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α- D -Gal-cyclophellitol cyclosulfamidate is a Michaelis complex analog that stabilizes therapeutic lysosomal α-galactosidase A in Fabry disease

Fabry disease is an inherited lysosomal storage disorder that is characterized by a deficiency in lysosomal α- D -galactosidase activity. One current therapeutic strategy involves enzyme replacement therapy, in which patients are treated with recombinant enzyme. Co-treatment with enzyme active-site stabilizers is advocated to increase treatment efficacy; a strategy that requires effective and selective enzyme stabilizers. Here, we describe the design and development of an α- D gal -cyclophellitol cyclosulfamidate as a new class of neutral, conformationally-constrained competitive glycosidase inhibitor that acts by mimicry of the Michaelis complex conformation. We found that D -galactose-configured α-cyclosulfamidate 4 effectively stabilizes recombinant human α- D -galactosidase (agalsidase beta, Fabrazyme®) both in vitro and in cellulo .


Introduction
Deficiency in α-galactosidase A (α-gal A, EC 3.2.1.22, a retaining glycosidase of the GH27 glycoside hydrolase family (www.cazy.org) 1 ) underlies Fabry disease. This inherited lysosomal storage disorder is characterized by toxic accumulation of glycosphingolipid globotriaosylceramide (Gb3) in lysosomes and its sphingoid base, globotriaosylsphingosine (Lyso-Gb3) in plasma and tissues 2,3 . Several mutations in the GLA gene encoding α-gal A can result in diminished protein levels and/or enzyme activity, leading to altered metabolite levels and a range of Fabry disease phenotypes. The accumulation of glycosphingolipid metabolites is thought to cause progressive renal and cardiac insufficiency and CNS pathology in Fabry patients 4 . Enzyme replacement therapy (ERT) for Fabry disease involves intravenous treatment with recombinant human α-gal A (agalsidase beta, Fabrazyme® or agalsidase alpha, Replagal®), but the clinical efficacy of this therapy is limited [5][6][7] . 1-Deoxygalactonojirimycin (Gal-DNJ 8, Migalastat®, Figure 1B) has recently been approved as pharmacological chaperone (PC) for the treatment of Fabry disease in patients with amenable mutations 8 . Gal-DNJ 8 binds mutant forms of α-gal A, which are catalytically competent but otherwise targeted for degradation due to misfolding. Gal-DNJ 8 stabilizes the protein fold, allowing the mutant α-gal A to be trafficked to lysosomes. However, PC therapy for Fabry disease is limited to specific mutations and its efficacy is hotly debated [9][10][11][12][13] . For this reason, an attractive alternative therapeutic intervention strategy, proposed recently, comprises jointlyadministering recombinant enzyme and a pharmacological chaperone [14][15][16] . This strategy aims to stabilize the recombinant enzyme in circulation such that larger proportions may reach disease affected tissues; permitting the use of extended intervals between injections and lower enzyme dosages which should diminish side effects, improve patient's life-style and reduce treatment costs 17,18 . For this strategy to become clinical practice, allosteric enzyme stabilizers or orthosteric competitive α-gal A inhibitors that prevent enzyme unfolding and are displaced by the accumulated metabolites in the lysosome recovering the glycosidase activity, with good selectivity and pharmacokinetic/ pharmacodynamic properties, are required 16,18,19 . We argue that the discovery of such commodities would be greatly facilitated by the design of new inhibitor templates. Human α-gal A hydrolyzes its substrate following a Koshland double displacement mechanism, resulting in net retention of stereochemistry at the anomeric center of the produced galactopyranose 20,21 . The reaction coordinates by which α-gal A processes its substrate to form the intermediate covalent adduct are 4 C1 → 4 H3 ⧧ → 1 S3 with respect to the conformation of the galactopyranose moiety in the Michaelis complex → TS → covalent intermediate complex ( Figure 1A) [22][23][24] . This same reaction itinerary is also employed by GH31 retaining αglucosidases, with the difference that a glucopyranose, rather than a galactopyranose, is captured in the enzyme active site 25 . We have recently shown that α-glc-cyclosulfate 1 ( Figure 1B) potently, selectively and irreversibly inhibits retaining αglucosidases. Compound 1 in free solution predominantly resides in a 4 C1 chair conformation, thus mimicking the initial Michaelis complex conformation utilized by α-glucosidases 25 allowing facile interception by the catalytic nucleophile. We reasoned that α-gal-cyclosulfate 2 would covalently and irreversibly inhibit α-gal A with equal efficiency and selectivity following the same mode of action ( Figure 1B and 1C). Building on this concept, we further hypothesized that substitution of one or both of the cyclosulfate ring oxygens for nitrogen, as in compounds 3-5, would lead to competitive α-gal A inhibitors because of the decreased leaving group capacity of cyclosulfamidates/cyclosulfamides, when compared to cyclosulfates ( Figure 1D). Such compounds would then offer competitive enzyme inhibitors to be tested as stabilizers of recombinant α-galactosidase A for Fabry treatment. Here, we show the validity of this reasoning by revealing α-galcyclosulfamidate 4 as a first-in-class, effective and selective, competitive α-gal A inhibitor. Structural and computational analysis of the conformational behavior of compound 4 in solution and in the active site of human α-gal A supports our design and provides a molecular rationale why compound 4 is an effective α-gal A inhibitor. We also show compound 4 to be effective in stabilizing recombinant α-gal A in vitro and in cellulo and that sphingolipid levels in Fabry fibroblasts are effectively corrected by co-treatment with α-gal A and 4.

