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Structural Basis of Human Dimeric α-Amino-β-Carboxymuconate-ε-Semialdehyde Decarboxylase Inhibition With TES-1025

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Human α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) stands at a branch point of the de novo NAD ⁺ synthesis pathway and plays an important role in maintaining NAD ⁺ homeostasis. It has been recently identified as a novel therapeutic target for a wide range of diseases, including inflammatory, metabolic disorders, and aging. So far, in absence of potent and selective enzyme inhibitors, only a crystal structure of the complex of human dimeric ACMSD with pseudo-substrate dipicolinic acid has been resolved. In this study, we report the crystal structure of the complex of human dimeric ACMSD with TES-1025, the first nanomolar inhibitor of this target, which shows a binding conformation different from the previously published predicted binding mode obtained by docking experiments. The inhibitor has a K i value of 0.85 ± 0.22 nM and binds in the catalytic site, interacting with the Zn ²⁺ metal ion and with residues belonging to both chains of the dimer. The results provide new structural information about the mechanism of inhibition exerted by a novel class of compounds on the ACMSD enzyme, a novel therapeutic target for liver and kidney diseases.
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Structural Basis of Human Dimeric
α-Amino-β-Carboxymuconate-
ε-Semialdehyde Decarboxylase
Inhibition With TES-1025
Michele Cianci
1
*
, Nicola Giacchè
2
*
, Lucia Cialabrini
1
, Andrea Carotti
3
, Paride Liscio
2
,
Emiliano Rosatelli
2
, Francesca De Franco
2
, Massimiliano Gasparrini
1
, Janet Robertson
2
,
Adolfo Amici
4
, Nadia Raffaelli
1
and Roberto Pellicciari
2
1
Biochemistry and Structural Biology Laboratory, Department of Agricultural, Food and Environmental Sciences, Polytechnic
University of Marche, Ancona, Italy,
2
TES Pharma S.r.l, Perugia, Italy,
3
Department of Pharmaceutical Sciences, University of
Perugia, Perugia, Italy,
4
Department of Clinical Sciences DISCO, Section of Biochemistry, Polytechnic University of Marche,
Ancona, Italy
Human α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) stands at
a branch point of the de novo NAD
+
synthesis pathway and plays an important role in
maintaining NAD
+
homeostasis. It has been recently identied as a novel therapeutic target
for a wide range of diseases, including inammatory, metabolic disorders, and aging. So
far, in absence of potent and selective enzyme inhibitors, only a crystal structure of the
complex of human dimeric ACMSD with pseudo-substrate dipicolinic acid has been
resolved. In this study, we report the crystal structure of the complex of human dimeric
ACMSD with TES-1025, the rst nanomolar inhibitor of this target, which shows a binding
conformation different from the previously published predicted binding mode obtained by
docking experiments. The inhibitor has a K
i
value of 0.85 ± 0.22 nM and binds in the
catalytic site, interacting with the Zn
2+
metal ion and with residues belonging to both chains
of the dimer. The results provide new structural information about the mechanism of
inhibition exerted by a novel class of compounds on the ACMSD enzyme, a novel
therapeutic target for liver and kidney diseases.
Keywords: ACMSD, X-ray crystallography, TES-1025, decarboxylase, drug discovery, de novo NAD+ synthesis
INTRODUCTION
Human α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD, EC 4.1.1.45)
(Fukuoka et al., 2002) stands at a branch point of the de novo NAD
+
synthesis pathway, starting
from the essential amino acid tryptophan, and plays an important role in maintaining NAD
+
homeostasis. Given the benecial effects of replenished NAD
+
pools, there is an intense search for
strategies to increase intracellular NAD
+
by limiting NAD
+
consumption or increasing NAD
+
production (Katsyuba and Auwerx, 2017;Katsyuba et al., 2020). In this view, ACMSD inhibition is
emerging as a potent strategy to replenish NAD
+
levels by improving the coenzymes production
(Yoshino, 2019).
