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Targeting Metalloenzymes by Boron-Containing Metal-Binding

Pharmacophores
You-Cai Xiao,* Jun-Lin Yu, Qing-Qing Dai, Gen Li, and Guo-Bo Li*

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ABSTRACT: Metalloenzymes have critical roles in a wide range of

biological processes and are directly involved in many human
diseases; hence, they are considered as important targets for
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therapeutic intervention. The specific characteristics of metal

ion(s)-containing active sites make exploitation of metal-binding
pharmacophores (MBPs) critical to inhibitor development targeting
metalloenzymes. This Perspective focuses on boron-containing
MBPs, which display unique binding modes with metalloenzyme
active sites, particularly via mimicking native substrates or tetrahedral
transition states. The design concepts regarding boron-containing
MBPs are highlighted through the case analyses on five distinct
classes of clinically relevant nucleophilic metalloenzymes from
medicinal chemistry perspectives. The challenges (e.g., selectivity)
faced by some boron-containing MBPs and possible strategies (e.g., bioisosteres) for metalloenzyme inhibitor transformation are also
discussed.

■ INTRODUCTION
Metalloenzymes are a broad set of enzymes that use active site
metal ion(s) for catalysis; more strictly, metalloenzyme active
site metal ion(s) need to form stable coordination with active
site residues and be involved in related chemical reactions.1−3
The metal ion(s) usually acts as a Lewis acid or redox partner
and facilitates the catalyzed reaction by raising the ground state
energy of reaction partners, enhancing substrate binding or
stabilizing reactive intermediates.4 Metalloenzymes cover a wide
range of enzyme types including oxidoreductases, hydrolases,
transferases, lyases, isomerases and ligases, and are involved in
various physiological processes, e.g., metabolism and immune
modulation. By data-mining analyses of the Protein Data Bank
(PDB), we found that there are more than 3300 structurally
validated metalloenzymes. More importantly, a great number of
metalloenzymes have been directly associated with various
human illnesses, such as neoplastic, neurodegenerative,
inflammatory, cardiovascular, and infectious diseases, thereby
representing a rich target space for drug discovery and
development.5
To date, about 70 small-molecule inhibitors for metal-
loenzymes have been approved for clinical use.5,6 Most of
these inhibitors bind to make direct coordination interactions
with the active site metal ion(s) by specific chemotypes,
commonly termed as metal-binding pharmacophores
(MBPs).7−12 For example, the antihypertensive drug captopril
binds to coordinate the two zinc ions of angiotensin converting
enzyme (ACE) with its thiol group;13 the antifungal drug
fluconazole is positioned to make coordination with the heme-
iron of Saccharomyces cerevisiae CYP51 (ScCYP51) with the
triazole motif (Figure 1A). Several other representative MBPs
such as sulfonamide and hydroxamic acid often exist in approved
drugs (Figure 1A).14−16 MBPs are important for target binding
affinity but are associated with metalloenzyme selectivity; a
rational design or selection of proper MBPs is often the focal
point for metalloenzyme inhibitor development. Our previous
analyses of metalloenzyme-ligand interactions yielded a diverse
set of 469 curated MBPs,8 which may help provide a wealth of
ideas for inhibitor design and MBP expansion. Of these curated
MBPs, some have been considered as privileged moieties, e.g.,
hydroxamic acid and imidazole, coupling in several clinically
useful drugs, while some have not been widely explored such as
cyano and oxirane groups. Deep understanding of more
biocompatible, privileged MBPs is hence critical for drug
discovery targeting metalloenzymes.
The boron-containing MBPs are of particular interest to
researchers in developing new agents targeting metalloenzymes.
On the one hand, the boron atom with a vacant p orbital is
Received: September 28, 2021
Published: December 8, 2021

