Crystallography Studies

85

86 The work started with the optimization of the conditions for PPE crystallization (Figure 34). Co-crystallization of the compounds with the enzyme was attempted by two techniques, soaking of pre-formed PPE crystals with 3-Oxo-β-Sultam compounds and co-crystallization of 3-Oxo-β-Sultam compounds with PPE. Only the second technique yielded the desired crystals where a positive density corresponding to a 3-Oxo-β-Sultam compound near the active site of PPE was identified.

Figure 34 – Native PPE crystals obtained when screening different conditions for PPE crystallization.

One of the first compounds to be successfully co-crystallized with PPE was 94 (Figure 35). The compound structure was clearly defined, with the open-ring form after reacting with the catalytic serine from PPE. Surprisingly, the density map showed that PPE was inhibited by sulfonylation of the active site, a result which directly challenged previously published data.¹⁷⁰

The crystallization process was optimized, and an improved electronic density map technique was developed after refinement of the previous results. Compounds 106, 94, 95 and 96 were co-crystallized with PPE. The open 3-Oxo-β-Sultam ring was visibly bound to the active site serine for all enzyme-compound pairs. In all co-crystallization experiments the protein had a positive density corresponding to the density of the ligand in the protein’s catalytic center. In all the refined models obtained the electron density suggested that the enzymes were being inhibited by sulfonylation. This was observed due to the presence of two density spheres near the catalytic serine which clearly correspond to the oxygens of the sulfonyl group. The crystallographic structures were refined and are presented in Figures 36 and 37.

87

Figure 35 – Reaction of compound 94 with PPE via attack of the catalytic serine to the sulfonyl group of 94. Active site zoomed view of the complexes between PPE and compounds 94, 95 and 96 with PPE.

The results clearly showed that the mechanism involves formation of a bond between the catalytic serine oxygen and the sulfur from the 3-Oxo-β-Sultam, displacing an amide leaving group and leaving the enzyme sulfonylated. This was a surprising and promising result since it showed an unexpected mechanism of reaction between a serine hydrolase and the 3-Oxo-β-Sultam structure. Inhibition of serine hydrolases by sulfonylation is an underexplored strategy and these results might uncover some of the requirements and mechanisms underlying the inhibition of this type of enzyme.

88 For larger compounds like 106, electronic density was only well-defined at the catalytic center. This is probably explained by the flexibility and mobility of the distal part of the molecule containing the linker and the NBD fluorophore, given the superficial PPE pocket.

Figure 36 – Electron density map around the covalently bound 95 (A) and 96 (B) ligands of PPE, |2Fo-Fc| map is depicted in blue mesh and contoured at 1σ level. Color code: carbon in yellow, oxygen in red, nitrogen in blue, sulfur

in green, and Br atoms in dark red.

Using the COOT program, structures of native HNE were superimposed with the obtained structures for PPE-3-Oxo-β-Sultam crystals. The inhibitors were fitted into HNEs active site and bound to Ser-195, after which an energy minimized model for this complex was obtained. The results suggested that orientation of the ligand in the HNE-inhibitor complexes should be similar to the ones obtained for the PPE-inhibitor complexes.

After the exciting results obtained with PPE, the same experiments were performed for HNE. This enzyme proved to be significantly more difficult to co-crystallize with the 3-Oxo-β-Sultam compounds and only one crystal was obtained, for the complex of compound 102 with HNE. An X-ray structure of the complex at 2.6 Å resolution with Rcryst of 20.8 % was obtained. The open-ring compound was clearly defined in HNE’s active site, covalently bound to the catalytic serine via the sulfur atom, which was consistent with the mechanism found for PPE. The results showed that the sulfonyl moiety participates in hydrogen bonding with Gly-193, Ser-195 and His-63, contributing to complex stabilization, along with the expected accommodation of the two ethyl groups in the hydrophobic S1 pocket.

89

Figure 37 - Surface representation of HNE:102 (A) and PPE:94 (B) with ligands depicted in sticks (carbon in yellow, oxygen in red, nitrogen in blue, sulfur in green, and halide atoms in dark red (C) or light blue (D)); catalytic serine is

colored in orange. Zoomed view of active site of HNE:102 (C) and PPE:94 (D) with |2Fo-Fc| electron density map drawn in blue mesh (at 1σ contour) around the covalently bound 3-Oxo-β-Sultam ligands.

Comparison of the results for PPE and HNE showed that 94 seemed to interact more extensively with the surface of PPE than 102 interacted with the surface of HNE, which instead extended towards the solvent and showed greater structural flexibility (Figures 37 and 38).

Overall, for all tested compound-enzyme pairs, the mechanism of inhibition of PPE and HNE was shown to be sulfonylation of the active site serine. The three compounds crystallized with PPE showed similar crystal structures, with stabilization of the sulfone moiety by hydrogen bonding with neighboring aminoacids. These results suggest a unified mechanism for hydrolysis and serine hydrolase inhibition by 3-Oxo-β-Sultams.

Sulfonylation is an underexplored strategy in enzymatic inhibition and drug discovery, with the β-sultams as the only sulfur (VI)-nitrogen-based reactive groups to successfully result in selective enzyme inhibitors and ABPs.113,277-279

90

Figure 38 – Atomic interactions around the ligands in HNE:102 (A) and PPE:94 (B). Hydrophobic interactions are displayed in green.

The covalent modification of proteins using sulfur-based reactive groups has focused on sulfur (VI) fluoride exchange chemistry, including sulfonyl fluorides and aryl fluorosulfates.^280-282 The unanticipated preference of a catalytic serine to react with the sulfonyl center of the 3-Oxo-β-Sultam when a theoretically more reactive neighboring acyl center is present, which upon ring opening could expel the better sulfonamide anion leaving group, strongly suggests that the nature of the leaving group does not have a large effect on reactivity. The discovery that 3-Oxo-β-Sultams inhibit serine hydrolases by sulfonylation could provide an opportunity to expand the available pool of sulfonylating reactive groups in chemical biology and uncover new enzymatic mechanisms that could guide medicinal chemistry endeavors for drug discovery.