Structure-Activity Relationships in Metal-Binding Pharmacophores for Influenza Endonuclease

Article abstract of DOI:10.1021/acs.jmedchem.8b01363

Metalloenzymes represent an important target space for drug discovery. A limitation to the early development of metalloenzyme inhibitors has been the lack of established structure-activity relationships (SARs) for molecules that bind the metal ion cofactor(s) of a metalloenzyme. Herein, we employed a bioinorganic perspective to develop an SAR for inhibition of the metalloenzyme influenza RNA polymerase PA_N endonuclease. The identified trends highlight the importance of the electronics of the metal-binding pharmacophore (MBP), in addition to MBP sterics, for achieving improved inhibition and selectivity. By optimization of the MBPs for PA_N endonuclease, a class of highly active and selective fragments was developed that displays IC₅₀ values <50 nM. This SAR led to structurally distinct molecules that also displayed IC₅₀ values of ～10 nM, illustrating the utility of a metal-centric development campaign in generating highly active and selective metalloenzyme inhibitors.

Full text of DOI:10.1021/acs.jmedchem.8b01363

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Article
Structure-Activity Relationships in Metal-Binding
Pharmacophores for Influenza Endonuclease
Cy V. Credille, Benjamin Dick, Christine N. Morrison, Ryjul Wynn Stokes,
Rebecca Adamek, Nicholas C. Wu, Ian A. Wilson, and Seth M. Cohen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01363 • Publication Date (Web): 16 Oct 2018
Downloaded from http://pubs.acs.org on October 21, 2018
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Structure-Activity Relationships in Metal-Binding
Pharmacophores for Influenza Endonuclease
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Cy V. Credille,1 Benjamin Dick,1 Christine N. Morrison,1 Ryjul W. Stokes,1 Rebecca N. Adamek,1
Nicholas C. Wu,2 Ian A. Wilson,2,3 and Seth M. Cohen1*
1 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA
92093, United States
2 Department of Integrative Structural and Computational Biology, The Scripps Research
Institute, La Jolla, CA 92037, United States
3 The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA
92037, United States
*scohen@ucsd.edu
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ABSTRACT
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Metalloenzymes represent an important target space for drug discovery. A limitation to the early
development of metalloenzyme inhibitors has been the lack of established structure-activity
relationships (SARs) for molecules that bind the metal ion cofactor(s) of a metalloenzyme. Herein,
we employed a bioinorganic perspective to develop an SAR for inhibition of the metalloenzyme
influenza RNA polymerase PAN endonuclease. The identified trends highlight the importance of
the electronics of the metal-binding pharmacophore (MBP), in addition to MBP sterics, for
achieving improved inhibition and selectivity. By optimizing the MBPs for PAN endonuclease, a
class of highly active and selective fragments were developed that display IC50 values <50 nM.
This SAR led to structurally distinct molecules that also displayed IC50 values of ~10 nM,
illustrating the utility of a metal-centric development campaign in generating highly active and
selective metalloenzyme inhibitors.
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Introduction
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Metalloenzymes comprise over one-third of all known enzymes, are ubiquitous across all
domains of life, and are implicated in a wide variety of human diseases.1,2 As a result,
metalloenzymes represent prime target space for drug discovery; however, the clinical
development of metalloenzyme inhibitors is rather limited. In the past five years, only ~9% of
new molecular entities approved by the FDA target metalloenzymes, and <5% of all FDA
approved drugs inhibit metalloenzymes.1,2 Compounds that are able to interact strongly with an
active site metal center can effectively inhibit the catalytic activity of metalloenzymes, by
disrupting substrate access to the active site and preventing metal-mediated catalysis.3 Metal
binding inhibitors are reversible, but are capable of forming strong interactions due to the large
bond enthalpy of metal-ligand dative or coordinate covalent bonds.
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Within the context of metalloenzyme inhibitors, a shortcoming to the development of new
inhibitors has been an over-reliance on a very limited number of metal-binding pharmacophores
(MBPs).4,5 In addition, despite the importance of metal-ligand interactions in the development of
metalloenzyme inhibitors, relatively little work has been focused on the development and
optimization of MBPs, with a general lack of structural diversity in the MBP chemical space.6,7
Indeed, the only metalloenzyme targets where a substantial chemical diversity is present in terms
of the MBPs are inhibitors of HIV integrase (HIV IN) and HIV reverse-transcriptase associated
RNaseH (HIV RNaseH),8,9 with most of this structural diversity reported in the patent literature.10-
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However, despite the structural diversity in the patent literature against these targets, there is
little analysis into the effects of varied MBP cores on metalloenzyme inhibition. Furthermore,
these reports generally do not detail development of the MBP core nor efforts towards MBP
optimization. To address these shortcomings, MBP libraries, consisting of fragment-like
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compounds designed to bind metal ion cofactors in metalloenzyme active sites, have been
developed.13 These MBP libraries have been used in fragment-based drug discovery (FBDD) to
identify novel inhibitors of several metalloenzymes, including the influenza RNA-dependent RNA
polymerase PA subunit.13
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The influenza polymerase complex is an attractive target for new antiviral therapies,
particularly the polymerase PA endonuclease domain. This domain is both highly conserved
across influenza strains and serotypes and is indispensable for the viral lifecycle.14
Crystallographic and biochemical studies have shown that the polymerase PA N-terminal
endonuclease domain (PAN) contains a dinuclear metal active site which binds to two Mg2+ or
Mn2+ cations.15,16 The metal cations reside in a pocket comprised of a histidine (His41), an
isoleucine (Ile120), and a cluster of three acidic residues (Asp108, Glu80, Glu119) that all
coordinate to the active site metal ions (Figure 1).15,17 These metal ions are essential for catalysis,
and it has been shown that metal coordination by small molecules effectively inhibits endonuclease
activity.13,18-22 Indeed, nearly all reported inhibitors of endonucleases have been shown by X-ray
crystallography or modeling to coordinate to at least one active site metal center, including the
polymerase PA inhibitor Baloxavir marboxil, developed by Roche and Shionogi, which is
currently in Phase III clinical trials in the U.S. and has received regulatory approval in Japan.23
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Figure 1. Structure of the RNA-dependent RNA polymerase PA subunit active site (PDB ID:
5DES). The endonuclease active site employs two divalent metal cations to facilitate the
hydrolytic cleavage of the phosphodiester backbone of RNA. Protein secondary structure elements
are shown in cartoon representation (gray). Mn2+ cations are shown as purple spheres.
