the design of more selective inhibitors targeting HpαCA.



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Conclusions Analysis of the crystal structures of HpαCA in complex with a family of sulfonamide inhibitors, including the newly synthesized compound 3, allowed us to investigate the structural features of the enzyme-inhibitor interactions that modulate binding affinity, and to identify similarities and differences of the inhibitor binding sites in HpαCA and its human orthologue HCAII. Our study revealed that the binding mode of different sulfonamides correlates well with their inhibitory activities against HpαCA. Compound 3 displayed nanomolar inhibition (KI = 8 nM), the lowest that has ever been reported for any sulfonamide inhibitor of HpαCA. The structural comparisons showed that HpαCA possesses an additional small pocket in the active site (absent in HCAII) that serves as an alternative binding site for sulfonamides. Furthermore, the pocket that accommodates the R2/X group of sulfonamide inhibitors in HpαCA is more open and hydrophilic in nature, as compared to the respective hydrophobic pocket in HCAII. The structural features of HpαCA-sulfonamide interactions identified in our study can be exploited in the design of selective and more potent inhibitors of HpαCA, which may lead to novel anti-H. pylori drugs.

Experimental Section Synthesis of the inhibitors: EZA was purchased from Sigma-Aldrich. Compounds 1 and 2, and BZA were synthesized as reported earlier.28,29 To synthesize compound 3, 5 mmoles of compound 2 was dissolved in 15 mL of an aqueous 2.5 M solution of NaOH and cooled to 0-5°C in a salt-ice bath. 4-tert-butylphenylsulfonyl chloride (5 mmol) was added in small portions concomitantly with 10 mL of a cold 2 M NaOH solution, with the temperature maintained below


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10°C. The reaction mixture was stirred at room temperature for 5-10 hrs with monitoring by thin-layer chromatography, then the pH was adjusted to 2.0 with 5 N HCl, and the precipitated sulfonamides were filtered and recrystallized from aqueous ethanol. The purity of all compounds was >95% as determined by HPLC. 5-(4-tert-Butylphenylsulfonamido)-4-methyl-δ2-1,3,4-thiadiazoline-2-sulfonamide, 3: white crystals, mp 250-1°C (EtOH); IR (KBr) (cm-1) 1133 and 1175 (SO2 sym), 1310 and 1332 (SO2 as); 1H NMR (DMSO-d6) (δ, ppm; J, hertz) 0.96 (s, 9H, t-Bu), 3.42 (s, 3H, N-Me); 7.70 (d, 2H, AA'BB', 8.7), 7.84 (d, 2H, AA'BB', 8.7), 8.52 (s, 2H, SO2NH2), 13.657 (br s, 1H, SO2NH); 13C NMR (DMSO-d6) (δ, ppm): 13.23 (Me), 46.81 (N-Me); 61.69 (C from t-Bu), 128.21 (C2/C3 of Ph), 131.50 (C3/C2 of Ph), 141.23 (C1/C4 of Ph), 144.78 (C4/C1 of Ph), 162.69 (Cthiadiazoline), 164.52 (C-thiadiazoline); Anal. (C13H18N4O4S3) C, H, N.

Inhibition studies: Inhibitory constant of AZA, MZA, BZA, EZA, BRZ, DZA, compound 1 and compound 2 were reported earlier.19 The inhibitory activity of compound 3 was measured by following the initial rates of the HpαCA-catalyzed hydration of CO2 in buffer containing 20 mM Hepes (pH 7.5) and 20 mM Na2SO4 using an Applied Photophysics stopped-flow instrument as previously described.30 HpαCA was pre-incubated with compound 3 for 15 min at room temperature prior to the assay. The KI value was calculated by non-linear least-squares methods using PRISM 3. Protein purification, crystallization and X-ray diffraction data collection: HpαCA from strain 26695 was expressed in E. coli using the pET151/D-TOPO vector and purified by following the previously described procedure.31 Crystals of the HpαCA complex with AAZ were obtained by hanging-drop vapor diffusion method from buffered PEG 1.5 K solutions as