In detail, ACMSD catalyzes the decarboxylation of 2-amino 3-carboxymuconate 6-
semialdehyde (ACMS), an intermediate in the de novo NAD
+
synthesis pathway, to 2-
aminomuconate-6-semialdehyde (AMS), through a metal-mediated, O
2
-independent, non-
Edited by:
Gianluca Molla,
University of Insubria, Italy
Reviewed by:
Giorgio Giardina,
Sapienza University of Rome, Italy
John Tanner,
University of Missouri, United States
*Correspondence:
Michele Cianci
m.cianci@univpm.it
Nicola Giacchè
ngiacche@tespharma.com
These authors have contributed
equally to this work and share rst
authorship
Specialty section:
This article was submitted to
Structural Biology,
a section of the journal
Frontiers in Molecular Biosciences
Received: 13 December 2021
Accepted: 24 February 2022
Published: 07 April 2022
Citation:
Cianci M, Giacchè N, Cialabrini L,
Carotti A, Liscio P, Rosatelli E,
De Franco F, Gasparrini M,
Robertson J, Amici A, Raffaelli N and
Pellicciari R (2022) Structural Basis of
Human Dimeric α-Amino-β-
Carboxymuconate-ε-Semialdehyde
Decarboxylase Inhibition With TES-
1025.
Front. Mol. Biosci. 9:834700.
doi: 10.3389/fmolb.2022.834700
Frontiers in Molecular Biosciences | www.frontiersin.org April 2022 | Volume 9 | Article 8347001
ORIGINAL RESEARCH
published: 07 April 2022
doi: 10.3389/fmolb.2022.834700
oxidative decarboxylation reaction (Li et al., 2006), which
proceeds through a metal-bound hydroxide (Huo et al.,
2013,2015). AMS can either undergo spontaneous
cyclization of the pyridine ring to form picolinic acid (PIC)
or be oxidized to 2-aminomuconate, which is further
metabolized so that it can enter the tricarboxylic acid
(TCA) cycle. Otherwise ACMS, if not metabolized by
ACMSD, can cyclize spontaneously to quinolinic acid
(QUIN), which is further converted to the coenzyme NAD
+
(Figure 1). Because the cyclization of ACMS into QUIN is a
spontaneous reaction, the amount of ACMS undergoing this
conversion and therefore leading to the production of NAD
+
is
primarily determined by the activity of ACMSD (Fukuoka
et al., 2002). Thus, inhibition of ACMSD, which is primarily
and highly expressed in the liver and kidneys (Pucci et al.,
2007), would channel ACMS toward de novo NAD
+
biosynthesis, providing a novel way to replenish NAD
+
levels and re-establish NAD
+
homeostasis in pathological
conditions, particularly in liver- and kidney-associated
diseases.
ACMSD is catalytically inactive in the monomeric form and
active in the homodimeric form since the neighboring subunit
contributes with one of the two substrate-binding arginine
residues (Huo et al., 2013). Furthermore, recent experiments
using size-exclusion chromatography coupled with small-angle
X-ray scattering (SEC-SAXS) analysis have evidenced a protein
concentration-dependent activity of the enzyme, revealing that its
quaternary structure is in a dynamic equilibrium among the
monomeric, dimeric, and higher-order oligomeric states (Yang
et al., 2019).
The rst small molecule inhibitors of ACMSD to be identied
were the anti-tuberculosis drug pyrazinamide (Saito et al., 2000)
and the phthalate monoester, such as mono (2-ethylhexyl)
phthalate (MEHP) (Fukuwatari et al., 2004) with weak and
nonselective activity. Subsequent efforts in understanding the
mechanism of recognition and binding of ligands to the active site
of the human enzyme resulted in the release of the rst co-crystal
complex of ACMSD (PDB code 2WM1) with the inhibitor 1,3-
dihydroxyacetonephosphate (DHAP, 1)(Garavaglia et al., 2009)
(Figure 2).
Subsequently, the structure of the human recombinant
ACMSD complex with the competitive inhibitor pyridine-2,6-
dicarboxylic acid (PDC, 2) (PDB code 4IH3) was published,
rening the previous ndings (Huo et al., 2015)(Figure 2).
Recently, the salycilic-derivative, nonsteroidal anti-
inammatory drug (NSAID) and FDA-approved drug
diunisal (3) was identied to inhibit ACMSD with an IC
50
of
13.5 µM, and its complex structure in the Pseudomonas
uorescens ACMSD has been resolved and published (PDB
code 7K12) (Yang et al., 2021)(Figure 2). A chronological
summary of all ACMSD structures that have been resolved
and published is reported in Table 1.
