10.1002/cmdc.202000680
ChemMedChem
FULL PAPER
compound 19 which belongs to the MA series had almost similar
inhibition potencies (i.e. Ki values of 92.8 nM and 95.2 nM).
Compound 19 was the only effective inhibitor of the VhCA α (Ki of
40.7 nM) whereas compound 1 showed the best inhibition value
against the VhCA β isoform (Ki of 535.1 nM).
inhibitor at least six traces of the initial 5–10% of the reaction have been
used for determining the initial velocity. The uncatalyzed rates were
determined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-
deionized water and dilutions up to 0.01 nM were done thereafter with the
assay buffer. Inhibitor and enzyme solutions were preincubated together
for 15 min at room temperature prior to assay, in order to allow for the
formation of the E-I complex. The inhibition constants were obtained by
non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff
equation, as reported earlier [17-19], and represent the mean from at least
three different determinations. All CA isofoms were recombinant ones
obtained in-house as reported earlier. [17-19]
ii) Compounds 1-20 showed rather complex inhibition profiles
against B. pseudomallei CAs (i.e. BpsCA β and BpsCA γ).
Compounds 1, 3, 13 and 14, resulted the best performing CAIs
against BpsCA β with Kis of 394.0, 501.7, 648.8 and 471.4 nM,
respectively, whereas the 4-OH derivative 5 and the 4-N,N-
dimethyl amino substituted 17 resulted almost equal in inhibiting
the such an isoenzyme when compared to the reference AAZ (Ki
values of 741.2, 742.8 and 745.0 nM respectively). Again, the
introduction of various moieties at the phenyl ring tail resulted
critical for the kinetic profile. For instance, the MA analogue of 1
(i.e. compound 11) showed its Ki reduced by 6.6-fold (KIs of 394.0
and 2605 nM respectively). Among the most effective BpsCA β
inhibitors are the fluorine containing 13 and 14. It is worth noting
that the regioisomer 14 was 1.4-fold more potent than 13,
whereas opposite kinetic trend, with comparable intensity, was
observed for the same regioisomers belonging to the SA series
(i.e. compounds 3 and 4). As for the BpsCA γ the CAI reference
AAZ was the most effective (KI of 149.0 nM), whereas 18 resulted
the most potent among the compounds synthesized (Ki value of
324.6 nM). A clear SAR for the BpsCA γ by means of the data in
table 1 resulted quite difficult although the MA series still
confirmed to be the most efficient in inhibiting such an isoform.
Overall, most of the compounds from the 1-20 series were found
selective and effective CAIs against the VhCA γ and BpsCA β
over their bacterial CAs expressed strains.
Keywords: bacterial carbonic anhydrases, benzene-
sulfonamides, V. cholerae, B. pseudomallei, inhibition studies
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Conclusions
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This study makes use of an ad hoc modular design strategy to
obtain a set of compounds useful to investigate for the first time
their ability to inhibit CAs expressed from V. Cholerae (i.e. α-CA
and β-CA) and B. Pseudomallei (β-CA and γ-CA). Most of the
compounds presented excellent and selective inhibition potencies
against VhCA γ and BpsCA β with Kis in the range of 82.5 – 191.4
nM and 394 – 742.8 nM, respectively. Although an exhaustive
SAR is not feasible at this stage, for the scope of this study is
acceptable to define as general trend that compounds 11-20,
which belong to the MA series, are far more efficient in inhibiting
the VhCA γ and BpsCA β when compared to their SA counterpart
(i.e. compounds 1-10). More importantly the data obtained in this
study represent a solid starting point for the design of potential
new therapeutics useful reduce bacterial resistance to
conventional and clinical used antibiotics.
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Experimental Section
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General
All compounds were obtained according to previous reported procedures
[15].
Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for
assaying the CA catalysed CO2 hydration activity [16] Phenol red (at a
concentration of 0.2 mM) has been used as indicator, working at the
absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer,
and 20 mM Na2SO4 (for maintaining constant the ionic strength), following
the initial rates of the CA-catalyzed CO2 hydration reaction for a period of
10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants. For each
3
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