Coordinating protein residues are colored by element and labeled and coordinating
water/hydroxide molecules are shown as red spheres. All coordination bonds are displayed as
dashed yellow bonds. This structure, as well as all other protein structures presented, were
generated in PyMOL.24
The influenza virus RNA polymerase has no proofreading capability, which results in a
high mutation rate of approximately one error per genome replication cycle.25 This results in each
infected cell producing on average ~10,000 new viral mutants during the course of infection.16
One primary advantage to a discovery campaign focused on metal binding is an intrinsic barrier to
antiviral resistance. Any mutation to the PAN metal coordinating residues (with the exception of
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substituting Glu119 with Asp, which coordinates identically to Glu119) results in total loss of viral
transcription activity and ultimately virulence.26,27 Hence, an inhibitor molecule that obtains
significant binding energy from metal coordination may be less susceptible to antiviral resistance,
as mutations that disfavor metal coordination will likewise disfavor substrate binding and/or
catalytic activity.
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Little work has been reported on the optimization of specific metal-ligand interactions for
PAN inhibitors,19 despite some chemical diversity among the various reported PAN inhibitor MBPs
both in terms of chemical structure and coordination motif. However, there are few clear structure-
activity trends that can be derived from the different reported MBPs.28 This lack of consensus
regarding preferred metal coordination interactions is not unique to PAN endonuclease, and has
been a limitation to the development of inhibitors of many metalloenzymes.10 A more detailed
understanding of the metal coordination preferences of the dinuclear metal center of PAN
endonuclease would allow for optimization of MBP domains and produce highly active
endonuclease inhibitors. To this end, a targeted FBDD screen was conducted employing a MBP
library.13 This screen included MBPs with the ability to coordinate to both metals of the dinuclear
metal active site simultaneously. Using a FRET-labeled DNA oligonucleotide substrate, the
endonuclease activity of the PAN subunit of H1N1 influenza A polymerase was measured in the
presence of MBPs to identify fragments that exhibit strong inhibition. Several MBP fragments
were found to have IC50 values of <500 nM. SAR elucidated from the library screen and
subsequent derivatization identified key chemical properties that were essential for tight binding
to the metal cofactors in the PAN active site. In addition, good selectivity for PAN inhibition over
other dinuclear metalloenzymes was also achieved. This strategy resulted in the identification of
an optimized chemotype for MBP-based inhibition of PAN, with several MBPs displaying IC50
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values of <50 nM. X-ray structural determination of MBPs in PAN were used to elucidate key
binding interactions. Ultimately, the identification of key metal binding preferences allowed for
the predictive development of other chemically distinct, highly active inhibitor chemotypes
illustrating the value of a bioinorganic, coordination chemistry-based fragment discovery approach
for metalloenzyme inhibitor development.
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Results and Discussion
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Fragments in the MBP library were screened for endonuclease inhibition against a
truncated N-terminal (PAN) endonuclease construct employing a FRET-labeled DNA
oligonucleotide, as previously reported.13,15,22 This construct is derived from an influenza A
(A/California/04/2009) H1N1 clinical isolate that has been modified such that residues 52-64 of
the N-terminal (PAN) endonuclease were replaced by a single glycine residue.18,29 This truncation
removes a highly disordered protein loop that engages in protein-protein interactions with other
subunits of the RNA polymerase complex. Loop removal was found to produce higher quality
protein crystals and improve the solution stability of the isolated PAN domain.18,29 Note that
several compounds used in this study were obtained from commercial suppliers (compounds 4-5,
7-14, 16, 19, 23, 31, and 34).
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Compounds that exhibited inhibition of PAN endonuclease at a fragment concentration of
200 µM and were predicted to simultaneously coordinate both metal centers in the endonuclease
active site (i.e., possessed a suitable triad of donor atoms)30 were further analyzed with dose-
response experiments to determine IC50 values. The activity of these MBPs is shown in Table 1.
To ensure that the observed activity against endonuclease was not due to metal-stripping, all
inhibition assays were performed with a large excess (2 mM) of both Mn2+ and Mg2+ metal cations
in the assay media. From these results, a characteristic pharmacophore model with a common
donor triad chemotype was identified that is shared by the most active inhibitors produced in this
study. As shown in Figure 2, compounds sharing this chemotype were predicted to coordinate to
the divalent metal centers as multicoordinate bridging ligands with the conserved phenolic oxygen
atom replacing the bound hydroxide anion observed in the native protein. To elucidate the mode
of binding, crystallographic studies were performed. The truncated PAN construct was co-
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crystalized or soaked with several ligands that share the active chemotype (Figure 2). All X-ray
structures validate the multi-coordinate bridged coordination mode hypothesis. Compound 3 was
observed to coordinate simultaneously to both metal centers with the carbonyl oxygen atom
coordinating to Mn-1 at the open coordination site, the phenolic oxygen replacing the bound
hydroxide anion as the bridging ligand, and the carboxylic acid group coordinating to Mn-2 (Figure
2). No steric clash was observed within the active site, and binding of compound 3 places each
metal ion in an octahedral coordination geometry. From this co-crystal structure, it is inferred that
the majority of the binding enthalpy for 3 is derived from metal coordination and not from other
interactions with the protein active site. This is corroborated by the analogous binding mode and
similar inhibitory activity of compound 1 (Figure 2).