Results
Synthesis of α-D-galactose configured cyclosulfate 2 and cyclosulfamidate 4. Benzoylated diol 11 (see SI and Scheme S1 for its synthesis) was treated with thionyl chloride and subsequently oxidized with ruthenium trichloride and sodium periodate to afford protected cyclosulfate 12. α-Gal-cyclosulfate 2 was obtained after benzoyl removal using methanolic ammonia (Scheme 1A). The synthesis of cis-1-amino-6-hydroxy cyclohexane 18, a key intermediate in the synthesis of α-gal-cyclosulfamidate 4 proceeded through oxazolidinone 17, which was obtained from trans-azido alcohol 13 (itself obtained from perbenzylated galacto-cyclophellitol, see the SI) as depicted in Scheme 1B. Hydrolysis of the carbamate in 17 and N-Bocylation provided 19, which was transformed into fully protected cyclosulfamidate 20. Global deprotection finally yielded the target compound 4. The synthesis of compounds 3, 5 and 9 ( Figure 1B) and intermediates follows related strategies, as is described in the Supporting Information. Free energy landscapes (FELs) of inhibitors report the conformational behavior in solution well, and can therefore be used to predict the selectivity for GH active sites 26 . We calculated conformational FELs of compounds 2-5 using ab initio metadynamics ( Figure 2A, S1 and S2). The FEL of α-galcyclophellitol cyclosulfate 2 is strongly biased towards 4 C1, with a secondary minimum around B2,5. This B2,5 conformer is unlikely to be enzyme active-site-reactive as it exhibits an equatorial C1-O bond ( Figure S2). The 4 C1 minimum of the substrate extends towards the TS-like 4 H3 conformation, indicating that cyclosulfate 2 in a 4 H3 conformation could be transiently populated on-enzyme, favoring the nucleophilic attack and formation of a glycosyl-enzyme adduct. The FELs of 3-5 show that substitution of the cyclic sulfate trap by cyclic sulfamidates (3 and 4) or sulfamide (5) does not significantly affect the conformational preferences. The local B2,5 minimum in 4 is more pronounced, probably due to a hydrogen bond between the 2-OH and one cyclosulfamidate oxygen ( Figure S2).

