Phosphonamidates are the first phosphorus-based zinc binding motif to show inhibition of β-class carbonic anhydrases from bacteria, fungi, and protozoa

Article abstract of DOI:10.1080/14756366.2019.1681987

A primary strategy to combat antimicrobial resistance is the identification of novel therapeutic targets and anti-infectives with alternative mechanisms of action. The inhibition of the metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) from pathogens (

Full text of DOI:10.1080/14756366.2019.1681987

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Phosphonamidates are the first phosphorus-based
zinc binding motif to show inhibition of β-class
carbonic anhydrases from bacteria, fungi, and
protozoa
Siham A. Alissa, Hanan A. Alghulikah, Zeid A. Alothman, Sameh M. Osman,
Sonia Del Prete, Clemente Capasso, Alessio Nocentini & Claudiu T. Supuran
To cite this article: Siham A. Alissa, Hanan A. Alghulikah, Zeid A. Alothman, Sameh M.
Osman, Sonia Del Prete, Clemente Capasso, Alessio Nocentini & Claudiu T. Supuran (2020)
Phosphonamidates are the first phosphorus-based zinc binding motif to show inhibition of β-class
carbonic anhydrases from bacteria, fungi, and protozoa, Journal of Enzyme Inhibition and Medicinal
Chemistry, 35:1, 59-64, DOI: 10.1080/14756366.2019.1681987
To link to this article: https://doi.org/10.1080/14756366.2019.1681987
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
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Published online: 30 Oct 2019.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 59–64
https://doi.org/10.1080/14756366.2019.1681987
SHORT COMMUNICATION
Phosphonamidates are the first phosphorus-based zinc binding motif to show
inhibition of b-class carbonic anhydrases from bacteria, fungi, and protozoa
Siham A. Alissaa, Hanan A. Alghulikaha, Zeid A. Alothmanb, Sameh M. Osmanb, Sonia Del Pretec,
Clemente Capassoc , Alessio Nocentinid and Claudiu T. Supurand
b
aChemistry Department, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia; Chemistry Department,
c
d
College of Science, King Saud University, Riyadh, Saudi Arabia; Istituto di Bioscienze e Biorisorse, CNR, Napoli, Italy; NEUROFARBA Department,
ꢀ
Sezione di Scienze Farmaceutiche e Nutraceutiche, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Sesto
Fiorentino (Firenze), Italy
ABSTRACT
ARTICLE HISTORY
Received 28 August 2019
Revised 5 October 2019
Accepted 10 October 2019
A primary strategy to combat antimicrobial resistance is the identification of novel therapeutic targets and
anti-infectives with alternative mechanisms of action. The inhibition of the metalloenzymes carbonic anhy-
drases (CAs, EC 4.2.1.1) from pathogens (bacteria, fungi, and protozoa) was shown to produce an impair-
ment of the microorganism growth and virulence. As phosphonamidates have been recently validated as
human a-CA inhibitors (CAIs) and no phosphorus-based zinc-binding group have been assessed to date
against b-class CAs, herein we report an inhibition study with this class of compounds against b-CAs from
pathogenic bacteria, fungi, and protozoa. Our data suggest that phosphonamidates are among the CAIs
with the best selectivity for b-class over human isozymes, making them interesting leads for the develop-
ment of new anti-infectives.
KEYWORDS
Virulence; resistance;
carbonic anhydrase;
inhibition; phosphonami-
date; selectivity
1. Introduction
of their growth, which in turn leads to a significant anti-infect-
ive effect10.
Virulence is labelled as the ability of a microorganism to infect the
host and cause a disease. Virulence factors are molecules that
assist the pathogen to colonise the host at the cellular level1.
These factors can be either secretory, membrane associated or
cytosolic and facilitate the microorganism metabolic, physiological,
Unlike mammals and human whose genome uniquely encode
for a-class CAs, the genome of many pathogenic bacteria (e.g.
Vibrio cholerae11, Francisella tularensis12, Burkholderia pseudoma-
lei13, the Gram-negative bacteria provoking cholera, tularaemia,
and melioidosis), fungi (e.g. Candida glabrata14, Cryptococcus neo-
formans15, or Malassezia globosa16, respectively responsible of can-
didiasis, cryptococcosis, and dandruff), and protozoa (e.g.
