Structural elucidation of L-ascorbic acid-6-hexadecanoate – a

4.3.3 Structural elucidation of L-ascorbic acid-6-hexadecanoate – a potent hyaluronidase inhibitor

Most parts of this section were included in a recent publication by Botzki et al.⁶¹.^§ Supported by X-ray analysis of the hylSpn-L-ascorbic acid complex¹³³ hydrophobic interactions with Trp292 and Tyr408 were supposed to play an im-portant role for binding of vitamin C (4.1) in the catalytic site of the bacterial hyaluronidase from S. pneumoniae (hylSpn). Hence, vitamin C derivatives with increased hydrophobic properties should lead to stronger inhibitors. This hy-pothesis was confirmed as demonstrated in chapters 4.3.1 and 4.3.2. Subse-quently, the potent inhibitor of hylB4755 L-ascorbic acid-6-hexadecanoate (4.21), a highly effective antioxidant¹⁵⁸ and glutathione-S-transferase inhibitor¹⁵⁹, was investigated on its inhibitory effect on hylSpn and in cooperation with the work group of M. J. Jedrzejas a crystal structure of S. pneumoniae hyaluronate lyase, co-crystallized with 4.21 was determined at a 1.65 Å resolution (pdb-file:

1W3Y)⁶¹. The X-ray structure should shed light on the enzyme-inhibitor interac-tions resulting in proposals for more potent inhibitors. The crystallization ex-periments were performed by M. Nukui and M. J. Jedrzejas (Children’s Hospital Oakland Research Institute, Oakland, California 94609, USA). The X-ray struc-ture of the complex was solved by D. Rigden (National Center of Genetic Re-sources and Biotechnology, Cenargen/Embrapa, Basília, D.F. 70770-900, Bra-zil).

4.3.3.1 Inhibition of hyaluronidases caused by ascorbic acid and L-ascorbic acid-6-hexadecanoate: a comparison

As already mentioned, it was reported that L-ascorbic acid inhibited the activitiy of hyaluronate lyase of S. pneumoniae (hylSpn) with an IC50 value of about 6 mM but no inhibitory effect could be observed on the bovine testicular hyalu-ronidase measured under the same reaction conditions¹³³. These results were revised using our slightly different turbidimetric assay protocol using Neoper-mease^®, a bovine testicular hyaluronidase, hylSpn and additionally, the

§ Note: The author of this doctoral thesis made substantial contribution and is co-author of this publication by Botzki et al.

rial hyaluronidase from S. agalactiae strain 4755, the bacterial enzyme prefer-entially used in this doctoral project. At first, we had to standardize the experi-ments for all three enzymes by performing the turbidimetric assay with equiac-tive concentrations of all enzymes at pH 5. The enzyme activities in presence of 4.1 and 4.21 were determined as described elsewhere⁶¹.

In agreement with the result described in the previous sections, the bacterial en-zymes were weakly inhibited by vitamin C (4.1) with IC50 values of 6 mM for hylB4755 and 35 mM for hylSpn, respectively, and 4.1 was inactive on the bovine enzyme up to concentrations of 100 mM. Consequently, vitamin C turned out to be a weak and selective inhibitor of the bacterial enzymes under the used reac-tion condireac-tions. As depicted in Fig. 4.7, vitamin C ester 4.21 displayed a strong inhibitory effect on hylB4755 with an IC50 value of 4 µM. On the related bacterial enzyme hylSpn and the bovine testicular hyaluronidase IC50 values of 100 µM and 56 µM, respectively, were determined. Compared to the parent compound 4.1, 6-O-palmitoyl-L-ascorbic acid (4.21) is an up to 1500 times more potent inhibitor of bacterial and bovine hyaluronidases (see Table 4.4).

log c in mol / l

-6,0 -5,5 -5,0 -4,5 -4,0 -3,5 -3,0

enzymatic activity in %

0 20 40 60 80

100 hylB₄₇₅₅

BTH hylSpn

Fig. 4.7: Inhibition of hylB4755, hylSpn and Neopermease^® (BTH) by L-ascorbic acid-6-hexa-decanoate (4.21)

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Table 4.4: IC50 values ± SEM of vitamin C (4.1) and L-ascorbic acid-6-hexadecanoate (4.21) determined on equiactive concentrations of S. pneumoniae hyaluronate lyase, S. agalactiae hyaluronate lyase strain 4755 and BTH (Neopermease^®) using a turbidimetric assay at pH 5

Compound hylB4755, IC50 [µM] hylSpn, IC50 [µM] BTH, IC50 [µM]

4.1 6100±100 34800±300 inactive (< 100 mM)

4.21 4.2±0.13 100.0±2.00 56.5±0.13

Furthermore, 4.21 is about 25-fold more active on hylB4755 than on hylSpn whereas 4.1 revealed only a 6-fold selectivity. Moreover, the selectivity of 4.21 for the bacterial enzyme vs. BTH is only weak. With respect to the selectivity for the enzymes further explorations are necessary since the obvious selectivity cannot be quantitatively confirmed without the data for the Km values of the ap-plied HA substrate.