Structural analysis of α-gal-cyclosulfate 2 and α-galcyclosulfamidate 4 in complex with agalsidase beta.
Firstly, in order to confirm the covalent inhibition by the cyclic sulfate, the X-ray structure of agalsidase beta in complex with 2 (PDB:6IBM) was determined to 1.99 Å, revealing a single molecule of 2 covalently bound to the enzyme active site ( Figure  2B). The observed electron density unambiguously shows that α-cyclosulfate 2 has reacted by attack of the catalytic nucleophile, Asp 170, to form a covalent enzyme-inhibitor complex. This covalent complex adopts a 1 S3 conformation, consistent with the conformation of the covalent intermediate in the α-galactosidase reaction itinerary ( Figure 1A). Upon nucleophilic attack to the cyclic sulfate, the sulfate forms hydrogen bonds with Asp231 and Cys172, the latter suffering a shift in position.
Armed with the knowledge that 2 indeed forms a covalent adduct to agalsidase beta, we moved onto ascertain if the cyclosulfamidate 4 would, as envisaged, function as a non- covalent, active-centre-directed, inhibitor of the enzymereplacement enzyme. In contrast to cyclosulfate 2, cyclosulfamidate 4 (PDB: 6IBK determined to 2.07 Å) was indeed shown to reversibly bind the catalytic site ( Figure 2B). As expected, the α-galactose configured cyclosulfamidate 4 adopts a 4 C1 "Michaelis-like" complex conformation in the active site. Interestingly, the NH from the cyclosulfamidate moiety forms a hydrogen bond with the acid/base Asp231. Thermostability of agalsidase beta in the presence of αcyclosulfamidate 4 and Gal-DNJ 8.
Competitive α-galactosidase inhibitors, including Gal-DNJ 8, are currently investigated in clinical studies as stabilizers of recombinant enzyme, where they are deployed to enhance enzyme replacement efficacy. In such a treatment regime, enzyme and active site inhibitor are administered jointly 11,14,16 . The stability of a recombinant enzyme relative to the temperature is considered to reflect well its stability in body circulation 31 , and can be measured with ease, also in the presence of competitive inhibitors designed to stabilize protein fold 31,32 . Accordingly, we performed thermal stability assays (TSAs) on agalsidase beta alone and in the presence of increasing concentrations of 2, 4 or 8.
Thermal melting profiles of lysosomal α-gal A revealed that αgal A is most thermostable at pH 5.5 ( Figure 3A and S4 Surprisingly, we observed no thermostabilization effect on α-gal A in the presence of α-gal-cyclosulfate 2, despite this compound being an irreversible α-galactosidase inhibitor. Possibly, the sulfate group does not provide the optimal enzyme-ligand interactions to induce stabilization of α-galactosidase when the ring is in 1 S3 conformation adopted by covalently bound 2, compared to the 4 C1 conformations adopted by both 4 and Gal-DNJ 8.

Stabilization of agalsidase beta by α-cyclosulfamidate 4 in cell culture medium.
Agalsidase beta shows poor stability in plasma at pH of 7.3 -7.4, with only ~25 % of the hydrolytic activity being retained after incubation of enzyme at 1 µg/mL in human plasma for 30 minutes 33 . Given the stabilizing effect observed for 4 in the above described TSAs, we investigated the ability of this compound to stabilize agalsidase beta in culture medium at physiological pH compared to Gal-DNJ 8 34 .We first investigated the stabilization effect of the inhibitors in culture medium alone, as surrogate measure for plasma stability ( Figure 3B and 3C). Incubation of agalsidase beta (25 µL of 2.5 µg/µL) in cell culture medium (Dulbecco's Modified Eagles Medium/Nutrient Mixture F-12 (DMEM/F12), supplemented with 10 % fetal calf serum and 1 % penicillin/streptomycin) led to 80 % loss of activity within 15 min, in line with the poor stability of this enzyme in blood plasma. To assess the stabilizing effects of 2, 4 and Gal-DNJ 8 in cell culture media, agalsidase beta was incubated with increasing concentrations of these compounds, followed by capture of the enzyme on concanavalin A (ConA) sepharose beads, washing to remove bound inhibitor, and quantification of residual α-galactosidase activity with 4MU-αgal substrate. Media stabilization of agalsidase beta followed the same trend as observed in TSAs, with 2 failing to stabilize the enzyme and instead irreversibly inhibiting agalsidase beta. In contrast, α-gal-cyclosulfamidate 4 and Gal-DNJ 8 both prevented inactivation of agalsidase beta in cell culture media (pH 7.2) -~75 % residual α-gal-A activity was retained after incubation with 4 (at 500 µM) or Gal-DNJ 8 (at 50 µM) ( Figure  3C).
In situ treatment of cultured fibroblasts from patients with Fabry disease. We next investigated whether the stabilizing effect of αcyclosulfamidate 4 towards agalsidase beta produced an improvement in the cellular uptake of the enzyme by fibroblasts. We performed in situ studies in 5 different primary cell lines obtained from adult male volunteers: wild-type (WT, Control) representing normal α-gal A activity, 2 classic Fabry mutant fibroblasts (R301X and D136Y) with no α-gal A activity and 2 atypical variant Fabry mutants (A143T and R112H) with substantially lowered residual α-gal A activity. Fibroblasts were incubated with 0.5 % DMSO (untreated) or either 4 (200 µM) or Gal-DNJ 8 (20 µM) (in blue), agalsidase beta (100 ng) or with a combination of both enzyme and stabilizing agent (in green) ( Figure 5A). After 24 h treatment, the cells were harvested and homogenized, and the intracellular α-gal A activity of the corresponding cell lysates was measured. WT cell line presented normal α-gal A activity whereas untreated classic Fabry patients (R301X and D136Y) and variant mutation samples (A143T and R112H) showed reduced enzymatic activity. None of the cell lines, not even classical Fabry fibroblasts R301X and D136Y, showed significant increase in α-gal A activity when incubated with 4 (200 µM) or 8 (20 µM) alone for 24 h. Of note, Gal-DNJ 8 is known to enhance α-gal A activity in R301Q lymphoblasts after in situ 4-day treatment of a 100 µM daily dose. 34 Treatment with agalsidase beta showed a considerable increase in α-gal A activity in all the studied cell lines. This effect was improved in  most cases with the combinatorial treatment of agalsidase beta and 4 or 8 after 24 h incubation. We also measured α-gal A activity in media in order to confirm that the increase in α-gal A activity in cell lysates is due to stabilization of the enzyme ( Figure 5B). Thus, culture media were collected before harvesting the cells and α-gal A activity was measured after ConA purification. α-Gal A activity in media was at least double in all the cell lines treated with α-cyclosulfamidate 4 (200 µM) or Gal-DNJ 8 (20 µM), consisting with these compounds preventing α-gal A degradation during cell culture.