© 2021 American Chemical Society https://doi.org/10.1021/acs.jmedchem.1c01691

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Figure 1. (A) Representative metal-binding pharmacophores (MBPs) of FDA-approved drugs targeting metalloenzymes.13−17 (B) Boronic acid and
its diverse binding modes with protein targets, including metalloenzymes.
unique in its chameleonic capacity to interact with various
protein targets (Figure 1B),18 including relatively challenging
metalloenzymes for their flexibility, dynamics, and electronic
structure of metal ion(s)-containing active sites in metal−ligand
interactions.19 On the other hand, the boron-containing MBPs
have diverse interaction modes with active site metal ion(s)
(Figure 1B); some have been clinically validated, e.g., the
benzoxaborole of Crisaborole (a drug approved for psoriasis
treatment in 2014) as an MBP to inhibit phosphodiesterase 4 by
reacting with the active site metal ion-activated water
molecule.20 Besides, boron-containing MBPs are not prone to
being metal chelators and usually do not promiscuously destroy
metalloenzyme active sites. Moreover, the therapeutic potentials
of boron-containing MBP compounds remain to be ex-
ploited.21−26 We herein provide an overview on boron-
containing MBPs targeting five distinct types of clinically
relevant nucleophilic metalloenzymes and their common
inhibition principles, with the hope to aid metalloenzyme
inhibitor transformation and boost drug discovery targeting
metalloenzymes.
■
TARGETING HUMAN PHOSPHODIESTERASES
We describe work on boron-containing MBPs targeting
metalloenzymes, beginning with disease-related phosphodies-
terases (PDEs). PDEs are a family of enzymes that are
responsible for regulating the intracellular level of the second
messenger molecules cAMP and cGMP.27,28 PDEs are
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promising targets for several human diseases, including heart
failure, depression, asthma, inflammation, cancer, and so on.27,29
The PDE family includes 11 isoenzyme classes. Taking PDE4
isoenzyme, for example, it is encoded by four genes (PDE4A,
PDE4B, PDE4C, and PDE4D) that widely exist in the brain,
leukocytes, and bones.30 These PDE4 enzymes are important
regulators for inflammation and have been the focus of extensive
drug development projects for the treatment of autoimmune
diseases.31−33 PDE4 consists of an N-terminal regulatory
domain, a C-terminal domain, and a conserved catalytic domain
that lies at the interface of these two domains; each PDE4
isoform has a unique N-terminal region (Figure 2A). The
catalytic domain of PDE4 contains two metal ions; usually, one
metal ion is Zn2+, and the other is Mn2+, Mg2+, Zn2+, or Ca2+.34
The two metal ions are bridged by an activated hydroxide, which
is likely to perform a nucleophilic attack on the phosphorus of
cAMP, triggering the cAMP hydrolysis (Figure 2B). The
hydrolysis product AMP was observed by crystallographic
analysis to have coordination interactions with the active site
metal ions (Figure 2C), offering a structural basis for the
hydrolytic mechanism and inhibitor design.
Boron-containing compounds were first reported as PDE4
inhibitors by Akama et al. in 2009.35 They found that 5-
phenoxybenzoxaboroles were particularly active against PDE4;
for example, compound 1 (Crisaborole, AN2728)) showed an
IC50 of 110 nM to PDE4 (Figure 3A). Crisaborole also has
inhibitory activity against PDE1A3, PDE3C, and PDE7AZ but
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Figure 2. Overall architecture and catalytic mechanism of PDE4. (A) Domain organization of PDE4. (B) Chemical mechanism of PDE-mediated
cAMP hydrolysis. (C) View of a crystal structure of PDE4B complexed with AMP (PDB ID 1ROR),34 showing the binding features of AMP with metal
ions and catalytically important residues.
has no or very low inhibition to other PDE isozymes at 10 μM.
Crisaborole was approved by FDA for treatment of atopic
dermatitis in 2014. The inhibitory activity could be increased to
60 nM by introducing another cyano group at the adjacent
position (2, AN2898).20 Crystallographic analyses of the
PDE4:2 complex (PDB ID 3O0J)20 revealed that the
benzoxaborole motif of 2 binds to interact with the dimetal
center, receiving the nucleophilic attack by the bridging
hydroxide to form the sp3 hybridized boronate form. All the
three boronate oxygen atoms are involved in coordination with
the dimetal ions, forming a classical trigonal-pyramidal geometry
(Figure 3B). The 3,4-dicyanophenoxy ring makes π−π
interactions with Phe446, partly contributing to stabilize the
specific coordination mode. More interestingly, 2 was observed
to have a similar binding mode as that of AMP, i.e., sp3
hybridized boronate and phosphate making a highly similar
coordination manner to the active site metal ions and 3,4-
dicyanophenoxy and adenosine making π−π interactions with
Phe446, reflecting that 2 is likely to mimic the binding nature of
AMP (Figure 3C). This comparison also led to a conclusion that
the sp3 hybridized boronate and phosphate are bioisostere
MBPs, which can be used for related structural transformation
and drug design.
Extended medicinal chemistry efforts were carried out for this
benzoxaborole series to improve the inhibitory potency or other
features, such as selectivity and toxicity. When introducing a side
chain onto 1 to occupy the PDE4 adenine-binding pocket
mainly formed by the side chains of Phe446, IIe410, and Gln443,
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compounds 3 and 4 showed 3- and 100-fold improvement
activities compared with 1, respectively (Figure 4).20 The acetyl
group of 4 may have additional hydrogen bonding interactions
with Gln443, affording approximately 30-fold potency improve-
ment over 3. The tetrahedral character of the sp3 hybridized
boronate is essential for PDE4 inhibition, which was confirmed
by the observation that compound 5 (AN2913) lacking a boron
atom has weak PDE4 inhibition (Figure 4).
Zhang et al. proposed a soft-drug strategy for the design of
benzoxaborole PDE4 inhibitors, with the aim to improve the
therapeutic index.36 Changing the cyano-substituted benzox-
aborole 6 (IC50 = 180 nM) to an ester group led to a more potent
inhibitor 7 (IC50 = 47 nM), whereas further hydrolysis to the
carboxylic acid form (8) yielded much less PDE4 inhibition
(IC50 > 10 μM) (Figure 4). In this case, the ester compound 7
exerts its therapeutic effects in the target area of the skin, and
interestingly, it will be converted into inactive inhibitor (8)
when penetrating the skin and reaching the circulation system
(e.g., hydrolyzed by plasma esterases). The inhibitor 7 was
observed to have good skin bioavailability and stability, broad in
vivo anti-inflammatory activity, and improved safety properties
with less systemic side effects.36 Although this strategy is able to
decrease PDE inhibition in other tissues, it could not exclude the
possibility of this benzoxaborole series inhibiting other targets
other than PDEs.
Recently, Xu’s group designed various bicyclic substituents at
the benzoxaborole 5-position to occupy the adenine pocket and
discovered several potent PDE4 inhibitors. For example,
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Figure 3. Inhibition mode of benzoxaboroles to PDE4. (A) Chemical structures of 1 (AN2728) and 2 (AN2898). (B) View of a crystal structure of
PDE4:2 complex (PDB ID 3O0J)20 reveals a sp3 hybridized boronate as an MBP to coordinate with the two active site metal ions. (C) Superimposition
of PDE4:2 and PDE4:AMP (PDB ID 1ROR)34 revealing that 2 is likely to mimic the nature of AMP binding to PDE4.

Figure 4. Structural transformation of benzoxaborole PDE4 inhibitors.

Figure 5. Representative oxaborole PDE4 inhibitors.

compound 9 displayed 136-fold improved inhibitory activity
(IC50 = 0.42 nM) compared with Crisaborole (Figure 4).37 In
addition, 9 exhibited high selectivity over PDE3A, PDE3B, and
PDE10A. In vivo studies indicated that 9 manifested greater
efficacy than Crisaborole at the same dosage (P < 0.05) both in
the PMA-induced mouse ear edema model and the calcipotriol-
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induced mouse atopic dermatitis model. Moreover, 9 displayed
a favorable safety property in repeated oral dose toxicity and
phototoxicity studies, which is a good drug candidate worthy of
further clinical investigation.
Interestingly, a series of novel 4-biaryl-substituted oxaboroles
were reported recently as highly potent PDE4 inhibitors (Figure
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Figure 6. Overall architecture and catalytic mechanism of ATX. (A) Domain organization of ATX. (B) ATX-medicated hydrolysis of LPC to LPA. (C)
A view of the mouse ATX:LPA complex structure (PDB ID 3NKM)41 revealing binding features of LPA to mATX active site zinc ions and long and
narrow subpockets.
5).38 The representative oxaboroles 10, 11, and 12 showed IC50
values of 0.05, 0.12, and 0.24 nM, respectively. These
compounds could modulate inflammatory cytokine levels and
could be used for treating inflammatory disorders such as atopic
dermatitis, arthritis asthma, and fibrosis (details regarding in vivo
studies are not disclosed).
Compared with other MBPs (e.g., pyridazin-3-ol), the
benzoxaboroles or oxaboroles are promising MBPs targeting
PDE4 and can uniquely mimic the AMP phosphate binding with
PDE4 active site metal ions.20 Although almost all PDE
isoenzymes have the dimetal active site, the benzoxaboroles
manifest selectivity for PDE4, implying that the target selectivity
is not solely dependent on MBPs, but rather the entire
compound chemical structure, particularly with respect to
interactions with unique N-terminal regions of PDE isoenzymes.
Notably, MBPs-containing inhibitors are not the only manner
for targeting PDE4; several non-MBP inhibitors have been
developed as highly potent, selective PDE4 inhibitors,8,28 and
those, including Roflumilast, Cilomilast, and Apremilast, have
been approved for clinical uses.
■
TARGETING HUMAN AUTOTAXIN
Another example of using boronic acid as an MBP to mimic the
substrate phosphate group is human autotaxin (ATX)
inhibitors. ATX belongs to the nucleotide pyrophosphatase
and phosphodiesterase (NPP) family, and it is the only family
member that has lysophospholipase D activity, i.e., responsible
for the hydrolysis of lysophosphatidyl choline (LPC) to the
bioactive lysophosphatidic acid (LPA).39,40 LPA can activate the
LPA-specific G protein-coupled receptors (GPCRs), including
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LPA1-6, to evoke a wide range of cellular responses, including
cell proliferation, survival, differentiation, and migration.
As shown in Figure 6A, ATX consists of four domains, and the
core catalytic domain has an α/β fold and binds two Zn2+ ions.41
One Zn2+ ion is coordinated by His315, His474, and Asp311, and
the other Zn2+ is coordinated by Asp171, Thr209, and His359. The
proposed catalysis mechanism for LPC hydrolysis by ATX
involves a nucleophilic attack on the LPC phosphorus atom by
Thr209 oxygen anion, resulting in a choline and a pentacovalent
reaction intermediate, and the latter undergoes a water-
mediated hydrolysis to give the product LPA (Figure 6B).42
LPA was observed to bind with mATX active site zinc ions by its
phosphate group,41 providing insights for inhibitor design
(Figure 6C).
Since ATX is highly associated with several human diseases
including cancer, great efforts have been made for the
development of new ATX inhibitors for therapeutic inter-
vention.43−45 Boron-containing compounds have played an
important role in the early stage of ATX inhibitor development.
In 2010, Ovaa’s group campaigned a high-throughput screening
of 40 000 molecules and identified the thiazolidinedione 13 as a
selective ATX inhibitor (Figure 7A).46,47 Inspired by the case of
the proteasome inhibitor bortezomib which uses boronic acid to
react with N-terminal threonine oxygen nucleophile,48 they
replaced the carboxylic acid of 13 to boronic acid (14), leading
to a significant increase in ATX inhibition; shifting boronic acid
from the meta-position to para-position (15) further improved
the inhibitory activity to the single-digit nanomolar range (IC50
= 5.67 nM), while to the ortho-position greatly decreased the
activity to an IC50 larger than 5 μM (Figure 7A). The 3-
benzyloxyboronic acid analogues 17 and 18 showed no
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Figure 7. Thiazolidinedione-boronic acid based ATX inhibitors. (A) Key structure−activity relationship for thiazolidinedione-boronic acid ATX
inhibitors. (B) View of the ATX:15 complex structure (PDB ID 2XRG)49 revealed the binding mode of 15, explaining the structure−activity
relationship shown in part A. (C) Comparison analyses reveals that 15 likely mimics the reaction intermediate, and boronate and phosphate groups are
high-quality MBP bioisosteres.