TES-1025 (CAS. 1883602-21-8, 2-[3-[(5-cyano-6-oxo-4-
thiophen-2-yl-1H-pyrimidin-2-yl) sulfanylmethyl]phenyl]
acetic acid, PubChem CID: 137142885) is a potent and
selective human α-amino-β-carboxymuconate-ε-
semialdehyde decarboxylase (ACMSD) inhibitor with an
IC
50
of 13 nM. The compound has been selected as the rst
low-nanomolar inhibitor of human ACMSD with a suitable
overall balance of good physicochemical properties and
in vitro safety prole, identied after the discovery,
synthesis, and biological evaluation of a series of 2-
thiopyrimidone-5-carbonitriles as the rst class of small-
molecule drug-like ACMSD inhibitors. Proof-of-concept
studies for the rst time revealed that the inhibition of
ACMSD by TES-1025 led to the modulation of intracellular
NAD
+
levels with consequent in vivo enhancement of de novo
NAD
+
biosynthesis via ACMSD target engagement (Pellicciari
et al., 2018).
On the basis of the discovery of TES-1025 and related analogs,
we have established valuable tools for a better understanding of
the therapeutic applications of ACMSD inhibitors for disorders
such as mitochondrial dysfunctions and metabolic and renal
diseases, associated with the dysregulation or reduced NAD
+
levels. In vivo efcacy data obtained with TES-1025 in preclinical
murine models of liver and kidney diseases (Katsyuba et al., 2018)
suggested ACMSD as a promising novel therapeutic target to
improve health in pathological settings such as that of acute
kidney injury (AKI) (Kellum and Prowle, 2018;Manrique-
Caballero et al., 2021).
In this study, we report the crystal structure of the complex of
human dimeric ACMSD with TES-1025 (4), (Figure 2) the rst
potent and selective ACMSD inhibitor.
MATERIALS AND METHODS
Expression and Purication of hACMSD
Expression of the recombinant protein was achieved as described
previously (Pucci et al., 2007). Purication was performed as
described previously (Garavaglia et al., 2009), with some
modications. In detail, a pellet of Pichia pastoris cells
expressing the enzyme and derived from 400 ml culture was
resuspended in 80 ml of lysis buffer consisting of 10 mM
potassium phosphate, pH 7.0, 50 mM NaCl, 1 mM DTT,
5 mM 2-mercaptoethanol, and 1 mM PMSF and aprotinin,
leupeptin, chymostatin, pepstatin, and antipain at 0.002 mg/ml
each. After disruption by two cycles of French press (SML-
Aminco, Urbana, IL, United States) at 1,000 psi, the
suspension was centrifuged at 40,000 × g, for 30 min at 4°C.
The supernatant was made to 10 mg/ml by dilution with lysis
buffer, and streptomycin sulfate was added dropwise at a nal
concentration of 1%. After 30 min stirring on ice, the sample was
centrifuged at 20,000 × g for 10 min at 4°C, and the supernatant
was applied to 4 ml TALON Superow resin (Cytiva,
United States) equilibrated with 50 mM potassium phosphate
pH 7.4 and 50 mM NaCl (buffer A). After washing with 20 mM
imidazole in buffer A, elution was performed with 350 mM
imidazole in buffer A.
Kinetics Studies
The inhibitory effect of TES-1025 on ACMSD activity was
determined by the coupled spectrophotometric assay (Pucci
et al., 2007). Briey, pre-assay mixtures consisting of different
Frontiers in Molecular Biosciences | www.frontiersin.org April 2022 | Volume 9 | Article 8347002
Cianci et al. X-Ray Structure of hACMSD With TES-1025
concentrations of hydroxyanthranilic acid (from 5 to 20 µM) and
an excess amount of recombinant R. metallidurans
hydroxyanthranilic acid dioxygenase, in 50 mM 4-
morpholinepropanesulfonic acid, pH 6.5 and 100 mM
ammonium iron sulfate, were incubated at 37°C, with
monitoring ACMS formation at 360 nm. After the reaction
was complete, 30 nM ACMSD and TES-1025 (from 0.5 to
40 nM) were added. The enzyme activity was calculated by the
initial rate of the absorbance decrease subtracted from that of a
control mixture in the absence of ACMSD. The K
i
value was
calculated from the initial velocity data using the Dixon equation
for tightly bound competitive inhibitor (Segel, 1993):
[It]Ki1+[S]
KmV0
Vi
+[Et]p1Vi
V0,(1)
where V
i
is the initial velocity at a given [S] in the presence of the
inhibitor, V
o
is the initial velocity at the same [S] in the absence of
the inhibitor, K
m
is the MichaelisMenten constant for the
substrate, [I
t
] and [E
t
] are the total amount of the inhibitor
and enzyme, respectively, and K
i
is the apparent K
i
value. The
intercept on the y-axis of the replot of apparent K
i
values against
[S] gives the K
i
value.