Gb3 and LysoGb3 levels are corrected by α-cyclosulfamidate 4.
Generally, Fabry patients present elevated Gb3 which is further metabolized by acid ceramidase into LysoGb3 in lysosomes 35 . LysoGb3 constitutes a signature of Fabry disease and allows diagnostic monitoring of disease progression 2,3,36-38 , and has been linked to neuronopathic pain and renal failure through its effect on nociceptive neurons and podocytes [39][40][41][42] . We investigated whether co-administration of α-cyclosulfamidate 4 and Gal-DNJ 8 with agalsidase beta would also have a positive effect in correcting these toxic metabolite levels. Gb3 and lysoGb3 levels from in situ treated cells were measured by LC-MS/MS ( Figure 6). Normal Gb3 and lysoGb3 levels observed in wild-type cells range around 2000 pmol/mL and 2 pmol/mL of Gb3 and lysoGb3, respectively. Cultured fibroblasts from classic Fabry patients (R301X and D136Y) treated with agalsidase beta resulted in a reduction of Gb3 and lysoGb3. This reduction was similar when fibroblasts were treated with the combination of α-cyclosulfamidate 4 (200 µM) or Gal-DNJ 8 (20 µM) and agalsidase beta. Variant Fabry A143T cell line presents normal Gb3 and lysoGb3 basal levels, whereas in R112H fibroblasts, these metabolites are increased and not corrected by agalsidase beta itself or inhibitor-agalsidase beta combination treatment ( Figure S6).  Fibroblasts were treated every 24 h with new medium and/or enzyme with or without inhibitor, and medium samples were collected for α-gal A activity quantification (See supporting information, Figure S7). α-Gal A activity was 3-4 times higher in fibroblasts treated with the combination of recombinant α-gal A and α-cyclosulfamidate 4 (200 µM) or Gal-DNJ 8 (20 µM) than those treated with agalsidase beta alone ( Figure 7A). This increase in α-gal A activity correlates with the reduction of Lyso-Gb3 from ~14 pmol/mL to ~4 pmol/mL in the cell lysates of Fabry (classic R301X) fibroblasts ( Figure 7B). We finally studied whether the amount of required ERT could be decreased when stabilized with 4 or Gal-DNJ 8 and still produce a similar effect. WT and Fabry (classic R301X) fibroblasts were treated with agalsidase beta at 200 ng/mL and 100 ng/mL. A reduction in toxic metabolites can be achieved in 4 days with half the concentration of enzyme (100 ng/mL) when either αcyclosulfamidate 4 (200 µM) or Gal-DNJ (20 µM) is added ( Figure  7C,D), with a similar reduction of toxic Lyso-Gb3 from ~10 pmol/mL to ~5-6 pmol/mL in the cell lysates of Fabry (classic R301X) fibroblasts ( Figure 7E).