improvement in the inhibitory activity compared with 15 On the basis of the ATX:15 complex structure, a series of
(Figure 7A). Co-crystallography revealed that the boronic acid imidazolidine-2,4-dione based boronic acid inhibitors were
of 15 (PDB ID 2XRG) is transformed to a sp3 hybridized further developed (Figure 8).49 The representative compound
boronate by forming a covalent bond with the nucleophilic 19 showed similar inhibitory activity against ATX compared
hydroxyl group of Thr 209 and one boronate hydroxyl
with 15. Interestingly, the Z and E isomers of 19 showed similar
coordinated with the active site zinc ions, which are essential
for ATX catalytic activity (Figure 7B).49 This is unusual but is a nanomolar potency against ATX (Figure 8), reflecting the
particularly interesting binding mode for MBPs, reflecting the considerable tolerance of the ATX active site to accommodate
specific features of boronate to both nucleophilic residues and structurally distinct inhibitors.
metal ions. Further superimposition of ATX:15 and ATX:LPA
revealed that 15 perfectly mimics the binding mode of the
substrate reaction intermediate, e.g., sp3 hybridized boronate of
15 and phosphate of the substrate intermediate positioned to
form similar covalent bonding with Thr209, similar coordination
interactions to Zn1, and similar hydrogen bonding interactions
with Asn230 (Figures 6C and 7C). This comparison highlights
that the substrate or transition state-mimicking strategy is highly
effective to identify potent inhibitors and further reveals that the
boronate (in sp3 form) and phosphate groups are high-quality
bioisosteres, particularly in developing metalloenzyme inhib-
itors. Figure 8. Imidazolidine-2,4-dione-boronic acid ATX inhibitors.