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Table 1. Select MBP fragments with activity against PAN endonuclease.
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a pIC50 is defined as pIC50 = −log(IC50) and is included to provide a linear comparison of relative
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inhibition activity.
Ligand efficiency (LE) provides a measure of binding energy per non-
hydrogen atom in the fragment molecule.
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Figure 2. Top left: Representative chemotype shared by active MBP fragments of influenza PAN
highlighting the coordination motif of these MBPs to the dinuclear active site metal ions. Top
right: X-ray co-crystal structure of compound 3 (PDB ID: 6DCZ) in the PAN active site. Mn-2
is also coordinated by two water molecules at the open coordination sites (not shown). Bottom:
X-ray co-crystal structure of compounds 1 (left; PDB ID: 6DZQ), 2 (middle; PDB ID: 6DCY),
and 3 (right; PDB ID: 6DCZ) in the PAN active site. All ligands were found to coordinate in a
similar manner. Compound 2 coordinates Mn-2 with the electron density (blue mesh is the 2Fo-
Fc map contoured to 2σ) corresponding to the carboxylic acid moiety found to be diffuse above
the metal center. The poorer coordination ability of compound 2 is likely due to internal steric
pressure exerted by the methyl group residing alpha to the carboxylic acid.
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The inhibitory activity of compounds such as 1 is outstanding given the small molecular
weight of the fragment (ligand efficiency of 0.84). Generally, hits identified in FBDD screening
campaigns have IC50 values >100 M, and not infrequently >1 mM.7,31 More striking is the
comparison between 1 and the related MBPs allomaltol and pyromeconic acid (Table 1, IC50 =
17.1 µM and 22.5 µM, respectively), which were previously identified as PAN hits.13 It was
observed that the addition of only three atoms (in the form of the coordinating carboxylate group)
to these inhibitor scaffolds (to generate compounds 1 and 3, respectively) resulted in a ~500-fold
increase in activity.32 Interestingly, not every MBP that can present a donor atom triad to the PAN
metal centers was found to display such tight binding affinities (e.g., compounds 4, 6, 8, Table 1),
and suggests that the PAN metal centers have specific ligand preferences, which have not been
previously identified for this metalloenzyme.
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Based on the excellent activity observed with MBPs such as 1, other fragments were
synthesized to obtain SAR to elucidate what aspects of these molecules were responsible for their
tight binding affinity. Beyond probing simple steric effects or attempting to introduce new protein
contacts, changes to the electronic character of the MBP ring system were explored. The effect of
changes to donor atom identity, Lewis basicity, and isosteric replacement of coordinating moieties
were also investigated. Multiple structural variants of the identified chemotype were ultimately
designed and synthesized, and the effect of modulating each coordinating moiety was assessed by
both biochemical and structural experiments to understand the observed trends.
Conversion of a pyrone to a pyridinone scaffold has been shown to improve activity in
previously reported endonuclease inhibitors.13 Pyridinones have a greater aromatic character than
pyrones, which can result in greater electron density on the oxygen donor atoms. This change in
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ligand electronics generally results in greater ligand basicity, which can lead to stronger
coordination to the hard Lewis acidic metal centers found in endonucleases.33 To probe this effect,
a series of analogs of 1 were synthesized and investigated (Table 2). As expected, conversion of
a pyrone oxygen to a pyridinone nitrogen (17) resulted in an approximately 3-fold increase in
activity relative to 1. In addition, no change in the activity of compounds 1 and 17 was observed
as a function of the presence or absence of excess metal ions in solution (up to 25 mM). These
results conclusively demonstrates that the high affinity of these inhibitor molecules is derived from
a strong binding preference for the dinuclear Mn2+ PAN metal centers and not from any general
affinity for free Mn2+ in solution.
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The position of the nitrogen atom within the ring system strongly effects inhibitory activity.
While 3,4-hydroxypyridinones (17) did show an improvement in activity, 2,3-hydroxypyridinones
(18) and 4,5-hydroxypyrimidinones (19) showed markedly less activity than the 3,4-
hydroxypyridinone or the parent hydroxypyrone fragments, with compounds 18 and 19 showing a
25-fold and 100-fold loss in activity, respectively (relative to 17). Furthermore, N-hydroxy-1,2-
hydroxypyridinones (5) were even less active, with a >400-fold loss in activity when compared to
17. Analysis of the pKa of the phenolic and N-hydroxy protons in these compounds revealed that
the donor oxygen in 5 is less basic (pKa ~7.0 calculated;33 pKa = 7.51 reported)34 than the equivalent
donor oxygen in compounds 16 (pKa ~9.5 calculated)33, 17 (pKa ~11.3 calculated)33, 18 (pKa ~9.2
calculated)33, or 19 (pKa ~7.5 calculated)33. This change in ligand basicity is likely the electronic
driving force behind the variation in inhibitory activity, with the most basic ligand 17 being the
most active. Interestingly, the overall inhibitory activity (pIC50) of these fragments was found to
be linearly correlated to the calculated pKa of the bridging oxygen atom (r = 0.96, n = 6, p < 2×10-3;
Figure 3). It was also found that catechol-based fragments, which contained no endocyclic
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heteroatoms (e.g., 7), were far less active than the parent pyrone or pyridinone fragments in spite
of their high ligand basicity. Compound 7 is likely too basic to fully deprotonate at physiological
pH (pKa ~9.8, 14.3 calculated;33 pKa = 10.30, 13.48 reported)35 and hence may be unable to
efficiently coordinate to the metal centers in the PAN active site. Compound 7 also ultimately
coordinates as a trianionic species, as opposed to the other MBPs in Table 2, which all coordinate
as dianions.19 This difference in overall charge of the final coordination complex (-2 vs -1) is
substantial, and the vast disparity in activity between compounds 7 and 1 may also be attributed to
the formation of a highly anionic coordination center.