Protein Crystallization and Data Collection
TES-1025 was developed by TES Pharma as reported by
Pellicciari et al., (2018). For the crystallization trials, the
TALON pool containing the puried protein was diluted ten-
fold with 50 mM potassium phosphate and 5 mM 2-
mercaptoethanol and then concentrated by ultraltration,
using an Amicon Ultra Centrifugal Filter (cutoff 10 kDa,
Merck, Millipore), at 4°C, to a nal protein concentration of
4.5 mg/ml. For all crystallization trials, the sitting drop vapor
diffusion method was applied. The concentrated enzyme was
incubated at the 1:10 molar ratio with a stock solution of 50 mM
TES-1025 in dimethyl sulfoxide, for 1 hour at room temperature,
and 1.25 μL of proteinligand solution was mixed with an equal
volume of reservoir solution, and it was equilibrated against
100 µL of the reservoir solution. The best crystals were
obtained in reservoir solution containing 100 mM
Na(CH
3
COO), pH 5.7, 22% (w/v) PEG 4000, and they grew to
their nal size in few weeks at 18°C.
FIGURE 1 | Role of ACMSD as the branching point in the kynurenine pathway leading to the de novo NAD
+
biosynthesis. ACMSD catalyzes the decarboxylation of
the ACMS intermediate to AMS metabolite toward total oxidation in the tricarboxylic acid (TCA) cycle. Inhibition by TES-1025 favors the ux from tryptophan through
ACMS toward quinolinic acid (QUIN) and increases NAD
+
production.
FIGURE 2 | Molecular structures of published co-crystalized ACMSD
inhibitors.*: diunisal has been resolved in P. uorescens ACMSD.
Frontiers in Molecular Biosciences | www.frontiersin.org April 2022 | Volume 9 | Article 8347003
Cianci et al. X-Ray Structure of hACMSD With TES-1025
Crystals were transported to the synchrotron in plates,
mounted in nylon loops, and ash-frozen directly at 100 K in
a nitrogen gas stream. Diffraction data were collected at the
European Synchrotron Radiation Facility (ESRF, Grenoble,
France) at beamline ID30A-3 (MASSIF-3) (von Stetten et al.,
2020).
Structure Determination, Renement, and
Analysis
The diffraction data were integrated and scaled with the XDS/
XSCALE program package (Kabsch, 2010). The crystals belong to
the space group P2
1
2
1
2, with unit cell a = 153.3 Å, b = 92.5 Å, and
c = 103.9 Å. Starting phases for solving the crystal structure were
obtained with molecular replacement using PHASER (Adams
et al., 2002) with monomer A of the hACMSD structure as a
starting model reported by Huo et al. (2015) (PDB code 4IH3)
after atom randomization to avoid any bias as a search model.
Automated model building was accomplished by the PHENIX
(Adams et al., 2002) suite, followed by manual tting of the side
chains and solvent molecules into electron density maps
performed using COOT (Emsley and Cowtan, 2004) and
PHENIX suite (Adams et al., 2002), while monitoring R
work
,
R
free
, and Ramachandran plot with PROCHECK (Laskowski
et al., 1993) and related geometrical parameters. The Fourier
difference electron density OMIT maps at 3σwere inspected to
verify the presence of TES-1025. The models were checked with
the PDB REDO web server (Joosten et al., 2014). Model
coordinates and structure factors of the X-ray crystal structure
of ACMSD co-crystallized in the presence of TES-1025 were
deposited in the Protein Data Bank (PDB) under the accession
code: 7PWY. Data collection, processing, and nal renement
statistics are given in Table 2. Ligand interaction diagram is
generated by the tool of Maestro of the Schrodinger suite 20171
(https://www.schrodinger.com). The images produced in this
article were generated using CCP4mg (McNicholas et al.,
2011) and PyMOL software (https://www.pymol.org).