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described earlier.22 To obtain HpαCA crystals in complex with BZA, EZA, compound 1 or compound 3, the crystals grown in the presence of AAZ were soaked for 3-4 hrs in a stabilizing solution containing 30% PEG 1.5 K, 240 mM di-basic ammonium citrate and 1.2 mM ZnCl2, and then soaked overnight in a stabilizing solution containing 5 mM BZA, EZA, compound 1 or compound 3. The crystals were then cryo-protected in a stabilizing solution supplemented with 20% (v/v) glycerol and 5 mM respective ligand and flash-cooled in liquid nitrogen for data collection. X-ray diffraction data were collected at cryogenic temperatures using the MX1 and MX2 beamlines of the Australian Synchrotron. All data processing and scaling were performed using iMOSFLM32 and SCALA,33 respectively, from the CCP4 software suite.34 A summary of the data processing statistics is presented in Table 1. The crystals of all complexes were isomorphous, belonged to space group P21, had the β value close to 90° and displayed pseudomerohedral twinning with the twin law (h, -k, -l) detected by using PHENIX Xtriage.35 Structure determination and analysis: The crystal structures of the HpαCA complexes with BZA, EZA, compound 1 and compound 3 were determined using MR (PHASER)36 with the coordinates of the HpαCA dimer in complex with AAZ (PDB ID 4YGF)22 as a search model, with ions, AAZ and water molecules excluded. MR revealed that the asymmetric unit of all complexes contains four dimers. Refinements were carried out using PHENIX,37 with the twin law (h, -k, -l) and torsion non-crystallographic symmetry restraints applied. COOT38 was used to inspect the electron density maps and rebuild the models manually where necessary. The difference Fourier maps clearly revealed phase-unbiased electron density for one zinc ion and respective bound inhibitors in each subunit of all complexes except in chain F and chain D of the BZA and EZA complexes, respectively. The topology and restraints files of the inhibitors were generated using the COOT Ligand Builder interface and PHENIX eLBOW,39 respectively.


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Refinement of the structures using PHENIX and iterative model building using COOT was continued until the R and Rfree converged. The average B factors for the Zn ions and the respective inhibitors molecules in the final refined model were close to those of the surrounding protein atoms, suggesting that the ions and inhibitor molecules are bound at close to full occupancy. An automatic water molecules search was performed using PHENIX but waters were only retained after refinement if their B-factor remained below 50 Å². The validity of the final models was confirmed using MOLPROBITY.40 The final model refinement statistics are summarized in Table 2. The structure figures were produced using PYMOL.41 Molecular modeling: Compound 2 was modeled in the active site of HpαCA using the coordinates of its complex with compound 1 as a template. BRZ and DZA were modeled using the HpαCA/EZA complex coordinates as a template, by superposing the sulfonamide moiety and the five-membered ring of BRZ and DZA onto those of EZA and selecting a conformationally allowed rotamer of the ethylamine and methoxypropyl groups that does not clash with protein atoms. The models were generated and analysed using COOT. ASSOCIATED CONTENT Supporting Information HpαCA and HCAII inhibition data for the sulfonamide compounds discussed in this study; figures showing a comparison of the compound 1 and AAZ binding modes in HpαCA, and the superposition with the respective HCAII/compound 1 complex; molecular formula strings (CSV). PDB ID Codes