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While hydroxypyridine and hydroxypyrimidine MBP scaffolds have been explored as
inhibitors of endonucleases,13,19,21,22 no systematic study of the effects of their stereoelectronic
differences on enzymatic inhibition has been performed previously. Surprisingly, the subtle
differences in the electronic character of these structurally analogous MBPs has been shown to
have a substantial impact on inhibitory activity. Similar relationships between small changes in
ligand electronics and inhibitory activity have likewise not been well explored in other
metalloenzyme systems;30 however, this new evidence would suggest that the exploration of such
relationships would be extremely valuable in the development of metalloenzyme inhibitors.
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Table 2. Structure and activity of a series of MBP derivatives.
a
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Compound
Number
IC50 (nM)
pIC50
Phenol pK a
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O
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43 ± 9 nM
7.4
9.7
O
O
OH OH
O
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O
3
5
68 ± 11 nM
7500 ± 1200 nM
6400 ± 900 nM
17 ± 3 nM
7.2
5.1
5.2
7.8
6.4
5.8
9.6
7.0
O
OH OH
O
N
O
OH OH
O
O
O
7
9.8; 14.3
11.2
9.2
OH
OH OH
HN
17
18
19
O
OH OH
NH
430 ± 20 nM
1600 ± 400 nM
O
OH OH
7.5
a Calculated pKa values33
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Figure 3. Inhibition values (pIC50) of MBP isologues of compound 1 plotted against the pKa of
their bridging phenolic oxygen atom. A linear correlation (r = 0.96, n = 6, p < 2×10-3) was
observed between pKa and pIC50, illustrating a ligand electronics-based justification for the
observed disparity in inhibition values of otherwise structurally homologous inhibitors.
Despite high structural similarity, a peculiar disparity in inhibitory activity was observed
between several highly active MBPs (i.e., 1, 3, 17, 18) and compound 2. Compounds 1 and 2 are
structurally nearly identical (the only difference is the position of the carboxylate moiety), but
display a ~100-fold difference in activity. A potential explanation is the steric pressure exerted on
the carboxylic acid moiety by the α-methyl group of 2, which precludes an ideal coordination
geometry. The difference in bridging atom type (phenolic vs. carbonyl oxygen) could also be an
underlying reason for the disparity in activity, as has been previously suggested for related
inhibitor molecules in HIV integrase (HIV IN).30 Structural determination using X-ray
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crystallography showed that 1 and 2 shared a similar mode of binding, except the electron density
corresponding to the carboxylic acid moiety of compound 2 is diffuse above that of the Mn-2 metal
ion (Figure 2). This suggests that the carboxylic acid does not coordinate to the metal center with
a preferred orientation, is not co-planar with the aromatic ring, and only makes weak ionic
interactions with the Mn-2 metal ion. To further evaluate this effect, compound 20 was
synthesized (Table 3). Comparison of 17 and 20 shows a similar, ~200-fold difference in activity
as observed with compounds 1 and 2 giving evidence that steric pressure by a proximal methyl
group is a central cause of the dramatic decrease in activity. To ensure that the disparity did not
arise from a steric clash that was not observed crystallographically, compound 21 was synthesized.
Compound 21 should exert the same steric pressure on any active site protein residue or water
network as compound 20, but can also stabilize the carboxylic acid moiety in the proper orientation
for metal binding through an intramolecular hydrogen bond. Compound 21 was found to exhibit
activity similar to compounds 1 and 17. These data suggest that intramolecular steric pressure on
coordinating moieties can cause dramatic differences in the ability of MBP ligands to effectively
coordinate active site metal centers. Thus, care must be taken during structural elaboration of
MBP leads that employ coordinating carboxylic acid moieties to prevent inadvertent perturbations
to the desired modes of metal coordination. Despite the very strong binding enthalpy of
coordination bonds, dative interactions are highly dependent on proper bond angle and
orientation.36
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17
18
19
20
21
22
23
24
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Table 3. Structure and activity of MBP inhibitor molecules.