RESULTS
The human ACMSD crystal structure in complex with TES-
1025 at 2.5 Å resolution was rened to nal R
work
and R
free
values of 0.210 and 0.252, respectively. The Ramachandran plot
shows more than 96% of the residues in the favored regions and
4% in the allowed regions (Table 2). The molecular
replacement solution of the hACMSD structure in complex
with TES-1025 comprised four monomers in the asymmetric
unit to form two homodimers. The average RMSD, calculated
with SUPERPOSE (Winn et al., 2011) over 320 residues of each
monomer, against the starting model, is 0.45 Å, to conrm that
theoverallfoldoftheenzymeismaintained.Inbrief,hACMSD
shows a molecular architecture comprising of 12 α-helices,
11 β-strands, and the connecting loops. Residues 1448 form
the small insertion domain that comprises a short α-helix and a
three-stranded anti-parallel β-sheet; the remaining protein
residues form a (α/β)
8
barrel domain and a two-α-helices
C-terminal extension.
TABLE 1 | Summary of all ACMSD X-ray structures deposited in the Protein Data Bank (PDB) (order for date of release).
PDB
code
Date of
release
Resolution
(Å)
Organism Conformation Mutation Metal
cofactor
Ligand References
2HBX 2006-09-19 2.50 Pseudomonas
uorescens
Dimer No Co
2+
Martynowski et al.
(2006)
2HBV 2006-09-19 1.65 Pseudomonas
uorescens
Dimer No Mg
2+
,Zn
2+
Martynowski et al.
(2006)
2WM1 2009-11-03 2.01 Homo sapiens Monomer (but dimer in the
lattice)
No Zn
2+
DHAP Garavaglia et al.
(2009)
4EPK 2012-08-22 2.60 Pseudomonas
uorescens
Dimer H228G Mg
2+
,Zn
2+
Huo et al. (2012)
4ERI 2012-08-22 2.00 Pseudomonas
uorescens
Dimer H228Y Mg
2+
,Zn
2+
Huo et al. (2012)
4ERA 2012-08-22 2.40 Pseudomonas
uorescens
Dimer H228Y Co
2+
Huo et al. (2012)
4ERG 2012-08-22 2.79 Pseudomonas
uorescens
Dimer Fe
3+
Huo et al. (2012)
4IGM 2014-05-07 2.39 Homo sapiens Dimer No Zn
2+
No references
4IGN 2014-05-07 2.33 Homo sapiens Dimer R47A Zn
2+
Huo et al. (2015)
4IH3 2014-05-21 2.49 Homo sapiens Dimer No Zn
2+
PDC Huo et al. (2015)
4OFC 2014-11-19 1.99 Homo sapiens Dimer No Zn
2+
Huo et al. (2015)
6MGS 2019-06-19 3.13 Pseudomonas
uorescens
Dimer No Co
2+
Yang et al. (2019)
6MGT 2019-06-19 2.77 Pseudomonas
uorescens
Dimer H110A Co
2+
Yang et al. (2019)
7K12 2021-01-13 2.17 Pseudomonas
uorescens
Dimer No Zn
2+
Diunisal Yang et al. (2021)
7K13 2021-01-13 1.83 Pseudomonas
uorescens
Dimer No Zn
2+
Diunisal-
derivative
Yang et al. (2021)
Frontiers in Molecular Biosciences | www.frontiersin.org April 2022 | Volume 9 | Article 8347004
Cianci et al. X-Ray Structure of hACMSD With TES-1025
A total of two homodimers are observed in the asymmetric unit.
Analysis of interface area, solvation energy gain upon interface
formation, and the total binding energy of the interface, calculated
using PISA (Krissinel and Henrick, 2007), conrms homodimers
A, B and C, D as biological units. hACMSD homodimers have been
reported previously (Garavaglia et al., 2009;Huo et al., 2015)
(Figure 3A). In homodimer AB, there are no disordered
regions, while in homodimer CD there are disordered regions
namely in chain C between residues 3037, 178185, and 242253
and in chain D between residues 242253.
Similar to previously published X-ray structures (Garavaglia
et al., 2009;Huo et al., 2015), the active site of hACMSD is located
at the C-terminal opening of the β-barrel and characterized by a
Zn
2+
metal ion coordinated with a distorted trigonal bipyramid
geometry, by residues His6, His8, His174, His 224, the moiety of
Asp291, and a conserved water molecule. A water molecule bridges
the Zn
2+
metal ion with the guanidino moiety of Arg235 belonging
to the neighboring subunit (chain B) of the hACMSD dimer.