Finally, it can be concluded that additional hydrophobic enzyme-inhibitor inter-actions enhance the inhibitory effect as confirmed by the distinctly higher in-hibitory activity of 4.21 on all investigated enzymes compared to 4.1.

4.3.3.2 The binding mode of 6-O-palmitoyl-L-ascorbic acid to S. pneumo-niae hyaluronidase

In order to determine the binding mode of compound 4.21 the X-ray structure of hylSpn co-crystallized with L-ascorbic acid-6-hexadecanoate was determined at 1.65 Å resolution (pdb-file: 1W3Y)⁶¹. This information should be useful to create a model for the interactions of the inhibitor with amino acid residues inside the active site of the enzyme and should facilitate the further development of more potent inhibitors. The hylSpn part of the complex structure contains an N-termi-nal α-domain and a C-terminal β-domain connected by a 10-residue linker and has only slight changes when compared with the native hylSpn⁶⁸ and the vita-min C-hylSpn¹³³ crystal structures. The active site of the enzyme is situated in the predominant cleft between α- and the β-domains. The catalytic mechanism as proposed^79,160-162 has been supported by structural determinations66,67,71,160

and mutagenesis studies^67,71,163. Catalysis is implemented at the narrowest part of the catalytic cleft in which Asn349, His399 and Tyr408 are mainly responsible

for it. Additionally, a hydrophobic patch consisting of Trp291, Trp292 and Phe343 contributes to precise positioning of the substrate.

The electron density at the binding site enabled the satisfactory localization of 4.21 within the active site of hylSpn as shown in Fig. 4.8. The final X-ray struc-ture offered additional difference electron density and elevated B-factors of the inhibitor (Table 4.3) suggesting the possibility of alternative binding modes of 4.21. However, despite repeated attempts, only the conformation shown was well supported by the density. Surprisingly, the lactone ring of the inhibitor seems to bind in a ring-opened form which was evident from electron density, although the vitamin C portion of 4.21 binds to the same region as intact vitamin C¹³³. Possibly, the ring-opened form was emerged during the crystallization time (Fig. 4.8). It is well-known that oxidation of L-ascorbic acid (4.1) gives L-dehy-droascorbic acid, which is rapidly degraded under physiological conditions (pH 7.4) via L-diketogulonate to yield L-erythrulose and oxalate¹⁶⁴. It is suggested that 4.21 is modified in a similar manner, but only the hydrolyzed lactone moiety was modeled into the electron density. However, it is unlikely that ring-opening

Y408

H399

T400 F343

W291 W292

N290

Fig. 4.8: Binding mode of L-ascorbic acid-6-hexadecanoate (4.21) at the active site of hylSpn. Distances for possible Hydrogen Bonds: N290-O5: 1.65 Å; Y408-COO: 1.78 Å

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occurs during the performance of the enzymatic assay with respect to the much shorter time needed for the assay.

As shown in Fig. 4.8, 4.21 binds within the catalytic site of hylSpn explaining the inhibitory activity. With the exceptions of a hydrogen bond between the carb-oxylate group of the inhibitor and the hydroxyl of Tyr408 and others between the hydroxy groups at C-4 and C-5 and Asn290 the majority of proteinhibitor in-teractions are hydrophobic. The hydrophobic face of the vitamin C portion lies flat on the side chain of Trp292, a member of the hydrophobic patch. Such an arrangement is quite common in complexes of carbohydrate-binding proteins with their ligands¹⁶⁵. The palmitoyl moiety fits in a mainly hydrophobic surface crevice. With the exception of three terminal carbon atoms for which density did not permit modeling the aliphatic chain matches very well with the existent electron density. The palmitoyl group forms hydrophobic interactions with Trp291 and Phe343, both contributing to the hydrophobic patch along with His399 and Thr400.

H399 Y408

N290

W291

W292

Fig. 4.9: Illustration of the binding site of 4.21 (carbon atoms are colored in yellow, oxygen atoms are colored in red and nitrogen atoms are illustrated in white) with Connolly surface (sample radius: 1.4 Å). The color encoding range from brown (high lipophilicity) to blue (polar).

None of the interactions is sufficiently close and geometrically suitable to be considered as a weak hydrogen bond involving methylene carbons of the palmitoyl group. Comparing the binding modes of 4.21 with that of a substrate-based hexasaccharide (pdb-file:1LOH)⁷⁰ there is an obvious overlapping of the binding sites. But the surface cleft that accommodates the palmitoyl group is not directly involved in substrate binding. Interestingly, one of the three well-ordered cryoprotectant xylitol molecules bound to hylSpn fits close to the end of the palmitoyl moiety. The distance between the penultimate resolved carbon of the palmitoyl group and atom O5 of xylitol is only 4.0 Å (Fig. 4.9). This position of the bound xylitol suggests that the affinity of the present inhibitor might be en-hanced through addition of matching groups at the aliphatic end of the palmitoyl moiety.