Number Compound
IC50 (nM)
pIC50
Number Compound
IC50 (nM)
pIC50
Number Compound
IC50 (nM)
pIC50
5.9
HN
6
N
O
N
20
3900 ± 900 nM
5.4
25
3800 ± 1100 nM
5.4
30
1200 ± 400 nM
O
O
O
O
O
N
OH
O
7
OH OH
NH OH
8
9
O
H
O
N
NH
21
22
23
24
85 ± 17 nM
420 ± 60 nM
~200000 nM
2700 ± 600 nM
7.1
6.4
26
>50000 nM
37 ± 8 nM
< 4.3
7.4
31
32
33
1700 ± 400 nM
11 ± 1.8 nM
5.8
8.0
8.1
O
HO
OH
OH
O
O
OH
NH OH
OH OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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29
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32
33
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HN
H
O
N
27
O
O
O
HO
S
N
O
N
OH
OH
N
OH OH
HN
H2
N
O
< 3.7
5.6
28
29
~19000 nM
540 ± 80 nM
4.7
8.0 ± 1.1 nM
NH2
N
OH OH
HO
O
O
O
O
6.3
O
O
N
OH
NH2 OH
Donor atom identity was also found to play a key role in MBP activity against PAN
endonuclease. A small comparison of MBP fragments with similar structures, but different donor
atom sets, is detailed in Table 3. It was found that oxygen donor atoms were preferred to sulfur
donor atoms, with compound 22 displaying a 10-fold decrease in activity relative to 1. This trend
is consistent with a hard-soft theory of Lewis acids and bases, where hard Mg2+ or Mn2+ Lewis
acid metal centers are expected to form a more stable complexes with harder Lewis base oxygen
donor atoms.37 Some MBPs containing nitrogen donor atoms were found to exhibit good activity
against endonuclease (14), but only compounds containing borderline hard-soft Lewis basic
pyridine-like aryl nitrogen atoms as the coordinating atom were found to be active. Compounds
containing softer Lewis basic alkyl or aniline donor nitrogen atoms were found to have very little
inhibitory activity against endonuclease. For example, 23 (IC50 >200 µM, Table 3) was found to
be a very poor inhibitor of PAN. While these trends are not unexpected based on principles of
inorganic chemistry, such structural relationships are not often explored, optimized, or reported
during the development of metalloenzyme inhibitors, despite the clear effect of incorporating the
correct donor atom type on improving inhibition values.
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Conversion of the coordinating carboxylic acid to N-H or N-alkyl amides was found to
greatly impair inhibitory activity (24, 25) compared to the carboxylic acid (Table 3). This trend
was observed with all acid-to-amide conversions, regardless of the activity of the parent compound
(e.g., 18, 26). This transformation does not preclude metal coordination nor cause a
conformational change in mode of binding; however, it does greatly alter the electronics of the
coordinating moiety and the overall charge of the resulting coordination complex. This change to
the electronic character of the coordinating carboxylate is presumed to be the underlying cause for
the observed decrease in activity. This trend stands in contrast to that observed in HIV IN.
Structurally related hydroxypyridinone and hydroxypyrimidinone inhibitors of HIV IN with good
inhibitory activity have been reported that contain both carboxylic acid and amide metal-
coordinating groups, and the inclusion of a metal-coordinating 4-fluorobenzyl amide is a
characteristic motif found in many HIV IN inhibitors including the FDA-approved inhibitors
raltegravir and dolutegravir.1,30 However, it should be noted that this 4-fluorobenzyl amide group
also makes key interactions with active site nucleotides, and these favorable interactions may
compensate for any potential loss in binding enthalpy from less favorable metal binding.
Isosteric replacement is a common practice in medicinal chemistry to improve activity or
overcome pharmacokinetic liabilities of specific functional groups, such as carboxylic acids.38 The
effects of isosteric replacement in metalloenzyme inhibitors have not been widely studied.39 To
further probe the carboxylic acid moiety, a small series of hydroxypyrone and hydroxypyridinone
core scaffolds containing carboxylic acid isosteres were synthesized.40 Compound 27 maintains
an acidic tetrazole coordinating functional group and was found to have activity (IC50 = 37 nM)
similar to that of 1. However, structurally similar compounds that were less acidic, such as amides
24 and 25, or azoles 29 and 30 (Table 3), were all found to be between 10- and 100-fold less active.
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Interestingly, the structurally similar but overall basic N-hydroxy amidine 28 (IC50 = 19 µM) was
found to be substantially less active than all related isosteres, indicating a strong preference for
acidic coordinating ligands in the PAN metal active site.
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To illustrate the utility of a bioinorganic approach to SAR in the development of
metalloenzyme inhibitors, we applied the obtained SAR to the development of other, chemically
distinct PAN inhibitors. to highlight the applicability of a bioinorganic-guided inhibitor
development paradigm beyond single targets or single chemotypes. The obtained SAR suggests
that a tri-oxo, dianionic MBP that is aromatic and somewhat electron rich should be an ideal ligand
for the PAN metal centers. With this information, we were able to synthesize an unrelated MBP,
α-hydroxytropolone (32), that matches the proposed SAR, but is chemically distinct from 1 or 17.
Indeed, 32 was found to inhibit PAN with an exceptional IC50 value of 11 nM (Table 3).
Hydroxytropolone compounds 32 and 33 share similar ligand basicity as compound 17 despite
belonging to a distinct chemotype. The calculated pKa of the phenolic oxygens of 32 are ~10.1
and ~12.7,33 respectively, and the calculated pKa of the phenolic oxygens of 33 are ~9.8 and
~11.9,33 respectively, compared to the phenolic oxygen of 17 (pKa = ~11.2). The calculated pKa
values of these compounds compared to their inhibitory activity are consistent with the trends
observed with pyridinone carboxylate MPB inhibitors detailed in Table 2 and Figure 3, with
relatively basic and electron rich donor oxygen atoms that result in exceptional inhibitory activity.