The presence and positioning of the TES-1025 molecules was
indicated by the Fourier difference 2F
o
-F
c
maps at 1σ, the Fourier
difference F
o
-F
c
OMIT maps at 3σlevel, and the polder maps at
4σlevel in each active site of hACMSD homodimer AB
(Figure 3). In homodimer CD, no electron density was
observed that could be attributed to a TES-1025 molecule. The
molecule TES-1025 located in monomer A was rened with full
occupancy, and the molecule located in monomer B was rened
with a partial occupancy of 0.80. The correlation coefcients for
the polder map (Liebschner et al., 2017), calculated by omitting
both TES-1025 molecules were CC (1, 3) = 0.89, i.e., larger than
CC (1, 2) = 0.54 and CC (2, 3) = 0.56. When omitting only TES-
1025 molecule in chain A, we obtained CC (1, 3) = 0.91, i.e., larger
than CC (1, 2) = 0.62 and CC (2, 3) = 0.55. When omitting only
TABLE 2 | Data collection and renement statistics.
Wavelength (Å) 0.967
Space group P 2
1
2
1
2
Cell parameters (a, b, and c, Å) 153.4, 92.6, 103.9
Resolution range (Å) 45.892.50 (2.502.58)
a
Total reections 395,775 (30,276)
a
Unique reections 51,945 (4,423)
a
Redundancy 7.6 (6.8)
a
Completeness (%) 99.9 (100.0)
a
Mean I/sigma(I) 11.5 (1.6)
a
R
mergeb
0.11 (11.1)
a
R
pimc
0.063 (0.688)
a
CC1/2 0.999 (0.766)
a
CC* 1.00 (0.926)
a
Reections used in renement 51,611 (5,113)
a
Reections used for R
free
2,565 (292)
a
Wilson B-factor (Å
2
) 52.35
R
workd
0.210 (0.310)
a
R
freed
0.252 (0.358)
a
Total no. of atoms
d
10,647
Macromolecules 10,296
Ligands 62
Water molecules 289
Protein residues 1,289
RMSD
d
Bond length (Å) 0.003
Angles (°) 0.55
Ramachandran
d
Favored (%) 96.54
Allowed (%) 3.46
Outliers (%) 0.00
Average B-factor
d
68.28
Macromolecules 68.48
Ligands 73.64
Solvent 59.83
a
Values in the highest resolution shell.
b
Rmerge
hkl
j
|IjI|/
hkl
j
Ij, where I is the intensity of a reection, and I is the mean
intensity of all symmetry-related reections j.
c
Rp.i.m.
hkl
{[1/(N1)]1/2
j
|IjI|}/
hkl
j
Ij, where I is the intensity of a reection, and I is
the mean intensity of all symmetry-related reections j, and N is the multiplicity (Weiss,
2001).
d
Calculated with PHENIX suite (Adams et al., 2002), R
free
is calculated using 5% of the
total reections that were randomly selected and excluded from renement.
FIGURE 3 | A) Ribbon diagram of the dimeric structure of human
ACMSD in complex with TES-1025: dark blue, monomer A; light blue,
monomer B. Fourier difference maps of TES-1025 molecules bound to
monomers A and B, respectively: (B,C) 2F
o
-F
c
map (light blue for the
protein and blue for the ligand), contoured at 1σlevel; (D,E) F
o
-F
c
omit map
(green for the ligand) contoured at 3σlevel; (F,G) F
o
-F
c
polder map (green for
the ligand) contoured at 4σlevel.
Frontiers in Molecular Biosciences | www.frontiersin.org April 2022 | Volume 9 | Article 8347005
Cianci et al. X-Ray Structure of hACMSD With TES-1025
TES-1025 molecule in chain B, we obtained CC (1, 3) = 0.84,
i.e., larger than CC (1, 2) = 0.59 and CC (2,3) = 0.57. Since the CC
(1, 3) is larger than CC (1, 2) and CC (2, 3), then the density
observed corresponds to the atomic features of the TES-1025
molecules.
The active site of each subunit binds one TES-1025 molecule
with the same set of interactions. The carboxylic moiety of the
ligand coordinates the Zn
2+
metal ion (Zn···O distance is 2.5 Å)
and establishes an H-bond with the indole moiety of Trp191
residue (the H···O distance is 2.6 Å, and the N-H···O angle is
135.8°) while interacts with Asp291 through a water molecule.