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Figure 9. Thiazolinone-boronic acid ATX inhibitors.
In 2013, Nagano’s group developed a sensitive and specific
ATX fluorescence probe, TG-mTMP, and used it to screen ATX
inhibitors from a large chemical library.50 They identified the
thiazolinone 20 as a potent ATX inhibitor with an IC50 value of
180 nM (Figure 9). Substitution on the piperazine nitrogen of
20 with benzylcarbonate and benzylboronate afforded 21 and
22, respectively; both compounds showed improved inhibitory
potency. Compound 22 had promising pharmacologically
related features in subsequent in vivo studies.
Recently, a new class of ATX inhibitors that use
benzoxaborole as an MBP were reported (Figure 10).51 The
Figure 10. Benzoxaborole ATX inhibitor.
most potent compound 23 manifests IC50 of 0.13 μM to ATX,
comparable to 15 (IC50 = 0.088 μM) determined in the choline
release assays. Notably, 23 has improved druggable properties,
including favorable plasma protein binding feature, pharmaco-
kinetics, solubility, and rat/human plasma stability.51 Since ATX
is a promising drug target, many medicinal chemistry efforts
have been involved in seeking potential drug candidates, partly
including developing different MBPs, e.g., phosphate, phospho-
thioate, benzoxazolidinone, benoxazolone, and carboxylic acid,
in addition to boronic acid.52 Further studies of boron-
containing MBPs are needed to consider its in vivo efficacy
particularly in developing drug candidates for ATX-driven
disease models.
■ TARGETING HUMAN ARGINASES
The boron-containing MBPs are also reported to target
arginases, a class of dinuclear metalloenzymes, which use the
nucleophilic hydroxyl for catalysis, similar to PDEs and ATX.
Arginases are Mn2+-dependent metalloenzymes initially de-
scribed by Kossel and Dankin in 1904;53 two isoforms of
arginases in mammals, arginase I (Arg I) and arginase II (Arg II),
have been known to date.54,55 Both isoforms have similar kinetic
and biochemical properties but differ from their quantitative
requirement of Mn2+ for catalysis and subcellular localization.
Arg I is a cytosolic enzyme primarily existing in hepatocytes,
while Arg II is a mitochondrial enzyme expressed in small
intestine, kidney, brain, monocytes, and macrophages.56,57 Arg I
and Arg II play critical roles in L-arginine metabolism,
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responsible for converting L-arginine into ornithine and urea.58
The high expression or unusual activity of Arg I and Arg II has
been observed in various pathological states, e.g., cancer,
atherosclerosis, renal disease, inflammatory disease, and neuro-
degenerative disease;59 in particular, recently both isoforms have
been identified as major immunosuppressive players,60,61
suggesting that they are promising targets for immunologic
therapy and related disease treatment.
Arg I and Arg II are typically homotrimeric enzymes stabilized
by two Mn2+ ions per monomeric structure, with an α/β fold
consisting of an eight-strand β-sheet surrounded by several α-
helixes (Figure 11).62 Both arginase isoforms contain a dinuclear
Mn2+ active site. The main feature is the active site Mn2+ ions
bridged by an activated hydroxide, which is responsible for
substrate hydrolysis. The activated hydroxide is likely to attack
the guanidyl carbon of L-arginine, leading to production of L-
ornithine and urea, as observed by crystallography (Figure 11).
According to the active site features of arginases, the boron-
containing chainlike MBP was introduced to bind with
arginases, and several generations of boron-containing inhibitors
have been evolved, which are discussed as follows.64−66
Christianson’s lab pioneered the use of boron-containing MBP
to inhibit arginases by mimicking the proposed “tetrahedral”
substrate transition state and reported the first boron-containing
arginase inhibitor, 2-(S)-amino-6-boronohexanoic acid (24,
ABH) (Figure 12A).67 ABH acted as a “suicide” substrate and
showed a slow binding feature with an IC50 value of 1.45 μM to
human arginase I (hArg I).68 The X-ray crystal structure of the
hArg I:ABH complex (PDB ID 2AEB)69 reveals that the boronic
acid of ABH undergoes nucleophilic attack by the hydroxide ion
that is activated by binuclear Mn2+ to yield a tetrahedral sp3
hybridized boronate form, which likely mimics the L-arginine
tetrahedral intermediates during the hydrolytic process (Figure
12A). The observed hydrogen bonding networks reflect the high
selectivity nature of arginase in recognition of the L-arginine
substrate as well as the substrate-mimicking inhibitor (e.g.,
ABH) (Figure 12B).
The success of boron-containing MBP inspired researchers to
develop new MBPs to interact with the arginase active site Mn2+
ions (Figure 13). Shin et al. used sulfonamide as an MBP, which
is structurally similar to sp3 hybridized boronate; the obtained
compound 25 showed inhibition to Arg I with a Ki value of 90
μM, significantly weaker than ABH (Figure 13). They later
identified that using aldehyde as the MBP, i.e., 26, could increase
Arg I inhibition (Figure 13). Interestingly, the rArg I:26 complex
structure (PDB ID 1T5F)70 revealed that the aldehyde of 26 is
likely attacked by the hydroxide ion to form a gem-diol, similar
to that observed for boron-containing ABH, reflecting the
nucleophilic activity nature of the Arg I active site. Replacement
by a carboxylate or nitro group did not result in improved
inhibitory activity to arginases (Figure 13). Notably, they
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Figure 11. Structural and catalytic mechanism of arginase-medicated L-arginine hydrolysis. The dotted-line box shows details of the binding modes of
the L-arginine hydrolytic products with Rattus norvegicus Arg I (rArg I) from a crystal structure of the rArg I:L-ornithine:urea complex (PDB ID
1HQG63).
expanded 2-aminoimidazole as the MBP to yield 27, which
showed a Ki value of 4 μM to hArg I. The 2-aminoimidazole
MBP may have interactions with the carboxyl-rich, negatively
charged active site of hArg I, thereby worthy of further
investigation not confined to hArg I. Despite these MBP
bioisostere replacements, no more potent inhibitors were
obtained, suggesting that the boronic acid might be optimal
MBP for arginases. Optimization for potency and pharmacoki-
netic properties for boronic acid containing compounds to
target arginases is the main purposes in most medicinal
chemistry works.
The early studies were mainly focused on the modification of
the ABH alkyl chain (Figure 14).71,72 Replacement by sulfur in
the alkyl chain afforded 30, showing an IC50 of 5 μM to rat liver
arginases (RLA). Introduction of a phenyl ring and a double
bond in the alkyl chain yielded 31 and 32, respectively (Figure
14), which did not have an improved inhibitory activity to RLA
compared to ABH (rArg I IC50 = 0.8 μM) (Figure 14).
On the basis of the hArg I:ABH complex structure, the Cα-H
atom of ABH was considered to have the possibility for
structural modifications to obtain more potent arginase
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inhibitors, since it is solvent-exposed and oriented toward a