Leismania donovani chagasi which provokes leishmaniasis17)
encode for b-class enzymes. Actually, the seven different CA
classes do not show significant structural homology with each
other. Furthermore, CAs of the same class but belonging to genet-
ically distant species, show differences at the level of their cata-
lytic site18. Therefore, a probable and realistic possibility to
develop selective CA inhibitors exists. b-CAs exist as oligomers
formed by two or more identical subunits such as dimers, tet-
ramers, and octamers19.
and morphological adaption in the host1,2
.
Virulence factors are deeply implicated in the onset of pharma-
cological resistance to anti-infectives. Preventing the implementa-
tion of these processes is a strategy deemed successful to date to
overcome drug-resistance3. Targeting virulence factors might
indeed reduce, eliminate, and/or reverse the evolutionary selective
pressures that induce the pathogen to develop resistance, which
represents a main threat to human health nowadays. The metal-
loenzymes carbonic anhydrases (CAs, EC 4.2.1.1) have been shown
to play critical roles in the virulence of many pathogens among
which bacteria, fungi, and protozoa4,5
.
CAs are a superfamily of phylogenetically ubiquitous metal-
loenzymes, present in Prokaryotes and Eukaryotes, classified in
seven genetically unrelated families, a, b, c, d, f,ꢀg, and h4–8. By
catalysing the reversible hydration of CO2 to HCO3 and Hþ, these
enzymes are implicated in many physiological processes which are
basic for life5,9. In microorganisms, CAs are involved in photosyn-
thesis (cyanobacteria), biosynthesis of DNA, amino acids and fatty
acids, and proliferation, survival and differentiation of pathogens
both in the hosts and in the environmental niches4,10. Compelling
data exist in the literature, which strongly indicate that interfer-
Sulphonamides and bioisosteres are the most potent class of
CA inhibitors (CAIs) and have been, probably exhaustively,
exploited to date in the design of inhibitors against almost every
known CA9. Nonetheless, these chemotypes commonly show a
weak isoform-selectivity both within the subset of human CAs and
between isoforms from distinct classes, such as a- and b-5. As a
result, many other chemotypes (phenols20,21, mono- and dithiocar-
bammates22,23, boroles24, carboxylates,25 N-nitrosulfonamides26,27
have been investigated as inhibitors of b-CAs to identify selective
)
ence with CA activity in various parasites leads to an impairment modulators for targeting the pathogens encoding for enzymes of
CONTACT Siham A. Alissa
Riyadh 11671, Saudi Arabia; Claudiu T. Supuran
Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Sesto Fiorentino (Firenze), Italy
saalissa@pnu.edu.sa
Chemistry Department College of Science, Princess Nourah bint Abdulrahman University, P. O. Box 84428,
claudiu.supuran@unifi.it NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche,
ꢀ
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

60
S. A. ALISSA ET AL.
this class. Among these, N-nitrosulfonamides, phenols, and natural The uncatalyzed rates were determined in the same manner and
polyphenols stood out as selective inhibitors for b-class CAs from subtracted from the total observed rates. Stock solutions of inhibi-
pathogens over human ubiquitous CAs20,21,27
.
tor (0.1 mM) were prepared in distilled–deionised water and dilu-
A recent paper by us showed for the first time that phospho- tions up to 0.01 nM were done thereafter with the assay buffer.
namidates are able to inhibit human CAs in the micromolar range Inhibitor and enzyme solutions were preincubated together for
acting a sulphonamide biomimetics additionally possessing a 1 h at room temperature prior to assay, in order to allow for the
chiral binding mode (Figure 1)28. Up to now, no one scheduled formation of the E–I complex. The inhibition constants were
testing phosphorus based binding motifs against b-class CAs. obtained by nonlinear least-squares methods using PRISM 3 and
Considering the interesting results reported earlier against these the Cheng–Prusoff equation, as reported earlier, and represent the
enzymes, here we report an inhibition study with benzenephos- mean from at least three different determinations30,31. All CA iso-
phonamidates against b-CAs from pathogenic bacteria, fungi, and forms were recombinant ones obtained in-house as reported
protozoa working out their structure–activity relationship (SAR) in earlier32,33
.
comparison to hCA I and II previously reported inhibition.
3. Results and discussion
2. Methods
3.1. Chemistry
2.1. Chemistry
A straightforward strategy was used to synthesise the compounds
The synthesis of phenylphosphonic diamide 2 and alkyl phospho- starting from phenylphosphonic dichloride 128 (Scheme 1).
namidates 3–10 was reported earlier by our group28.