The pyrimidine ring interacts through the carbonyl group with
Arg243 (the H···O distance is 2.5 Å, and the N-H···O angle is
143.6°), belonging to the neighboring subunit (chain B) of the
functional dimer of ACMSD, while a second H-bond is
established with the catalytic residue Trp188 (the H···O
distance is 2.7 Å, and the N-H···O angle is 132.5°). The
negatively charged nitrogen of the TES-1025 pyrimidine ring
engages a chargecharge interaction with the positively charged
nitrogen of the Arg47 (the N···N distance is 3.6 Å). Also, further
interactions with solvent water molecules are dened. The 2-
thiophene ring ts into a hydrophobic cavity generated by
Trp176, Phe46, Met180, and Trp191; this latter residue also
makes PiPi stacking with the phenyl ring of TES-1025.
(Figure 4).
The kinetic analysis of the inhibition exerted by TES-1025 on
ACMSD was performed by assaying the enzyme activity in the
presence of varying concentrations of the inhibitor and substrate.
The IC
50
value of the inhibitor at 10 µM substrate concentration
is reported to be about 13 nM (Pellicciari et al., 2018). This value
is very close to the concentration of ACMSD which is used in the
activity assay, implying that a signicant portion of the total
inhibitor in the assay mixture is enzyme-bound. Therefore, to
investigate the inhibition kinetics, the Dixon method (Segel,
1993) was used as described in Materials and Methods. The
best t with the experimental data was obtained by using the
Dixon Equation 1 for a tightly bound competitive inhibitor
(Figure 5). A K
i
value of 0.85 ± 0.22 nM was calculated (inset
in Figure 5).
DISCUSSION
The structural basis of human dimeric α-amino-β-
carboxymuconate-ε-semialdehyde decarboxylase inhibition
with TES-1025 is severalfold. Overall, the X-ray structure of
the ACMSDTES-1025 complex described in this study
experimentally conrmed the competitive inhibition mode
displayed by the ligand, in which the catalytic Arg47 and
the Zn
2+
metal ion together with Trp191 and chain B are
FIGURE 4 | Ligand interaction diagram of TES-1025 in the ACMSD active site of monomer A, reporting the ligandprotein type of inte ractions and involved residues
(diagram calculated and generated by Maestro, Schrodinger suite).
Frontiers in Molecular Biosciences | www.frontiersin.org April 2022 | Volume 9 | Article 8347006
Cianci et al. X-Ray Structure of hACMSD With TES-1025
engaged in unique specic interactions with the meta
carboxylate group and the pyrimidine ring of the ligand.
These two interactions lock the head and the tail of the
ligand, respectively. Moreover, the pyrimidine ring strongly
interacts with the pocket residues side chains through a
network of hydrogen bonds mediated by water molecules,
thus stabilizing the central core of the ligand. This strong
network of interactions generated by TES-1025 ensured the
low nanomolar potency inhibition of the enzyme and
conrmed the previously published data of structure and
activity relationship (Pellicciarietal.,2018). The kinetic
analysis conrmed that TES-1025 is a competitive inhibitor
with a Ki value in the low nanomolar range.
In the article by Pellicciari et al. (2018), the investigation of the
induced-t docking pose of TES-1025 into the hACMSD catalytic
site coming from the 4IH3 X-ray (Huo et al., 2015) was presented.
The results indicated that the majority of the binding afnity was
due to the ionic interaction between the m-carboxy group of the
FIGURE 5 | ACMSD activity as a function of inhibitor concentration in the presence of xed concentrations of the substrate traced with diamonds (5 μM), circles
(10 μM), squares (15 μM), and triangles (20 μM). In the inset, the replot of apparent K
i
versus [S] is shown.
FIGURE 6 | TES-1025-binding (pink sticks) modes in the docking model (A), TES-1025 (green sticks) in the crystal structure (B), and superposition of the two
ligand poses (C), together with the protein ribbons and Zn
2+
ions. ACMSD chains A and B are shown in yellow and pink ribbons for the induced t result, while are in blue
and cyan ribbons for the X-ray image, respectively. The Zn
2+
metal ion is displayed as a gray ball. The relevant residues of the binding site are labeled and shown as sticks.
Hydrogen bond and salt bridge interactions are indicated with orange dashed lines.