region of protein surface that is uncharted with regard to
inhibitor binding. To this end, a series of α,α-disubstituted ABH
analogues (33−38, Figure 15) were developed, which were
called second-generation arginase inhibitors. Initial attempts to
introduce simple alkyl substituents on the amino acid Cα atom,
such as CH3 and CHF2, led to 33 and 34;73 although these
compounds did not show significantly enhanced binding affinity
to arginases, the work demonstrated that the arginase active site
can accommodate substituents on the ABH Cα-H position.
Later, by introducing larger substituents at the Cα-H position,
the new ABH analoguess showed improved inhibitory activities.
For example, compound 35 had IC50 values of 223 and 509 nM
to hArg I and hArg II, respectively,68 and it had an oral
bioavailability (F) of 28% and significantly reduced the infarct
size in a rat model of myocardial ischemia/reperfusion injury.68
The hArg I:35 complex structure (PDB ID 4HWW)68 revealed
that the Cα-H substituents occupied the hArg I solvent pocket
without affecting the hydrogen bond formation between the
amino acid group and hArg I; additional hydrogen bonds were
observed by a water-bridging the piperidine nitrogen atom with
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Figure 12. Inhibition mode of ABH with hArg I. (A) Design of L-
arginine-mimicking boron-containing chainlike MBP. (B) View of a
crystal structure of hArg I:ABH (PDB ID 2AEB69) revealed the binding
mode of the sp3 hybridized boronate form of ABH with hArg I.
Asp181 and Asn183 (Figure 15). More interestingly, incorporation
of a methyl on amine group (36) led to a nearly 4-fold increase in
potency compared with 35.68 This probably resulted from a gain
in entropy due to the exclusion of a water molecule (W2) that
was observed in the hArg I:35 structure (PDB ID 4HWW)68 but
not in the hArg I:36 structure (PDB ID 4HXQ)68 (Figure 15).
Further introduction of a two-carbon bridge in the piperidine
ring led to 37 and 38, which showed improved activity against
both hArg I and hArg II.74 Unfortunately, these compounds had
low oral bioavailability in rats. Recently, the OncoArendi
Therapeutics company disclosed two new arginase inhibitors, 39
(with a sulfamido side chain) and 40 (with a guanidine side
chain) (Figure 15).75 Both compounds had improved inhibitory
Figure 13. Several reported Arg I inhibitors with structurally different MBPs.
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activity to arginases compared with ABH but still had poor oral
bioavailability.
Based on the second-generation Arg I inhibitors, Van Zandt’s
group developed a series of novel ring-constrained ABH
analogues, e.g., 41−43 (Figure 16A), which were called third-
generation arginase inhibitors.76 The representative pyrrolidine-
based compound 41 showed IC50 values of 110 and 440 nM to
hArg I and hArg II, respectively; the introduction of a
constrained pyrrolidine ring is able to reduce the conformational
entropy, and the pyrrolidine nitrogen has water molecule (W1)-
bridging hydrogen bonding interactions with Asp200 (Figure
16B). Further introducing a piperidine ring on the pyrrolidine
ring led to 42, which showed 40-fold increased inhibition
activities. The cocrystallography revealed that in addition to the
boronic acid involving coordination, the piperidine moiety of 42
is positioned to form direct electrostatic interactions with hArg
II Asp200 (Figure 16C). Further optimization of 42 generated 43,
which showed single-digit nanomolar activities against both
hArg I and hArg II.
Very recently, further modifications of the monopyrrolidine
ring to a fused 5,5-bicyclic ring system such as 44 and 45 were
reported. Compound 44 had improved potency over ABH, with
an IC50 value of 29 nM to hArg I in a thioornithine generation
(TOGA) assay (Figure 17).77 X-ray crystallography revealed
that the 5,5-bicyclic pyrrolidine ring nitrogen atom of 44 makes
direct electrostatic interactions with Asp181 of hArg I (PDB ID
6V7C).77 When dosed orally, 44 was demonstrated to have
serum arginase inhibition and concomitant arginine elevation in
a syngeneic mouse carcinoma model, albeit with low
bioavailability (F = 7%, in mice). Greater oral bioavailability
was observed for 45 (F = 18%, in mice) with fluorinated
pentalane bearing hydroxyl substitution (Figure 17).
The nonexcellent pharmacokinetics, especially with low oral
bioavailability and fast clearance of earlier inhibitors, prompted
researchers to develop the fourth-generation arginase inhibitors.
One of the successful methods to improve oral bioavailability
was based on the peptide transporter (PEPT1/2 transporter)
targeted drug design. The peptide-based analogues with boronic
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Figure 14. Early research by modification of ABH.
Figure 15. Second-generation human arginase inhibitors and the binding modes of 35 (PDB ID 4HWW)68 and 36 (PDB ID 4HXQ)68 with hArg I.
acid moieties were reported by Calithera Biosciences.78 The
representative inhibitors are compounds 46−48, which were
obtained by coupling of the endocyclic nitrogen atom with
various natural amino acids. Compound 46 (Numidargistat, CB-
1158) is a potent inhibitor for hArg I (IC50 = 86 nM) and hArg II
(IC50 = 296 nM) (Figure 18); the other two compounds, 47 and
48 (without reported IC50 data), also showed good plasma-drug
concentrations (AUC). Numidargistat showed high oral
bioavailability in rats (52%) and mice (91%), moderated
clearance (10 mL/min/kg), and good tolerance in rats. Thus,
Numidargistat has been allowed for clinical trials in 2016 to treat
patients with metastatic solid tumors as monotherapy and in
combination with chemo- and immunotherapy.
As summarized above, recent trends in developing new
arginase inhibitors have moved toward exploration of the core
support scaffold rather than the boronic acid MBP. The unique
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nature of boron-containing MBP, i.e., an electron-deficient
boron atom, facilitates perfect interactions with arginase
dimanganese ions and catalytically important residues. The
amino acid-mimicking strategy is particularly commendable,
which not only led to a drug candidate (i.e., Numidargistat) for
cancer treatment but also provided a new idea to address related
pharmacokinetic issues for boronic acids as well as other
chemotypes.
■
TARGETING CARBONIC ANHYDRASES
Boron-containing MBPs were also reported to target mono-
nuclear metalloenzymes, for example, carbonic anhydrases
(CAs). CAs are ubiquitous zinc metalloenzymes, present in
most living organisms (i.e., bacteria, archaea, and eukarya),
which catalyze the reversible hydration of carbon dioxide and
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Figure 16. Third-generation human arginase inhibitors. (A) Representative third-generation human arginase inhibitors. (B) View of crystal structures
of hArg II:41 (PDB ID 6Q37)76 and hArg II:42 (PDB ID 6Q39)76 complexes reveals the common boron-involving interactions with manganese ions
and catalytically important residues such as Asp202, Asn149, and Ser156.