Phenylphosphonic diamide 2 was obtained by reacting 1 with an
aqueous solution of 35% ammonia. Derivative 2 was converted to
alkyl phosphonamidates 3–10 by reaction with the proper alcohol,
as described in the literature34 (Scheme 1). Unlike the diamide
derivative 2, all alkyl phosphonamidates 3–10 were obtained as
racemic mixtures of R and S optical isomers.
2.2. Carbonic anhydrase inhibition
An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic activity of various
CA isozymes for CO2 hydration reaction29. Phenol red (at a con-
centration of 0.2 mM) was used as indicator, working at the
absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5, for
3.2. b-Class carbonic anhydrase inhibition
a-CAs) or TRIS (pH 8.3, for b-CAs) as buffers, and 20 mM Na2SO4 Phenylphosphonic diamide 2 and alkyl phosphonamidates 3–10
(for maintaining constant the ionic strength), following the initial were assayed as inhibitors of a panel of b-class CAs from patho-
rates of the CA-catalysed CO2 hydration reaction for a period of genic bacteria, fungi, and protozoa: VChb from V. cholerae, FtubCA
10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for from F. tularensis, BpsCAb from B. pseudomalei, Can2 from C. neo-
the determination of the kinetic parameters and inhibition con- formans, CgNce from C. glabrata, MgCA from M. globosa, and
stants. For each inhibitor, at least six traces of the initial 5–10% of LdcCA from L. donovani chagasi. A stopped flow CO2 hydrase
the reaction have been used for determining the initial velocity. assay was used including acetazolamide (AAZ) as standard
Figure 1. In silico predicted binding mode of (A) phenylphosphonic diamide 2, (B) (S)-4 and (C) (R)-4 to hCA II, as repoted in Nocentini et al.28
Scheme 1. Synthesis of benzenephosphonamidate derivatives.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
61
Table 1. Inhibition data of VChb, FtubCA, BpsCAb, Can2, CgNce, MgCA and LdcCA with phenylphosphonic diamide 2 and alkyl phosphonamidates 3–10 and the
standard sulphonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay29
.
KI (mM)a
Compound
R
VChb
FtubCA
BpsCAb
Can2
CgNce
MgCA
LdcCA
CA I
CA II
2
3
4
5
6
7
8
9
–
0.9
0.5
9.5
15.4
7.3
26.8
64.9
34.7
20.9
0.45
10.3
8.5
2.5
2.1
4.6
12.6
5.8
22.5
29.4
32.7
18.9
0.74
0.03
0.02
0.05
0.9
0.1
2.2
10.4
0.6
5.3
0.05
0.2
0.8
1.3
0.7
5.3
8.7
1.8
20.2
0.01
28.4
26.1
68.1
79.0
52.4
102.4
265.7
117.3
97.4
40
2.1
0.8
3.5
6.8
5.1
16.4
36.5
26.4
19.3
0.09
77.8
145.6
338.6
589.9
730
876.3
961.2
322.2
575.8
32.8
39.8
160.6
459.4
348.8
750
520.1
95.4
465.3
0.01
–CH3
–CH2CH3
–(CH2)2CH3
–CH(CH3)2
–(CH2)3CH3
–(CH2)2OCH3
–(CH2)2Cl
–CH2CCH
–
16.4
35.6
18.4
42.0
55.3
15.1
6.9
10
AAZ
0.77
0.01
0.25
aMean from three different assays, by a stopped flow technique (errors were in the range of 5–10% of the reported values).
inhibitor29. A longer preincubation between compounds 2–10 and
the enzymes (1 h) was necessary to observe their maximum inhibi-
tory activity in comparison to the 15 min incubation which is com-
monly used for sulphonamides inhibitors. It was previously shown
that phosphonamidates possess a significantly lower acidity (more
than 2 orders of magnitude) than the bioisosteres sulphonamides
which presumably induce an extended time needed to form the
E–I complex.
The inhibition profiles against the human ubiquitous CAs I and
II are displayed for comparison. The following structure–activity
relationships (SAR) can be assembled from the inhibition data
reported in Table 1.
Table 2. Selectivity index (SI) for target b-CAs over hCA II.