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The coordinates and structure factors for the HpαCA complexes with BZA, EZA, compound 1 and compound 3 have been deposited in the Protein Data Bank, www.rcsb.org, under accession codes 5TT8, 5TT3, 5TUO and 5TV3, respectively. Authors will release the atomic coordinates an experimental data upon article publication. Corresponding Author Information Phone: +61399029194; email [email protected] Acknowledgement J.K.M. is the recipient of the Post Publication Award (PPA), Monash University 2016. Abbreviations Used HpαCA, α-carbonic anhydrase of H. pylori; HCAII, human carbonic anhydrase II; MALT, mucosa-associated lymphoid tissue; CA, carbonic anhydrase; HpβCA β-carbonic anhydrase of H. pylori; AAZ, acetazolamide; EZA, ethoxzolamide; MZA, methazolamide; DZA, dorzolamide; BRZ, brinzolamide; BZA, benzolamide; MR, molecular replacement References: 1. Marshall, B. J.; Warren, J. R. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984, 1, 1311-1315. 2. Calvet, X.; Ramírez Lázaro, M. J.; Lehours, P.; Mégraud, F. Diagnosis and epidemiology of Helicobacter pylori infection. Helicobacter 2013, 18, 5-11. 3. Goh, K. L.; Chan, W. K.; Shiota, S.; Yamaoka, Y. Epidemiology of Helicobacter pylori infection and public health implications. Helicobacter 2011, 16, 1-9.


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Table 1: X-ray data collection statistics Complex

BZA

EZA

Compound 1

Compound 3

Space group

P21

P21

P21

P21

Cell dimension (a,b,c

42.2, 134.4, 165.9,

42.9, 138.8, 168.2,

41.8, 136.9, 166.3,

41.6, 133.9, 166.6,

(Å), β (º)

90.1

90.1

90.1

90.3

Observed reflections

129205

282181

213336

138343

Unique reflections

55072

96783

64612

39929

Resolution range (Å)

42.6 – 2.4 (2.5 – 2.4)

39.9 – 2.2 (2.3 – 2.2)

30 – 2.5 (2.6 – 2.5)

29.9 – 2.9 (3.0 – 2.9)

Rmerge1

0.077 (0.319)

0.067 (0.491)

0.047 (0.315)

0.108 (0.265)

Average I/σ(I)

8 (2.7)

9.8 (2.4)

16.6 (3.7)

8.3 (4.0)

Completeness (%)

78.3 (81.1)

97.1 (98.5)

99.8 (99.7)

99.8 (99.7)

Redundancy

2.3 (2.2)

2.9 (2.9)

3.3 (3.1)

3.5 (3.5)

   ∑ ∑ Ihi − Ih   1 Rmerge =  h i , where Ihi is the intensity of the ith observation of reflection h.

∑∑ I

hi

h

i



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Table 2: Properties of the final models

Hpα αCA-BZA

Resolution range (Å)

Hpα αCA-EZA

Hpα αCA-

Hpα αCA-

compound 1

compound 3

41.3 – 2.4

39.9 – 2.2

29.9 – 2.5

29.9 – 2.9

55054

96747

64588

39892

1726/14479/155

1751/15081/538

1756/14565/103

1785/14827/0

0.19/0.23

0.20/0.24

0.22/0.27

0.18/0.24

Average B (protein) (Å²)

43

45

52

46

Average B (water) (Å²)

29

35

36

-

Average B (Zn ions) (Å²)

40

38

39

35

38

39

47

37

0.01

0.01

0.01

0.01

1.6

1.4

1.3

1.6

Favored

93

96

94

93

Allowed

7

4

6

7

Outliers

0

0

0

0

No. of reflections Residues/atoms/waters R/Rfree

Average B (inhibitors) (Å²) Bond-length deviation from ideality (Å) Bond-angle deviation from ideality (º) Ramachandran space (%)