Figure 17. Bicyclic human arginase inhibitors.

Figure 18. Peptide-based human arginase inhibitor.

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Figure 19. Overall architecture and catalytic mechanism of hCA II: (A) Structure organization of hCA II (PDB ID 1CA2).86 (B) View of the hCA
II:HCO3− complex structure (PDB ID 2VVB)87 revealing the intermediate binding mode. (C) The proposed catalytic mechanism of hCA II-mediated
hydration of carbon dioxide and water to bicarbonate and protons.

Figure 20. Noncyclic boronic acid-based hCA inhibitors.

water to bicarbonate and protons (CO2 + H2O ⇌ HCO3− +
H+ ). 79,80 CAs have been classified into eight distinct
families,81,82 and all human CAs (hCAs) belong to the α-
families. hCAs consist of 12 isoforms, including cytosolic hCA I,
hCA II, hCA III, hCA VII, and hCA XIII, membrane-bound
hCA IV, hCA IX, hCA XII, hCA XIV), mitochondrial (hCA VA
and hCA VB), and secretory hCA VI. These enzymes have been
extensively studied as potential therapeutic targets, e.g., for
glaucoma, retinopathy, and hemolytic anemia cancer.83−85
We take hCA II as a canonical example for describing their
structure and catalytic mechanism. The core structure of hCA II
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consists of β-sheet with little α-helix content, and the active site
contains a Zn2+ ion at the bottom of an ∼15 Å deep cleft, where
the Zn2+ is bound by three His residues and a water molecule in a
tetrahedral geometry form; the water molecule is further
stabilized by forming hydrogen bonding interactions to the
neighboring residue Thr199 that is important for catalysis (Figure
19A).86 The proposed catalytic process for hCA II includes the
nucleophilic attack of Zn2+-bound hydroxide ion to CO2,
producing a bicarbonate ion (HCO3−) by coordination with
Zn2+ as observed by crystallography (Figure 19B), followed by
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attack of a water molecule to release the bicarbonate ion (Figure
19C).79
Currently, the vast majority of hCA inhibitors approved by
FDA use sulfonamide as an MBP to coordinate with the active
site zinc ion, such as methzaolamide, dorzolamide, and
brinzolamide.83 In recent years, boronic acids have been
reported as an alternative MBP to target hCAs. A series of
aromatic, arylalkenyl-, and arylalkyl boronic acids (e.g., 49−56)
were reported as hCA inhibitors, mainly targeting the cytosolic
hCA I and hCA II, and the membrane-bound hCA IX and hCA
XII (Figure 20).88 In 2017, Supuran et al. reported that the
antitumor agent bortezomib showed micromolar inhibitory
activity to hCA isoforms, further offering insights for under-
standing the anticancer activity of bortezomib and suggesting
that it may be worth developing more potent boron-containing
hCA inhibitors for cancer treatment.89
Winum’s group reported a series of urea- and thiourea
substituted benzoxaboroles as a new chemotype for hCA
inhibition90 (Figure 21A). Among the reported benzoxaboroles,
Figure 21. Benzoxaborole-based hCA inhibitors. (A) Representative
inhibitors 57 and 58. (B) View of the hCA II:57 complex structure
(PDB ID 4HWW)68 reveals the binding mode of 57 and the evidence of
the advantage of benzoxaborole to interact with the hCA II active site.
compounds 57 (hCA I, Ki = 98 nM; hCA II, Ki = 89 nM; hCA IX
Ki = 414 nM; hCA XII Ki = 69 nM) and 58 (hCA I, Ki = 417 nM;
hCA II, Ki = 1838 nM; hCA IX Ki = 93 nM; hCAX II Ki = 71
nM) manifested potent inhibition to several hCAs. As observed
by X-ray crystallography, the benzoxaborole of 57 is likely
attacked by hydroxide ion to form an sp3 hybridized state to
coordinate with the hCA II active site zinc ion (Figure 21B),
similar to that described above for PDEs, ATX, and arginases.
The two oxygen atoms of sp3 hybridized benzoxaborole form a
trigonal bipyramidal coordination geometry with the zinc ion,
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and interestingly, one oxygen atom makes additional hydrogen
bonding interactions with Thr199 (Figure 21B), with a similar
position as that of the Zn2+-bound hydroxide ion in hCA II
active form (Figure 19), offering evidence to demonstrate the
advantage of benzoxaborole as an MBP engaging with the hCA
II active sites.
In 2019, Larcher et al. reported the synthesis and character-
ization of a set of novel bis-benzoxaboroles from readily available
6-aminobenzoxaborole 58 (Figure 22).91 The bis-benzoxabor-
Figure 22. Bis-benzoxaborole-based hCA inhibitors.
oles could inhibit five hCA isoforms in the micromolar range,
including hCA I, hCA II, hCA IV, hCA IX, and hCA XII. The
bis-benzoxaborole 59 with a glutamic acid central core showed
the most potent inhibition against the tumor-associated isoform
hCA IX (Ki = 64 nM) compared with 6-aminobenzoxaborole 58
(Ki = 813 nM) (Figure 22).
The potential of CAs as anti-infective targets has also been
considered in recent years. Since the CA enzymes in the β-CA
genetic family are widespread and functionally important in
bacteria, fungi, and archaea, development of selective inhibitors
targeting β-CAs may lead to new anti-infective agents. The
above-mentioned boronic acids 49−56 and antitumor agent
bortezomib showed some inhibition but not strong activity to β-
CAs.92,93 In 2017, Winum’s group reported a series of 6-urea and
thiourea-substituted benzoxaboroles as β-CA inhibitors from
three pathogenic fungi (Can2 from Cryptococcus neoformans,
CgNce103 from Candida glabrata, and MgCA from Malassezia
globosa) (Figure 23).94 Most reported derivatives showed
Figure 23. Benzoxaborole-based β-CA inhibitors.
nanomolar inhibitory activities against Can2 and CgNce103
and micromolar inhibition against MgCA. Some benzoxaborole
derivatives, e.g., 60, showed selectivity to Can2 and CgNce over
hCAI/II. Recently, these types of benzoxaboroles were
investigated for their inhibition against three CAs encoded by
Vibrio cholera, including VchCAα, VchCAβ, and VchCAγ
(Figure 23).95 Compound 61 exhibited nanomolar inhibition
to VchCAs, to a certain degree, with selectivity over hCA II,
suggesting that it is worthy of further optimization to develop
new anti-infective agents.
According to the CA active site features, boron-containing
MBPs have the unique feature to interact with nucleophilic
water and have been used to successfully develop potent
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Figure 24. Proposed mechanism of MBL-mediated hydrolysis of carbapenem antibacterials.
inhibitors for human and pathogens’ CAs. Although current
approved drugs are established based on the sulfonamide MBP,
boronic acids have shown great potential in inhibitor develop-
ment, which might provide an alternative way to identify new
treatments.
■
TARGETING BACTERIAL
METALLO-β-LACTAMASES
In addition to the above-described human metalloenzymes,
boron-containing MBPs have been also used to target bacterial
metalloenzymes, e.g., metallo-β-lactamases (MBLs). The
production of MBLs in bacteria is considered as one of the
most important mechanisms of resistance to β-lactam
antibacterials.96−99 A variety of MBLs have been identified in
clinically isolated resistant bacteria, which were divided into
Ambler classes B1, B2, and B3. The MBL enzymes utilize one or
two zinc ions to catalyze the hydrolysis of β-lactam antibacterials
as summarized in Figure 24. Compared with serine β-lactamases
(SBLs), MBLs have broader substrate specificity and can
hydrolyze penicillins, cephalosporins, and so-called “last-resort”
carbapenems.100 MBLs also can destroy β-lactam SBL
inhibitors, including clavulanic acid, sulbactam, and tazobactam
and even the non-β-lactam inhibitors, e.g., avibactam is slowly
hydrolyzed by some MBLs.100,101 The rapidly rising anti-
bacterial resistance prompted discovery of several broad-
spectrum potent MBL inhibitors, including the typical
boronates.102,103
The bicyclic boronate 62 (Figure 25) was initially reported by
Ness et al. as a potent inhibitor to class A TEM1 (IC50 = 13
nM).104 Introduction of larger substituents (e.g., 63 and 64) at
the 3-NH2 of bicyclic boronate resulted in improved inhibition
to both SBLs and MBLs (Figure 25).105 Notably, 64 exhibited
nanomolar inhibition to a broad spectrum of SBLs and class B
MBLs (NDM-1, 10 nM; VIM-1, 7.9 nM; VIM-2, 0.5 nM; IMP-1,
2.5 μM) and could inhibit some PBPs.105 Compound 64
showed high permeability to Gram-negative outer membrane
and periplasmic accumulation and showed in vivo activity
combined with cefepime in both the murine neutropenic lung
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infection and ascending urinary tract infection models, which is
the first SBL/MBL dual-action inhibitor to enter clinical trials
(currently in phase III, ClinicalTrials.gov Identifier:
NCT03840148). Crystallographic studies revealed that the
dual SBL/MBL inhibition of 64 was achieved by mimicking the
common tetrahedral intermediates in the β-lactam hydrolysis
process (Figures 24 and 25).106 The CTX-M-15:64 (PDB ID
6SP6)106 structure showed that the catalytic Ser70 is covalently
bound to the boron atom of 64, forming sp3 hybridized species.
Similarly, the VIM-2:64 (PDB ID 6SP7)106 revealed that 64 was
attacked by the zinc-bridged hydroxide anion to form sp3
hybridized boronate, mimicking the tetrahedral intermediates.
Interestingly, recently, a cocrystal structure of NDM-1:64 (PDB
ID 6RMF)107 revealed a tricyclic form of 64, i.e., the cyclization
of the amide oxygen onto the boron atom, reflecting multiple
possible binding modes for boronates in targeting metal-
loenzymes.
The above-mentioned bicyclic boronates have potent broad-
spectrum inhibition to SBLs/MBLs and excellent druglike
features, triggering efforts to identify more new bicyclic
boronates (Figure 26) with the aim of discovering better drug
candidates. For example, introducing more rigid substituents at
the 3-position (e.g., 65) or substituents at the 7-position (e.g.,
66) of bicyclic boronates resulted in new patent chemotypes
with comparable inhibition to 64.108 Since the membrane
permeability for these bicyclic boronates is most likely related to
their molecular size, it is desirable to identify new bicyclic
boronates with small molecular sizes but with potent SBL/MBL
inhibitions (e.g., 67 and 68). In 2020, Qpex Biopharma reported
a very classic lead optimization case. They first investigated the
effects of different substituents at the 3- and 8-positions and
discovered fluoroboronate 69 as inhibitors to SBLs only.
Structural modifications led to 3-thiol analogues 70, which
showed much improved MBL inhibition. The broader spectrum
analogue 71 was obtained by removal of the 3-substituents.
Moreover, further optimization resulted in a metabolically stable
tricyclic boronic acid QPX7728 (72), which displayed an
ultrabroad spectrum of MBL/SBL inhibition, including class A
KPC2 IC50 = 1.9 nM), class B NDM-1 (IC50 = 32 nM), class B
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Figure 25. Bicyclic boronate MBL/SBL inhibitors. (A) Representative bicyclic boronate inhibitors. Crystal structures of (B) CTX-M-15:64 (PDB ID
6SP6)106 and (C) VIM-2:64 (PDB ID 6SP7)106 revealed the dual-action inhibition by mimicking the common tetrahedral intermediates in the β-
lactam hydrolysis process.

Figure 26. Newly reported bicyclic boronate-based MBL/SBL inhibitors.

VIM-1 (IC50 = 8 nM), class C AmpC (IC50 = 8.5 nM), and class
D OXA48 (IC50 = 0.28 nM), notably with little affects by porin
modification and the efflux pump (Figure 26).109 Crystallo-
17720
graphic studies revealed that 72 has a similar MBL inhibition
mode as 64, e.g., the sp3 hybridized form coordinating with the
active site zinc ions. More interestingly, the cyclopropyl of 72 is
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positioned to occupy the small hydrophobic pocket near the zinc

ions in both NDM-1 and VIM-2, revealing new insights for drug
design and structural optimization for MBL inhibitors (Figure
27). As 72 has excellent pharmacokinetics in the rat similar to β-

Figure 28. Benzoxaborole-based β-lactamase inhibitors (IC50, μM).