As compared to compounds 1 or 17, compound 32 is more rigid, in terms of metal-
coordinating atoms; in compounds 1 or 17, the coordinating carboxylic acid moiety can rotate
whereas in 32, the three coordinating oxygen atoms are geometrically constrained in the proper
orientation for metal binding. The improved activity of 32 over 1 or 17 is likely due in part to the
added rigidity of this MBP’s pre-organized binding pose for the two active site metal ions. The
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overall ligand geometry (i.e., two 5-membered chelate rings), which is likely mimetic of the shape
and charge of the 5-membered phosphate ester transition state intermediate, may also explain the
observed increase in inhibitory activity. It is also worth noting that α-hydroxytropolone derivatives
have been identified as inhibitors of other dinuclear DNA/RNA processing metalloenzymes, such
as HIV IN and HIV RNaseH.41 However, simple α-hydroxytropolone derivatives were shown to
have 100- to 1000-fold less inhibitory activity against HIV IN or RNaseH than compound 32
displayed against PAN,41 highlighting that even similar metalloenzymes can display very specific
ligand preferences and that ligand selectivity can be engineered into MBPs. Further analysis of
several MBPs that share a similar chemotype with 32 indicate that the electronic, as opposed to
steric, aspects of this compound are responsible for its very high activity. N-Hydroxyphthalamide
(16), which possesses a similar donor triad, but is mono-anionic and more acidic, and pyrogallol
(31), which also possesses a similar donor triad, but is trianionic and more basic, are both
significantly less active than 32. Consistent with this SAR, gallic acid (9), was found to be much
less active than the analogous α-hydroxytropolone compound 33. The structure of 33 bound to
PAN (Figure S1) shows that the ligand binds to the active site as a bridging multidentate ligand,
with the central carbonyl oxygen acting as a bridging atom and both hydroxyl moieties replacing
the coordinating water molecules observed in the native structure. This binding mode is analogous
to compounds such as 1. The carboxylic acid moiety of 33 was not observed to engage in any
protein or water network interactions at the resolutions obtained, and the high inhibitory activity
is attributed to the optimized metal coordination. The identification of this novel inhibitor scaffold
demonstrates that metal-centric SAR is useful for predictive development of metalloenzyme
inhibitors and that similar analysis can be broadly applicable for any metalloenzyme inhibitor
development.
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32
33
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Finally, to validate the activity and selectivity of these highly active MBP inhibitors, cell-
5
6
based antiviral activity assays and metalloenzyme cross-inhibition screens were performed.42
A
7
8
9
subset of the MBP (compounds 1, 3, 17, 18, 22, 27, 32, and 33) were examined, which showed
little antiviral activity in cell-based assays (Supporting Information; Figures S2 and S3), likely due
to their high ionizability and low molecular weight.43,44 This result is not unexpected for these
MBP fragments, which are intended as core scaffolds in FBDD for fragment growth efforts to
achieve more complete inhibitors that can display cellular activity and drug-likeness. A cross-
inhibition screen of compound 1 was performed as a representative example of the series (Table
S1). Compound 1 was screened against eight other relevant metalloenzymes, specifically: HIV
IN, human carbonic anhydrase II, MMP-2, human glyoxalyase 1, NDM-1, HDAC-6, human
arginase 1, and human methionine aminopeptidase 1. At a concentration of 200 µM, no substantial
cross-inhibition was observed with 1, even against other dinuclear metalloenzymes, such as NDM-
1 (Zn2+), human arginase 1 (Mn2+), human methionine aminopeptidase 1 (Mn2+), and HIV
integrase (Mg2+).30 Despite the small size of these fragment molecules, which increases the
likelihood of off-target binding, optimized PAN inhibitors were found to display surprising good
selectivity. The strong preference that this chemotype displays for PAN endonuclease over other
metalloenzymes is evidence that target selectivity can be effectively engineered into MBP
fragments by optimization for the specific coordination environment of a target metalloenzyme.
This finding is corroborated by other work investigating metalloenzyme inhibitor selectivity,45,46
and stands in contrast to the widely held belief that MBP-containing compounds are intrinsically
promiscuous. The data herein clearly suggest that the dogma that metal-coordinating
metalloenzyme inhibitors may not be sufficiently selective over off-target metalloenzymes is not
well substantiated.
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To further illustrate that target selectivity can be engineered by MBP optimization,
isologues of 1 were screened against the related dinuclear metalloenzymes human arginase 1
(Arg1), human methionine aminopeptidase 1 (MetAP1), and NDM-1 (Table 4). A total of 25
structurally related MBPs were screened against these three enzymes (200 µM MBP
concentration) and PAN endonuclease (500 nM MBP concentration). Clear and distinct ligand
preferences for each metalloenzyme are readily apparent (Table 4), despite the high degree of
structural similarity between these fragments. This highlights the effects of varied ligand
electronics on selectivity profiles in different metalloenzyme systems. Additional analysis shows
that trends in donor atom preference remain, with compound 22 (which contains a sulfur donor
atom) showing the expected preference for the soft Zn2+ metal centers of NDM-1 over the hard
Mn2+ of Arg1 and MetAP1. Arg1 also clearly shows a preference for more highly basic MBP
ligands, such as 7 and 28, which stands in contrast to the preferences displayed by PAN, despite
both metalloenzymes employing dinuclear Mn2+ centers. These data further substantiate the
hypothesis that activity and selectivity can both be achieved through proper optimization of metal-
ligand interactions.
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17
18
19
20
21
22
23
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25
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32
33
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Table 4. Single concentration cross-inhibition studies of MBPs against PAN endonuclease and
three other dinuclear metalloenzymes. Final inhibitor concentration employed in assays is
indicated in the table header for each enzyme. Percent inhibition is listed and colored from yellow
to red with increasing inhibition values.