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Figure Legends Figure 1: The structures of the two different classes of sulfonamide compounds discussed in this study. A representation of the generic structure is also shown. (A) Class 1 is based on a 5-amino1,3,4-thiadiazole-2-sulfonamide or 5-imino-4,5-dihydro-1,3,4-thiadiazole-2-sulfonamide scaffold with substituents R1 and R2. Oxygen atoms are colored red, nitrogen blue, sulfur yellow, carbon gray, and R1 and R2 groups cyan. (B) Class 2 has a thiophene-2-sulfonamide scaffold fused with group X. Figure 2: Stereoview of the structural superposition of the active site residues of chains G and C, showing the two different binding modes of compound 1. For clarity, the protein atoms and molecular surface of chain C are not shown. Binding mode A is shown in stick representation with carbon atoms colored cyan; binding mode B is shown as thin lines with carbons in green. The main chain and/or side chains of the protein residues that only form interactions with compound 1 in binding mode A are shown with carbons colored cyan, and those that only interact with compound 1 in binding mode B are shown with carbons colored green. The residues that form interactions with compound 1 in both binding modes are shown with carbons colored gray. The coordinating bond between the inhibitor and the catalytic zinc ion is shown as a black dashed line; hydrogen bonds are shown as blue dashed lines. Nitrogen, oxygen and sulfur atoms are colored blue, red and yellow, respectively. Figure 3: BZA binding in the active site of HpαCA and comparison to HCAII. Residue labels in black refer to HpαCA. (A) The simulated-annealing omit |2Fo-Fc| electron density map for BZA bound to HpαCA calculated at 2.4 Å resolution and contoured at 1.0-σ level. The BZA molecule is shown in all-atom ball-and-stick representation with carbon atoms colored green. Amino acid



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residues that interact with BZA are shown in stick representation with carbon atoms colored wheat. The catalytic zinc ion that is coordinated tetrahedrally is shown as a black sphere. Hydrogen bonds are shown as blue dashed lines. (B) Superposition of all chains (except chain F) present in the asymmetric unit of the crystal complex HpαCA/BZA showing variation in the position of BZA. (C) The two different BZA binding modes observed in the active site of chain E of HpαCA. The amino acid residues that interact with the phenyl ring only in the first binding mode are shown with carbon atoms colored cyan; those interacting only with the second mode green; and the residues that form hydrogen bonds with the 5-sulfonamido group of benzolamide in both confirmations are shown with carbon atoms colored white. The molecular surface was calculated with the inhibitor molecule excluded. (D) Superposition of the active site residues of the HpαCA/BZA complex (wheat) with those of the HCAII/BZA complex (green) showing the similar binding mode of BZA. The catalytic zinc ion and protein residues that form interactions with BZA are shown. The helix 130-136 in HCAII is drawn as a ribbon. The HCAII residues that form additional stabilizing interactions with BZA are shown using ball-and-stick representation and marked with boxed labels in green. Figure 4: The HpαCA complex with compound 3 superposed on the HpαCA complex with BZA. The compound 3 and BZA molecules are shown with carbon atoms colored light green and cyan, respectively. For clarity, only the side chains lining the inhibitor-binding pocket in the complex with compound 3 are shown (with carbon atoms colored wheat). The molecular surface was calculated with the inhibitor molecule excluded. The figure illustrates the similar mode of binding of compound 3 and BZA and shows the detail of the additional stabilizing interactions provided by the tert-butyl group. The catalytic zinc ion that is coordinated tetrahedrally is shown as a black sphere; hydrogen bonds are shown as blue dashed lines. The simulated-annealing omit


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|2Fo-Fc| electron density map for compound 3 bound to HpαCA was calculated at 2.9 Å resolution and contoured at 1.0-σ level. Figure 5: The binding site for EZA in its crystal complex with HpαCA and comparison with the HpαCA/AAZ and HCAII/EZA crystal complexes. Residue labels in black refer to HpαCA. (A) The simulated-annealing omit |2Fo-Fc| electron density for EZA is shown in light blue. The map was calculated at 2.2 Å resolution and contoured at 1.0-σ level. The EZA molecule is shown in all-atom stick representation with carbon atoms colored light green. Amino acid residues that interact with EZA are shown in stick representation with carbon atoms colored wheat. The tetrahedrally coordinated catalytic zinc ion is shown as a black sphere. Hydrogen bonds are shown as blue dashed lines. (B) Superposition of the EZA and AAZ complexes of HpαCA. The EZA and AAZ molecule are shown in all-atom stick representation with carbon atoms colored light green and cyan, respectively. For clarity, protein residues and protein surface are shown for the EZA complex only. The surface was calculated with the inhibitor molecule excluded, and colored according to the electrostatic potential. (C) Superposition of the active site residues of the HpαCA/EZA complex (wheat) with the corresponding complex of HCAII (green) showing the short helix 130-136 that stabilizes the ethoxy group of EZA in the HCAII/EZA complex. EZA molecule is shown using stick representation. The catalytic zinc ion and protein residues that form interactions with EZA are shown. The HCAII residues that form additional stabilizing interactions with EZA are shown using ball-and-stick representation and marked with boxed labels in green. Figure 6: Compound 2 modeled in the active site of HpαCA using coordinates for compound 1 in the respective complex as a template. Binding modes A and B are shown with carbon atoms colored cyan and light green, respectively. In mode A, the weaker binding of compound 2 in