SI (KI CA II/KI b-CA)
VChb FtubCA BpsCAb Can2 CgNce MgCA LdcCA
Compound
R
2
3
4
5
6
7
8
9
–
36.4
79.6
16.9
3.2
4.7
9.8
12.9
19.0
17.9
9.4
13.1
19.0
34.9
36.5
60.1
33.3
17.7
2.9
1093.3 656.0 1.2
1990.0 199.0 1.5
3212.0 200.8 2.4
510.4 353.4 5.8
3488.0 498.3 6.7
340.9 141.5 7.3
50.0 59.8 2.0
159.0 53.0 0.8
87.8 23.0 4.8
15.6
49.8
45.9
67.6
68.4
45.7
14.2
3.6
–CH3
–CH2CH3
–(CH2)2CH3 29.8
–CH(CH3)2 47.8
–(CH2)3CH3 28.0
–(CH2)2OCH3 8.0
–(CH2)2Cl
–CH2CCH
–
2.7
22.3
6.3
67.4
10
AAZ
24.6
24.1
0.02
0.01
0.01
1.0
1.0
/
/
What immediately stands out from the data in Table 1 is that
benzenephosphonamidates 2–10 act as significantly stronger
inhibitors against the tested b-class CAs than CA I and CA II. In
fact, low to submicromolar inhibition constants (KIs) were meas-
ured against VChb (KIs ¼ 0.5–64.9 mM), FtubCA (KIs ¼ 6.9–55.3 mM),
BpsCAb (KIs ¼ 2.1–32.7 mM), Can2 (KIs ¼ 0.02–10.4 mM), CgNce
(KIs ¼ 0.05–20.2 mM), and LdcCA (KIs ¼ 0.8–36.5 mM), whereas solely
0.9 to 0.1 mM). Switching the propyl (6) to a propargyl (10) pro-
duced instead a positive effect uniquely in the case of FtubCA (KI
from 35.6 to 6.9 mM). Enhancement of inhibitory potency against
FtubCA (KI from 35.6 to 15.1mM) and Can2 (KI from 0.9 to 0.6 mM) is
also provoked by swapping the terminal CH3 of the propyl of 5
with a chlorine atom as in 9, whereas the two compounds inhibit
CgNce almost equally (KIs of 1.3 and 1.8 mM).
The standard AAZ is a more effective inhibitor of all tested
b-CAs than phosphonamidates 2–10 here investigated (KIs in the
range 0.01–0.77 mM), with the exception of MgCA, against which it
showed a KI of 40 mM, whereas the most potent compounds 2
and 3 act with KIs of 28.4 and 26.1 mM. However, AAZ also inhibits
CA I and II in a medium to low nanomolar range, while KIs of
compounds 2–10 do not go below 30 mM against these human
isozymes. In Table 2, the selectivity index (SI) for the target b-CAs
over hCA II is reported. With the exception of MgCA, all tested
phosphonamidates show a remarkably selective inhibitory action
against all b-CAs over the most relevant human isozyme. While
the SI for VChb, FtubCA, LdcCA and BpsCAb (over hCA II)
span between 2.7 and 79.6, it is noteworthy stressing the up to
four-digits SI calculated for a subset of compounds against Can2
(1093.3–3488.0) and the three-digits SI that compounds 2–7
showed against CgNce (141.5–656.0).
AAZ weakly inhibits MgCA from M. globosa whereas phosphona-
midates 2–10 inhibit this enzyme less effectively than other
screened b-CAs and almost comparably with hCA II, leading to SI
ranging between 0.8 and 7.3. Selectivity indexes for the target b-CA
over hCA I are obviously even higher than those depicted in Table 2
because hCA I is less inhibited than hCA II by derivatives 2–10.
Significantly higher SI have been calculated with most phos-
phonamidates for b-class CAs over human isoform II in compari-
son to sulphonamide derivatives35, which are here represented by
AAZ (SI in the range <0.01–1.0, Table 2). These data include this
MgCA was inhibited in
a
medium micromolar range
(KIs ¼ 26.1–265.7 mM). In contrast, CAs I and II had been shown to
be target of compounds 2–10 in a medium to high micromolar
scale spanning from 77.8 to 961.2 mM and 32.8 to 520.1 mM,
respectively.