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15
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17
18
19
20
21
22
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Human MetAP1
PAN
Human
Arginase 1
(200 µM)
NDM-1
(200 µM)
Mn2+ isoform
Compound
Endonuclease
(200 µM)
(500 nM)
96
21
94
9
1
2
3
5
7
8
8
5
1
27
6
5
9
4
12
15
4
25
15
6
10
87
6
9
3
1
22
84
7
8
6
2
15
18
67
5
97
44
28
21
81
46
0
30
22
7
96
15
88
30
21
95
98
82
41
22
16
24
8
0
2
12
9
11
5
8
5
7
83
0
6
14
15
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
27
16
0
64
0
1
32
34
0
35
24
3
8
1
2
99
43
8
33
4
37
58
30
6
3
49
92
32
63
92
61
The results obtained in this SAR campaign indicate that both steric and electronic ligand
effects must be explored to optimize MBP inhibitory activity for a given metalloenzyme
microenvironment and that different metalloenzymes will exhibit different electronic and steric
preferences at their active site. It was also found that small chemical modifications that might
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otherwise be considered innocuous in conventional inhibitor design (e.g., the position of a non-
hydrogen bonding nitrogen atom in an aryl ring) were found to have a substantial effect on metal
coordination and hence the overall activity of a MBP. These findings suggests that a more
expanded view of which factors directly influence activity is absolutely necessary when
developing and optimizing inhibitors of metalloenzymes as opposed to other classes of targets.
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Conclusions
Based on the results described herein, SAR for metalloenzyme inhibitor development is
useful for inhibitors that directly interact with active site metal ions. As opposed to probing
primarily steric effects when preforming SAR analysis, the effects of modulating ligand electronics
on metal coordination must also be considered. Upon metal binding, an MBP forms a coordination
complex, which will display preferences for certain ligand interactions (like any inorganic
coordination complex).47-49 In the field of metalloenzyme inhibitor development, this scenario has
often been overlooked, due in part to a long-standing focus on perceived ‘privileged’ scaffolds
such as hydroxamic acids,4 but also because principles of inorganic chemistry are often not
considered during development. Nevertheless, these design principles can be readily applied to
the development of metalloenzyme inhibitors to afford selectivity and activity, even at the
fragment level (i.e., before hit-to-lead development). In this report, we have demonstrated that
consideration of SAR derived from MBP electronic effects can have substantial impact on
optimizing both activity and selectivity of metalloenzyme inhibitor fragments. As shown by this
MBP-FBDD approach, expanding the toolkit used by chemists working on metalloenzyme
inhibitor development will allow for improved progress toward highly active and selective
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inhibitors and may help to overcome the barriers that have limited the clinical success of this class
of drug target.
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Experimental Section:
General Experimental Details. All reagents and solvents were obtained from commercial
sources and used without further purification. All reactions, unless otherwise stated, were done
under nitrogen atmosphere. Reactions were monitored using glass-backed silica TLC plates
impregnated with a fluorescent indicator, absorbing at 254 nm. Silica gel column chromatography
was performed on a CombiFlash Rf Teledyne ISCO system using hexane, ethyl acetate, methylene
chloride, or methanol as eluent. Reverse phase column chromatography (C18 column) was
performed on the same instrument using 0.1% formic acid in methanol, acetonitrile, or water as
eluent. Separations were monitored by mass spectrometry via a Teledyne ISCO RF+ PurIon ESI-
1
MS or APCI-MS detector with 1 Da resolution. H and NMR spectra were obtained on a Varian
(400 MHz) spectrometers in the Department of Chemistry and Biochemistry at U.C. San Diego.
The purity of all compounds used in assays was determined to be ≥95% by HPLC-MS analysis.
Standard resolution MS was performed either at U.C. San Diego Molecular Mass Spectrometry
Facility or on the aforementioned Teledyne ISCO RF+ PurIon MS. HRMS analysis was performed
using an Agilent 6230 Accurate-Mass LC-TOFMS located at the U.C. San Diego Molecular Mass
Spectrometry Facility. Compounds 4-5, 7-14, 16, 19, 23, 31, and 34 were obtained from
commercial suppliers. Compounds 1,50 2,51 3,6 15,6 32,52 and 3353 were previously reported. X-
ray diffraction data for co-crystal structures were collected either on an in-house Bruker X8
Proteum diffractometer at 100 K, using a Bruker Microfocus Rotating Anode (MicroSar FR-592)
X-ray generator with a Bruker APEX II CCD detector at wavelength 1.54178 Ǻ (compound 2), or
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ALS 5.0.1 beamline using a single-crystal, cylindrically bent, Si(220) monochromator set to a
wavelength of 0.977 Ǻ with a Pilatus 6M 25 Hz detector (compounds 1, 33), or at ALS 5.0.2
beamline using a double-crystal Si(111) monochromator set to a wavelength of 1.00 Ǻ with a
Pilatus3 6M 25 Hz detector (compound 2). Statistics for data collection and refinement of the X-
ray crystal structures are listed in Table S2 and discussed in the supporting information.
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Synthesis of Compounds 1, 17, and 33. The chemical synthesis of the most active reported
pyrone, pyridinone, and α-hydroxytropolone MBP molecules is described in Scheme 1. Full
synthetic details of all other reported molecules is described in the Supporting Information.
O
O
H
N
H
N
OH
OH
OH
OBn
38
f
O
O
17
g
O
O
O
O
O
O
O
O
O
O
O
O
HO
Cl
OH
OBn
OH
OBn
e
OH
OH
c, d
b
f
a
OH
OH
OH
O
O
35
Allomaltol
36
34
37
1
h
O
O
O
O
O
HO
-OTf
O+
O
j
k
i
O
OH
OH
OH
O
O
O
O
O
HO
39
33
41
40
a
Scheme 1. Reagents and conditions: (a) thionyl chloride, CH2Cl2, rt, 6-8 h; (b) zinc dust, HCl,
water, 70 °C, 4-6 h; (c) NaOH, formaldehyde, water/MeOH, 0-20 ºC, 18 h; (d) benzyl bromide,
TBACl, 70 °C, 3 h; (e) NaCO3H, KI, TEMPO, TBACl, NaOCl, CH2Cl2/water, 5 °C, 2.5 h; (f)
5:5:1 HOAc/HCl/TFA, 40 °C, 18 h. (g) NH4OH, MeOH/water, 75 °C, 18-24 h; (h) methyl triflate,
DCM/chloroform, reflux, 18 h; (i) ethyl propiolate, TEA, chloroform, microwave 100 ºC, 25 min;
(j) BCl3, CH2Cl2, 0-20 °C, 30 min; (k) HBr/HOAc, neat, 95 °C, 6-8 h.