Discussion
ERT with intravenous administration of recombinant human α-Dgalactosidase (agalsidase beta, Fabrazyme® or agalsidase alpha, Replagal®) reduces the levels of Gb3 and lyso-Gb3 in some tissues of Fabry patients, but its clinical efficacy is still limited [5][6][7]43 . The limited enzyme stability in plasma is a major drawback, and it is for this reason that enzyme active site binders that stabilize recombinant enzyme in circulation are pursued -with Gal-DNJ 8 (Migalastat®) currently in use in the clinic as the benchmark. Here we report the design and synthesis of the first-in-class conformational glycosidase inhibitor and α-gal A stabilizing agent, α-cyclosulfamidate 4. We show that this compound reversibly and selectively inhibits agalsidase beta with an IC50 value of 67 µM and a Ki of 110 µM. Please do not adjust margins Please do not adjust margins µM) and Gal-DNJ 8 (at 20 µM) for 24 h shows no effect on α-Dgalactosidase activity. However, we observe an increased α-Dgalactosidase activity in all cells treated with the combination of agalsidase beta and stabilizing agents (4 at 200 µM and 8 at 20 µM) when compared to the cells treated only with agalsidase beta. This result also correlates with an increased α-D-galactosidase activity in the cell medium of the cells treated with enzyme and 4 or 8. The stabilizing effect is more pronounced when cells are treated for longer time (4 days), suggesting that the agalsidase beta complexed with α-gal-cyclosulfamidate 4 or Gal-DNJ 8 is stabilized in the cell medium, internalized and dissociated from the reversible inhibitor in the lysosomes. Finally, co-administration of α-cyclosulfamidate 4 or Gal-DNJ 8 with agalsidase beta highlights a similar correction of toxic Gb3 and lyso-Gb3 metabolite levels as with the ERT alone. Importantly, similar α-gal A activity and correction of toxic metabolites is achieved with half the concentration of agalsidase beta when this is stabilized by α-cyclosulfamidate 4 or Gal-DNJ 8. The synergy between Gal-DNJ 8 and the human recombinant α-gal A in cultured fibroblasts from Fabry patients has recently been demonstrated both in agalsidase alpha and beta 16,19 . This synergism, together with our agalsidase beta stabilization results, supports the idea that the efficacy of a combination treatment may be superior to ERT or PC alone for several reasons. Co-administration of ERT and active site inhibitor may be effective in all Fabry patients, independent of mutations in their endogenous α-gal A. Furthermore, stabilization of the recombinant human α-gal A by a stabilizing agent may reduce the required enzyme dosages or extend IV injections intervals, and therefore improve patient's life-style and reduce side effects and treatment costs.

Conclusions
In conclusion, we have developed a new class of α-D-galactosidase inhibitors based on cyclophellitol cyclosulfamidate as a conformational Michaelis complex isostere. Although cyclosulfamidate 4 is a 1000-fold weaker inhibitor of recombinant αgal A compared to Gal-DNJ 8 in vitro, it stabilizes α-gal A in cellulo at only 10 fold higher concentration, and we argue that non-basic, competitive glycosidase inhibitors are attractive starting points for clinical development as stabilizers of (recombinant) glycosidases in the context of lysosomal storage disorders. As well, compound 4 and its structural isosteres (3 and 5) comprise a new class of competitive glycosidase inhibitors that stabilize agalsidase beta for the first time, not by the glycoside configurational mimicry and basic nature that is the hallmark of iminosugars (including Migalastat®), but by configurational and conformational mimicry of the Michaelis complex. Michaelis complex or product-like conformational competitive inhibitors have been reported that act on other glycosidases, for instance, thio-oligosacharides [44][45][46] and kifunensine 47,48 . We believe that transferring the structural characteristics of our cyclosulfamidates to differently configured structural analogues may yield potent and selective competitive inhibitors targeting other glycosidases and that these may have biological or biomedical value in their own right, be it as stabilizing agents or as classical enzyme inhibitors.

Conflicts of interest
There are no conflicts to declare.