Using in silico techniques, phosphonamidates have been
reported to act as zinc-binding group in the interaction with the
active site of hCA II (Figure 1) and thus it is not unexpected that
the incorporation of gradually bulkier substituents on the phos-
phorus atom might decrease the inhibitory effectiveness of this
class of compounds because of steric hindrance induced nearby the
zinc ion. Nonetheless, the reduction of inhibition potency against
b-class CAs upon increasing the length of the alkyl chain was not
as significant as that reported against hCAs, and thus other binding
factors appear to take place within the binding site of b-CAs. In
detail, phenylphosphonic diamide 2 and methyl phosphonamidate
3 indistinctly showed the best inhibitory potency against VChb (KIs
of 0.9 and 0.5mM), BpsCAb (KIs of 2.5 and 2.1 mM), Can2 (KIs of 0.03
and 0.02 mM), CgNce (KIs of 0.05 and 0.2 mM), MgCA (KIs of 28.4 and
26.1mM), and LdcCA (KIs of 2.1 and 0.8 mM) among the reported
compounds. As a unique exception, FtubCA was found to be most
potently inhibited by the propargyl phosphonamidate 10 which
shows a KI of 6.9 mM. In the cases of BpsCAb and Can2, the ethyl
derivative 4 showed a comparable inhibition to compounds 2 and
3, with KIs of 4.6 and 0.05 mM, and further showed an interesting
submicromolar KI of 0.8 mM against CgNce. Branching of the alkyl
chain from propyl (5) to isopropyl (6) always produced a positive
effect on the inhibition potency, which was two-fold against VChb,
FtubCA, BpsCAb, and CgNce and nine-fold against Can2 (KI from new class of compounds among the CAIs most selective for

62
S. A. ALISSA ET AL.
b-class over human isozymes known to date. One could speculate
that the narrower active site pocket of b-CAs better accommo-
dates benzenephosphonamidates than the roomier binding cavity
of human CAs in terms of binding interactions. Indeed, a recent
paper by us showed that the binding mode of benzoxaboroles
within the active site of b-CAs Can2 and MgCA is strongly driven
by p–p interactions involving aromatic residues solely present in
b-class enzymes19. Analogue binding interactions might exist with
benzenephosphonamidates, which increase their inhibition effect-
iveness against b-CAs. Novel insights are emerging by novel crys-
tallographic studies currently ongoing with phosphonamidates
and hCAs, which will definitively clarify the binding mode of such
a chemotype with these metalloenzymes. Therefore, this know-
ledge will be extended to visualise the reasons for benzenephos-
phonamidates selective action against b- over a-class enzymes.
ORCID
Clemente Capasso
Claudiu T. Supuran
http://orcid.org/0000-0003-3314-2411
http://orcid.org/0000-0003-4262-0323
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4. Conclusions
The robust spread of antimicrobial resistance represents a main
threat to human health. A primary strategy to combat it consists
in the identification of novel therapeutic targets and anti-infec-
tives with alternative mechanisms of action. The inhibition of the
metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) from patho-
gens among bacteria, fungi, and protozoa was shown to produce
an impairment of the microorganism growth and virulence, whose
targeting is deemed to prevent the evolutionary selective pres-
sures that induce the pathogen to develop resistance. Unlike
mammals and thus human, the genome of many pathogenic bac-
teria, fungi, and protozoa encode for b-class enzymes, which show
significant structural diversities compared to a-class isozymes. As
phosphonamidates have been recently validated as human a-CA
inhibitors (CAIs) and no phosphorus-based zinc-binding group
have been assessed to date against b-class CAs, herein we report
an inhibition study with this class of compounds against b-CAs
from pathogenic bacteria, fungi, and protozoa. Compounds 2–10
showed low to submicromolar KIs against most tested b-CAs,
namely VChb (KIs ¼ 0.5–64.9 mM), FtubCA (KIs ¼ 6.9–55.3 mM),
BpsCAb (KIs ¼ 2.1–32.7 mM), Can2 (KIs ¼ 0.02–10.4 mM), CgNce
(KIs ¼ 0.05–20.2 mM), and LdcCA (KIs ¼ 0.8–36.5 mM). As human iso-
forms CAs I and II are instead solely inhibited in a high micromo-
lar range (32.8–961.2 mM), these data include phosphonamidates
among the CAIs most selective for b-class over human isozymes
known to date, making them interesting leads for the develop-
ment of new anti-infective agents. Crystallographic studies cur-
rently ongoing with phosphonamidates 2–10 and hCAs will
definitively clarify the mechanism of action of this class of com-
pounds probably shedding light on their selective efficacy against
b- over a-class enzymes
Disclosure statement
No potential conflict of interest was reported by the authors.
10. Supuran CT, Capasso C. Biomedical applications of prokary-
otic carbonic anhydrases. Expert Opin Ther Pat 2018;28:
745–54.
11. Del Prete S, Vullo D, De Luca V, et al. Sulfonamide inhibition
studies of the b-carbonic anhydrase from the pathogenic
Funding
This work was funded by the Deanship of Scientific Research at
Princess Nourah bint Abdulrahman University, through the
Research Groups Programme Grant no. (RGP-1440–0024).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
63
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a
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