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Allomaltol. Kojic chloride (35) (10 g, 62.3 mmol) and water (75 mL) were stirred at 40 °C. Zinc
dust (8.14 g, 125 mmol) was added and the reaction was stirred vigorously and heated to 60 °C.
Concentrated HCl (15.4 mL, 187 mmol) was added dropwise via an addition funnel over ~30 min.
Effervescing hydrogen gas was observed. The slurry was left to stir for 2-3 h after HCl addition
was complete, at which time the excess zinc was removed from the pale green reaction by hot
filtration. The filtrate was adjusted to pH 1 and extracted with CH2Cl2 3x50 mL. The extracts
were dried over magnesium sulfate and concentrated under vacuum, and the product was isolated
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by silica chromatography in 85% yield. H NMR (400 MHz, Acetone-d6): δ 7.82 (s, 1H), 6.21 (s,
1H), 2.28 (d, J = 0.5 Hz, 3H). ESI-MS Experimental: 125.22. Calculated for [C6H5O3]-: 125.03
3-Hydroxy-6-methyl-4-oxo-4H-pyran-2-carboxylic acid (1). Compound 37 was taken up in a
5:5:1 mixture of concentrated HCl:HOAc:TFA and was stirred at room temperature for 48 h. After
this time, acids were removed under high vacuum, the resultant solids were co-evaporated several
times with methanol. The remaining solids were then purified by C18 chromatography eluting in
1
a water/methanol system to yield the target compound as a white solid in 93 % yield. H NMR
(400 MHz, DMSO-d6): δ 6.34 (s, 1H), 2.28 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 174.66
(s), 166.15 (s), 164.07 (s), 149.36 (s), 135.69 (s), 112.39 (s), 20.07 (d, J = 4.3 Hz). HR-ESI-MS
Experimental: 169.0141. Calculated for [C7H5O5]-: 169.0142. Δ = -0.6 ppm.
3-Hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxylic acid (17). Compound 38 was
taken up in a 5:5:1 mixture of concentrated HCl:HOAc:TFA and was stirred at room temperature
for 48 h. Acids were then removed under high vacuum, and the resultant solids were co-evaporated
several times with methanol. The remaining solids were then purified by C18 chromatography
eluting in a water/methanol system to yield the target compound as a white solid in 74 % yield.
1H NMR (400 MHz, DMSO-d6): δ 6.92 (s, 1H), 2.48 – 2.38 (m, 3H). 13C NMR (126 MHz,
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DMSO-d6) δ 174.66 (s), 166.15 (s), 164.07 (s), 149.36 (s), 135.69 (s), 112.39 (s), 20.07 (d, J = 4.3
Hz). HR-ESI-MS Experimental: 168.0302. Calculated for [C7H6NO4]-: 168.0302. Δ = 0.0 ppm.
5-Methyl-2,7-dihydroxytropolone-4-carboxylic acid (33). Compound 41 (200 mg, 0.84 mmol)
was taken up in a 1:1 mixture of acetic acid and 50% HBr (15 mL) and was heated to 95 °C for
6-8 h. After this time, solvent was removed under high vacuum and the residual solids were
purified by reverse phase chromatography to afford 33 in 56% yield. 1H NMR (400 MHz,
CD3OD): δ 7.62 (s, 1H), 7.44 (s, 1H), 2.53 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 170.69
(s), 169.02 (s), 160.40 (s), 157.70 (s), 138.08 (s), 132.49 (s), 124.22 (s), 119.76 (s), 25.11 (s). HR-
ESI-MS Experimental: 195.0298. Calculated for [C9H7O5]-: 195.0299. Δ = -0.5 ppm.
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Kojic chloride (35). To a rapidly stirring suspension of kojic acid (34) (10 g, 70.4 mmol) in DCM
(250 ml) at room temperature was added thionyl chloride (5.9 mL, 81 mmol), dropwise over the
course of 25 min. Throughout the addition, the suspension tends to clump. When clumping
occurred, the addition was paused to allow the solution to return to homogeneity. After 4 h of
stirring, the suspension was filtered, and the solids were recrystallized from ethanol to afford kojic
1
chloride as white needles in 91% yield. H NMR (400 MHz, DMSO-d6): δ 8.12 (s, 1H), 6.56 (s,
1H), 4.65 (s, 2H). ESI-MS Experimental: 159.14. Calculated for [C6H6ClO3]+: 159.99
3-(Benzyloxy)-2-(hydroxymethyl)-6-methyl-4H-pyran-4-one (36). Allomaltol (1.26 g, 10
mmol) was added to a solution of NaOH (0.44 g, 11 mmol) in water (60 mL) and the mixture was
cooled to 0 °C. Formaldehyde (37%, 0.83 mL, 11.1 mmol) was added dropwise maintaining the
solution temperature at ~0 °C. The mixture was stirred and allowed to warm to room temperature
after addition of formaldehyde. A solid began to appear after 1.5 h, and methanol (30 mL) was
added to solubilize the solids and the mixture was left stirring 18 h. The reaction was then heated
to 40 °C and benzyl bromide (1.3 ml, 10.9 mmol) and tetra-N-butylammonium chloride (TABCl)
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