Quinone Methide-Based Organophosphate Hydrolases Inhibitors: Trans Proximity Labelers versus Cis Labeling Activity-Based Probes

Article abstract of DOI:10.1002/cbic.202000611

Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors. Typically, enzyme-mediated bond breaking releases a phenol product that rearranges into an electrophilic QM that in turn covalently modifies protein side chains. However, the factors that govern the reactivity of QM-based inhibitors and their mode of inhibition have not been systematically explored. Foremost, enzyme inactivation might occur in cis, whereby a QM molecule inactivates the very same enzyme molecule that released it, or by trans if the released QMs diffuse away and inactivate other enzyme molecules. We examined QM-based inhibitors for enzymes exhibiting phosphoester hydrolase activity. We tested different phenolic substituents and benzylic leaving groups, thereby modulating the rates of enzymatic hydrolysis, phenolate-to-QM rearrangement, and the electrophilicity of the resulting QM. By developing assays that distinguish between cis and trans inhibition, we have identified certain combinations of leaving groups and phenyl substituents that lead to inhibition in the cis mode, while other combinations gave trans inhibition. Our results suggest that cis-acting QM-based substrates could be used as activity-based probes to identify various phospho- and phosphono-ester hydrolases, and potentially other hydrolases.

Full text of DOI:10.1002/cbic.202000611

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Combining Chemistry and Biology
Accepted Article
Title: Quinone methide based organophosphate hydrolases inhibitors:
Trans proximity labelers versus Cis labeling activity-based probes
Authors: Artem Dubovetskyi, Kesava Phaneendra Cherukuri, Yacov
Ashani, Anna Meshcheriakova, Eitan Reuveny, Gili Ben-
Nissan, Michal Sharon, Laura Fumagalli, and Dan Tawfik
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To be cited as: ChemBioChem 10.1002/cbic.202000611
Link to VoR: https://doi.org/10.1002/cbic.202000611
01/2020

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10.1002/cbic.202000611
ChemBioChem
Quinone methide based organophosphate hydrolases inhibitors: Trans
proximity labelers versus Cis labeling activity-based probes
Artem Dubovetskyi1, Kesava Phaneendra Cherukuri1, Yacov Ashani1, Anna
Meshcheriakova1, Eitan Reuveny1, Gili Ben-Nissan1, Michal Sharon1, Laura Fumagalli2 and
Dan S. Tawfik1,*
1 Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot 76
100, Israel; 2Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, via
Mangiagalli 25, I-20133, Milano, Italy.
* Corresponding author; E-mail: dan.tawfik@weizmann.ac.il
Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors.
Typically, enzyme-mediated bond-breaking releases a phenol product that rearranges into
an electrophilic QM that in turn covalently modifies protein side-chains. However, the
factors that govern the reactivity of QM-based inhibitors, and their mode of inhibition,
have not been systematically explored. Foremost, enzyme inactivation may occur in cis,
whereby one of the QM molecules inactivates the very same enzyme molecule that
released it, or by trans, if the released QMs diffuse away and inactivate other enzyme
molecules. We examined QM-based inhibitors for enzymes exhibiting phosphoester
hydrolase activity. We tested different phenolic substituents and benzylic leaving groups,
thereby modulating the rates of enzymatic hydrolysis, phenolate-to-QM rearrangement,
and the electrophilicity of the resulting QM. By developing assays that distinguish between
cis versus trans inhibition, we have identified certain combinations of leaving groups and
phenyl substituents that lead to inhibition in the cis mode, while other combinations led
to trans inhibition. Our results suggest that cis acting QM-based substrates may be used as
activity-based probes for identifying various phospho- and phosphono-ester hydrolases,
and potentially other hydrolases.
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10.1002/cbic.202000611
ChemBioChem
Inhibitors, and mechanism-based inhibitors in particular, are a key element in the
enzymology toolbox, with broader uses spanning from cell biology to medicinal chemistry.
In this context, quinone methide-based inhibitors were described for many classes of
enzymes, foremost for hydrolytic enzymes[1-7]. Enzyme-mediated breakdown, typically
hydrolysis, of an inert substrate leads to the release of a phenol product that rearranges into
an electrophilic QM that then modifies nucleophilic side-chains within the enzyme’s active-
site (Figure 1).
Figure 1. A general scheme of enzyme mediated quinone methide (QM) release and
inhibition. Enzymatic cleavage of the substrate group (SG; 1) releases a phenolate leaving
group (or phenol, depending on the product’s pKa and whether the enzyme protonates the
leaving group; (2). This phenol/ate product subsequently rearranges into a QM (3) that reacts
with nucleophiles to form a covalent adduct (4). Occurrence of the last step with a side-chain
within the enzyme’s active-site leads to enzyme labeling and inactivation.
QM-based inhibitors could, in principle, act as activity-based probes[1],[2],[8-10], namely, as
mechanism-based inhibitors that enable selective labeling, isolation and identification of
enzymes[3], [11-13]. Single turnover suicide inhibitors are ideal activity-based probes, but these
are available for relatively few classes of enzymes (e.g. activated phosphonates for serine
hydrolases[14],[15]). In other cases, inhibition may be based on release of an electrophile that
reacts with a nucleophilic side-chain in the enzyme’s active site (e.g. Refs[11],[12]). However,
in oppose to single turnover suicide inhibitors, inhibition by QM-based inhibitors is statistical.
The occasional turnover may result in the QM being formed and covalently modified within
the very same active site in which it was generated (cis labeling). However, most turnovers
result in the dissociation of the phenol/ate product or of its rearranged QM product. Once
released from the active-site, the QM can be quenched by water molecules and other
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ChemBioChem
nucleophiles (e.g. by the buffer), but it may also react with other enzyme molecules present
in the same solution (trans labeling; Figure 2).
Figure 2. Cis and trans modes of inhibition by QM-releasing diethyl phosphoesters
(DEP-QMs). A. In the cis mode, the QM is formed and is covalently trapped within
the very same active site in which it was generated (ochre zone). B. Alternatively, the
phenol/ate, or its QM product, may diffuse out of the active site in which they were
generated and react with other enzyme molecules (causing inhibition, or merely
modifying their surfaces) or be quenched by buffer and/or water molecules (blue
zone). Inset: A series of leaving groups (X) and ring substituents (Y) were applied as
to modulate the rate of enzymatic cleavage of the DEP-QM substrate (kenz), the rate
QM formation (kqmf), and the rate of alkylation of the released QM (kNu) which in turn
leads to enzyme inhibition (kinact). The benzylic side-chain, R, was either H (series 1, 2
and 6) or methyl group (series 3, 4 and 5) but this modification had little effect on the
efficacy of inhibition (Figure S2).
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