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comparison to compound 1 is likely due to energetically unfavorable interaction between the additional aliphatic methyl group of compound 2 and partially negatively charged carbonyl oxygen atoms of the main-chain peptides of Ala192 and Pro193. In mode B, binding of compound 2 would be less tight than compound 1 due to an unfavorable proximity between its methyl group and the carbonyl oxygen of His84. Figure 7: The BRZ and DZA inhibitor molecules modeled into the active site of HpαCA using the crystal structure of the HpαCA/EZA complex as a template. The EZA, BRZ and DZA molecules are shown in all-atom stick representation with carbon atoms colored light gray, magenta and light green, respectively. Possible additional hydrogen bonds between BRZ/DZA and protein (not observed in the EZA complex) are shown. Figure 8: Superposition of the HpαCA/BRZ (A) and HpαCA/DZA (B) complexes, modeled using the crystal structure of the HpαCA/EZA complex as a template, with the crystal structures of the respective complexes of HCAII.25,26 The HpαCA and HCAII complexes are colored wheat and green, respectively. The catalytic zinc ion and protein residues that form interactions with BRZ and DZA are shown. The BRZ and DZA molecules are drawn using stick representation. Residue labels in black refer to HpαCA. The HCAII residues that form additional stabilizing interactions with BRZ and DZA are shown using ball-and-stick representation and marked with boxed labels in green. Figure 9: Classification of the HpαCA residues forming interactions with different classes of sulfonamide inhibitors discussed in this study and analysis of the differences with the respective sulfonamide binding site in HCAII. (A) The superposition of the crystal structures of HpαCA in complex with compound 1 (magenta), compound 3 (blue), AAZ (red), BZA (black) and EZA


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(orange) showing different pockets in and around the active site that accommodate these inhibitors. Only the protein moiety of the respective AAZ complex is shown for clarity. The HpαCA protein surface that contributes to binding of all inhibitors is colored green and comprises residues Val131, Val141, Leu190, Thr191, Ala192, Trp201. The residues that form interactions with some sulfonamides but not others are shown in stick representation and colored according to the pocket they are in. The residues colored red form the pocket that accommodates the alternative binding mode (mode B) of compound 1 and BZA. The residues colored cyan form the walls of the pocket that accommodates the R2 substituent of AAZ, BZA in the binding mode A, and compound 3. Residues Pro193 and Pro194 that interact with the R1 substituent (methyl group) of compound 3 and MZA, and with the fused bicyclic ring of EZA, are colored orange. (B) Superposition of the crystal structures of HpαCA (wheat) in complex with compound 3 (thin black lines) and HCAII (gray) highlighting differences between the sulfonamide binding sites in the two enzymes. The additional helix 130-136 in HCAII is shown in green. Its structural equivalent in HpαCA is a short loop colored blue. The residues involved in inhibitor binding that are not conserved between the two enzymes are shown using ball-and-stick representation and labelled in black and green in HpαCA and HCAII, respectively. The catalytic zinc ion is shown only in HpαCA as a light blue sphere.



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StructureâActivity Relationship for Sulfonamide Inhibition of

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