Figure 27. View of crystal structures of 72 complexed with (A) NDM-1

(PDB ID 6V1M)109 and (B) VIM-2 (PDB ID 6V1P)109 reveals its
common binding features to NDM-1 and VIM-2.

lactam antibiotics and has good oral bioavailability without the

need for a prodrug (a common strategy to improve the
bioavailability of boronic acid-based inhibitors), this compound
has been advanced to phase I clinical trials (ClinicalTrials.gov
Identifiers: NCT04380207 and NCT05072444), providing a
new drug candidate for combating SBL/MBL-mediated
antibacterial resistance.
Recently, our group disclosed a series of 3-aryl substituted
benzoxaborole derivatives (e.g., 73 and 74) as dual SBL and
MBL inhibitors (Figure 28).110 Since such benzoxaborole
compounds do not have corresponding negatively charged
features to interact with the positively charged residue in the
MBL active site, which is important for recognition of β-lactam
antibiotics, we designed a series of C3-acrylic acid substituted
benzoxaboroles by mimicking carbapenem antibiotics.111 Mean-
while, in order to obtain the optical isomers, we presented an
asymmetric Morita−Baylis−Hillman (MBH) cascade reaction
and synthesized a series of optical C3 acrylic acid substituted
benzoxaboroles.111 Some of these compounds (e.g., 75 and 76)
showed nanomolar inhibitory activities to KPC-2 SBL and
micromolar inhibitory activity toward various class B1MBLs
(Figure 28), providing a new starting point to develop
benzoxaborole-based MBL inhibitors.
In addition to the above-mentioned cyclic boronates, some
acyclic boronates have also been investigated to target SBLs
since the 1970s when boric acid (B(OH)3) was reported to
inhibit an B. cereus SBL.112 In 2019, Cendron et al. reported
benzo[b]thiophen-2-ylboronic acids (e.g., 77−81, Figure 29A)
as inhibitors of several SBLs and MBLs.113 Compound 80
17721
Figure 29. Benzo[b]thiophen-2-ylboronic acid-based MBL inhibitors.
(A) Representative benzo[b]thiophen-2-ylboronic acids as NDM-1
inhibitors; (B) crystal structure of NDM-1:80 (PDB ID 6Q30)113
shows a similar inhibition mode as that described for cyclic boronate
MBL inhibitors.
showed good inhibition to class A KPC2 (IC50 = 4.9 μM), class
B NDM-1 (IC50 = 3.0 μM), class C AmpC (IC50 = 0.42 μM),
and class D OXA24 (IC50 = 16 μM). Similar to above-described
cyclic boronates, these compounds inhibit NDM-1 by
coordinating with active site zinc ions using the sp3 hybridized
boronate form, as observed in the NDM-1:80 (PDB ID
6Q30)113 crystal structure (Figure 29B).
Recently, a series of acyclic boronic acid derivatives (e.g., 82
and 83) were reported as dual-action MBL/SBL inhibitors by a
pharmacophore fusion strategy; for example, 83 has broad-
spectrum inhibition to clinically relevant enzymes, including the
widespread KPC-2 (IC50 = 0.61 μM) and VIM-2 (IC50 = 0.44
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μM).114 As demonstrated by crystallography, the thiol of 82 is
positioned to coordinate with the active-site zinc ions of MBLs,
and the boronic acid is covalently bound with the catalytic serine
of SBLs (Figure 30), suggesting that pharmacophore fusion is
Figure 30. Acyclic boronic acid-based β-lactamase inhibitors.
another useful strategy to develop dual-action inhibitors
particularly for structurally and mechanistically distinct targets.
Taken together, this metalloenzyme case analyses indicate that
development of proper boron-containing MBPs is quite
important for targeting MBLs or other metalloenzymes.
■ BIOISOSTERES OF BORON-CONTAINING MBPS
The diverse metal coordination modes of boron-containing
MBPs prompt researchers to consider bioisosteres in metal-
loenzyme inhibitor transformation. The MBP bioisosteres were
summarized with respect to the boron hybridization types. The
sp2 boronic acid is possible to have different binding modes with
active site metal ions. For example, one hydroxyl group of sp2
boronic acid could enable coordination with one or two metal
ions via a monodentate manner; the two hydroxyl groups of sp2
boronic acid may be able to simultaneously form monodentate
coordination with two metal ions or bidentate coordination with
one metal ion (Figure 1B). Several chemical motifs are proposed
to have similar metal binding features, such as carboxylic acid,
nitrate, thioic acid, imidamide, imidazole, and tetrazole (Figure
31A), which could be referred to as bioisosteres of sp2
hybridized boronic acid MBPs. Some of these bioisosteres
have been used in metalloenzyme inhibitor development, e.g.,
the above-mentioned arginase inhibitors (Figure 13). More
specifically, given the fact that the boron atom as a Lewis acid
with a vacant p orbital could accept an electron from metal ion-
activated hydroxyl anion, the sp3 boronates display more
attractive features in binding with metal ions, particularly via
forming more diverse metal coordination geometries (Figure
1B). Those chemical moieties, including sulfonamide, sulfonic
acid, phosphoric acid, siliconic acid, and arsenic acid, could be
used as bioisosteres of sp3 boronates since they have similar
tetrahedral pharmacophore features when binding with metal
ions (Figure 31B). As highlighted above, the boronate and
phosphoric acid have been mutually used as bioisosteres in PDE
(Figure 3) and ATX (Figure 7) inhibitor transformation; the
boronate, phosphoric acid, and sulfonamide have also been
successfully applied as bioisosteres for MBL inhibitor develop-
ment.102,115 The unique advantage of these bioisosteres is that
they might be able to mimic native substrates or reaction
transition states of nucleophilic metalloenzymes. Although such
bioisostere-based strategy is useful in improving binding affinity,
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Figure 31. Proposed bioisosteres of (A) sp2 and (B) sp3 hybridized
boron MBPs.
selectivity, or achieving other purposes (e.g., intellectual
property), its full potential remains largely unexploited. It is
highly desirable to exploit new MBP bioisosteres or their new
applications in metalloenzyme inhibitor development and,
particularly, to compare their respective advantages to
eventually promote drug discovery targeting metalloenzymes.
■
CONCLUSION AND FUTURE PERSPECTIVES
The comprehensive analysis of various boron-containing MBPs
targeting five distinct types of nucleophilic metalloenzymes
clearly reveal their unique features in engaging with metal-
loenzyme active sites. Most notably, the boron-containing MBPs
can receive a nucleophilic attack from an active site metal-
activated water molecule and enable their sp3 form to mimic the
binding nature of native substrates or reaction transition states
(i.e., tetrahedral intermediates). For example, the benzoxaborole
MBP was observed to mimic AMP binding with the PDE4 active
site, and it was also successfully used to target ATX (e.g., 23),
CAs (e.g., 57−61), and MBLs (e.g., 73−76) by involving the
common sp3 boronate form to interact with these nucleophilic
metalloenzymes. This offers the important basis for boron-
containing MBPs used in inhibitor transformation among
structurally different metalloenzymes, and prompts them as
bioisosteres to replace those, such as phosphoric acid and
sulfonamide, to derive new metalloenzyme inhibitors.
Given that boron-containing MBPs have the possibility to
interact with multiple nucleophilic metalloenzymes and other
nucleophilic enzymes (e.g., serine hydrolases), it is tempting to
assume that such MBPs may face challenges such as selectivity in
metalloenzyme inhibitor development. Some possible strategies
discussed below may be useful to address these challenges. The
first is to incorporate boron-containing MBPs into a target-
specific, substrate-mimicking chemical scaffold. As described
above, the boronic acid as an MBP was fused into arginine-
mimicking chemical scaffolds to develop arginase inhibitors by
involving interactions with the active site Mn2+ ions and
catalytically important residues. The second is to introduce
target-specific substituents close to boron-containing MBPs,
albeit with possibly high requirements for the chemical
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synthesis, to enhance binding affinity and metalloenzyme
specificity. The MBL inhibitor QPX-7728 is a classical example,
which has a magic cyclopropyl-fused cyclic boronate and
manifests highly potent MBL inhibition. Coupling boron-
containing MBPs to target-specific peptide, antibody, or
aptamer could be an alternative to improve selectivity and
binding affinity to the metalloenzyme target. In addition,
emerging boronic ester prodrug strategies are likely to promote
boron-containing drug development, including metalloenzyme
inhibitors. Some prodrugs have been validated in clinical
settings, e.g., ixazomib citrate, which has improved pharmaco-
kinetics, pharmacodynamics, and therapeutic efficacy.116,117
The MBP bioisostere-based inhibitor transformation may be
also useful for addressing issues that are related to boron-
containing inhibitor stability and other pharmaceutical proper-
ties.
Moreover, currently, boron-containing MBPs are mainly used
to target nucleophilic metalloenzymes, and it remains underex-
plored how they target other types of metalloenzymes, which
will be worthy of further study. The topic on how to exploit new
boron-containing MBPs and new MBP-metalloenzyme associ-
ations would also be worthwhile. Clearly, new computation tools
such as metal ion-focused pharmacophore fitting,118 artificial
intelligence algorithms,119 or new strategies120 are needed.
Overall, this paper brings the knowledge of boron-containing
MBPs targeting metalloenzymes and perspectives of how to
develop better metalloenzyme inhibitors. It is hoped that more
efforts can be focused in this area to make new discoveries that
not only advance understanding in inhibitor design principles by
integrating medicinal chemistry, bioinorganic chemistry, and
structural biology but also contribute to developing new
clinically useful drugs targeting metalloenzymes.
■ AUTHOR INFORMATION
Corresponding Authors
Guo-Bo Li − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Sichuan Engineering Laboratory for Plant-Sourced
Drug and Sichuan Research Center for Drug Precision
Industrial Technology, West China School of Pharmacy,
Sichuan University, Chengdu 610041, China; orcid.org/
0000-0002-4915-6677; Email: liguobo@scu.edu.cn
You-Cai Xiao − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Sichuan Engineering Laboratory for Plant-Sourced
Drug and Sichuan Research Center for Drug Precision
Industrial Technology, West China School of Pharmacy,
Sichuan University, Chengdu 610041, China;
Email: xiaoliguo1987@scu.edu.cn
Authors
Jun-Lin Yu − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Sichuan Engineering Laboratory for Plant-Sourced
Drug and Sichuan Research Center for Drug Precision
Industrial Technology, West China School of Pharmacy,
Sichuan University, Chengdu 610041, China
Qing-Qing Dai − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Sichuan Engineering Laboratory for Plant-Sourced
Drug and Sichuan Research Center for Drug Precision
Industrial Technology, West China School of Pharmacy,
Sichuan University, Chengdu 610041, China
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pubs.acs.org/jmc Perspective
Gen Li − Key Laboratory of Drug-Targeting and Drug Delivery
System of the Education Ministry and Sichuan Province,
Sichuan Engineering Laboratory for Plant-Sourced Drug and
Sichuan Research Center for Drug Precision Industrial
Technology, West China School of Pharmacy, Sichuan
University, Chengdu 610041, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c01691
Notes
The authors declare no competing financial interest.
Biographies
You-Cai Xiao is an Associate Professor at the West China School of
Pharmacy, Sichuan University. He completed his Bachelor’s degree in
2009 from Sichuan University and continued to pursue his Ph.D. in
Medicinal Chemistry at the same university. He has worked as a
Postdoctoral Fellow at KTH Royal Institute of Technology under the
supervision of Prof. Cristina Moberg between 2014 and 2015 and as
Research Associate at The Wistar Institute in Prof. Joseph. M. Salvino’s
group from 2015 to 2018. His research interests include the design and
synthesis of boron-based protease and metalloenzyme inhibitors,
proteolysis targeted chimera (PROTAC) technology, and asymmetric
reactions for drug synthesis.
Jun-Lin Yu is a postgraduate student, majoring in Medicine Chemistry
at the West China School of Pharmacy, Sichuan University. He joined
Prof. Guo-Bo Li’s group in 2019 and has great interest in developing
new computational methods, including deep learning algorithms, for
drug discovery.
Qing-Qing Dai is a postgraduate student majoring in Medicinal
Chemistry at the West China School of Pharmacy, Sichuan University.
She joined Prof. Guo-Bo Li’s group in 2019 and mainly conducts
research on developing new computer-aided drug design methods and
deep learning models to aid in drug discovery.
Gen Li is a Ph.D. candidate majoring in Medicinal Chemistry at the
West China School of Pharmacy, Sichuan University. He joined Prof.
Guo-Bo Li’s group in 2018 and moved to Prof. Fener Chen’s Group in
2021. He has interest in computer-aided drug design and medicinal
chemistry.
Guo-Bo Li is an Associate Professor at the West China School of
Pharmacy, Sichuan University. He received his Ph.D. degree in 2014 at
Sichuan University supervised by Professor Shengyong Yang. He
worked as a postdoctoral researcher in Professor Yang’s group from
2014 to 2015, then in Professor Christopher J. Schofield’s group at
University of Oxford from 2015 to 2016, and joined in the West China
School of Pharmacy in 2016. He is currently interested in developing
new computational methods for medicinal chemistry and new
inhibitors/chemical probes targeting clinically relevant enzymes,
particularly metalloenzymes. He has authored or coauthored more
than 60 peer-reviewed publications.
■
ACKNOWLEDGMENTS
This work was partly supported by the National Natural Science
Foundation of China (Grants 82122065, 82073698, and
81874291), Sichuan Science and Technology Program (Grant
22GJHZ0253), 111 Project (Grant B18035), and Outstanding
Interdiscipline Project of West China Hospital of Sichuan
University (Grant ZYJC18024).
https://doi.org/10.1021/acs.jmedchem.1c01691
J. Med. Chem. 2021, 64, 17706−17727
Journal of Medicinal Chemistry
■
ABBREVIATIONS USED
ABH, 2-(S)-amnio-6-boronohexanoic acid; ACE, angiotensin
converting enzyme; AMP, adenosine monophosphate; ArgI,
arginase I; ArgII, arginase II; ATX, autotaxin; AUC, area under
the curve; CA, carbonic anhydrase; cAMP, cyclic adenosine
monophosphate; Can2, Cryptococcus neoformans carbonic
anhydrase; CgNce103, Candida globosa carbonic anhydrase;
cGMP, cyclic guanosine monophosphate; GPCR, G protein-
coupled receptor; hCA, human carbonic anhydrase; KPC2,
Klebsiella pneumoniae carbapenemase 2; LPA, lysophosphatidic
acid; LPC, lysophosphatidyl choline; mATX, mouse autotaxin;
MBH, Morita−Baylis−Hillman; MBL, metallo-β-lactamase;
MBP, metal-binding pharmacophore; MgCA, Malassezia
globosa carbonic anhydrase; NDM, New Delhi metallo β-
lactamase; NPP, nucleotide pyrophosphatase and phosphodies-
terase; PDB, Protein Data Bank; PDE, phosphodiesterase;
PMA, phorbol ester; RLA, rat liver arginase; SBL, serine β-
lactamase; ScCYP51, Saccharomyces cerevisiae CYP51; TOGA,
thioornithine generation assay; VIM, Verona Integron encoded
metallo-β-lactamase
■
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