Frontiers in Molecular Biosciences | www.frontiersin.org April 2022 | Volume 9 | Article 8347007
Cianci et al. X-Ray Structure of hACMSD With TES-1025
ligand with the Zn
2+
metal ion and the catalytic residues Arg47
and Trp191. Meanwhile, the pyrimidine ring was strongly
anchored to Lys41 and Lys44 residues by hydrogen bond and
ionic interactions. Furthermore, additional hydrophobic contacts
were also established between the thiophene and Met180. In this
view, the current X-ray structure of ACMSD in complex with
TES-1025 reports a different ligand disposition with respect to the
putative binding mode predicted by docking computation (ligand
RMSD calculated on non-hydrogen atoms is 6.42 Å when using
the protein backbone for the alignment). Indeed, upon TES-1025
binding, the side chain of Arg47, as resolved in the crystal
structure, is oriented to the middle of the pocket driven by the
interaction with the acidic oxygen of the pyrimidine ring of the
ligand. It is worth noting that this Arg47 orientation was observed
neither in the 4IH3 complex used for the in silico studies nor in
other released ACMSD X-ray structures (pdb codes: 2WM1,
4IGM, 4IGN, and 4OFC) (Garavaglia et al., 2009;Huo et al.,
2015). Moreover, the carbonyl moiety of the pyrimidine ring of
TES-1025 engages an H-bond with Arg243, belonging to the
neighboring subunit (chain B) of the functional dimer of
ACMSD. The crystallographic disposition showed a 180°
rotation of the pyrimidine ring around the Csp
2
-S bond that
completely abolishes the interactions present in the docking pose
with Lys44 and Lys41 (Figure 6).
The previous detailed works of the Aimin Liu lab (Huo et al.,
2013;Huo et al., 2015)conrmed the metal ion (Zn
2+
) dependence
of the human ACMSD for its catalytic activity and the active role of
Arg47 and Arg235 of the chain B for the interaction of the natural
substrate ACMS in the homodimeric functional complex. TES-1025
was able to interact with all the essential catalytic features of the site,
with only the substitution of Arg235 with Arg243, stabilizing a
homodimeric inactive complex and conrming the high afnity and
potency of the ligand. Moreover, the preferential recognition and
binding of the pyrimidine moiety of TES-1025 in ACMSD
supported the subfamilial similarities with the related enzyme 5-
carboxyl-uracil decarboxylase (IDCase) (Huo et al., 2015).
The asymmetric unit consists of two homodimers, namely AB
and CD. In AB, two TES-1025 molecules were observed, while in
CD no electron density was observed that could be attributed to a
TES-1025 molecule. The two homodimers display high dynamics
of the different chains too. Indeed, in homodimer AB, there are
no disordered loop regions, while in homodimer CD there are
disordered regions. Considering that upon ligand binding,
Arg243 interacts with the pyrimidine ring of TES-1025, and
the presence of the ligand is reected in the folding of the
residue region 240253 to an α-helix structure.
In summary, the determination of the crystal structure of the
human ACMSD homodimer with the rst nanomolar and
selective inhibitor TES-1025 reveals unforeseen interactions of
the functional groups of the small molecule with the catalytic side
chains and the metal ion within the active site, elucidating the
principles of its potent inhibitory mechanism. These results
further validate the selectivity of TES-1025 for the enzyme and
consolidate the knowledge about ACMSD as a promising
therapeutic target (Katsyuba et al., 2018;Kellum and Prowle,
2018;Kellum et al., 2020;Manrique-Caballero et al., 2021) for the
recovery of the de novo NAD
+
biosynthesis pathway and the
maintaining of NAD
+
homeostasis impaired in hepatic (Zhou
et al., 2016) and renal diseases (Poyan Mehr et al., 2018).
DATA AVAILABILITY STATEMENT
Structure factors and coordinates have been deposited in the
Protein Data Bank with the PDB code: 7PWY.
AUTHOR CONTRIBUTIONS
NG designed the research and wrote the manuscript. MC
designed the crystallographic experiments, collected and
analyzed data, and wrote the manuscript. AA, LC, FD, and
MG performed protein expression and purication, prepared
crystallographic samples, and performed kinetics experiments.
AC performed in silico ligand interaction analysis and
calculations and wrote the manuscript. PL and ER prepared
TES-1025. JR and AA edited the manuscript. NR and RP
designed the research and edited the manuscript.
ACKNOWLEDGMENTS
We thank the research staff of ESRF (Grenoble, France) for the
technical support. The synchrotron MX data were collected at
ESRF (Grenoble, France) under the beam time award MX-
1949.
REFERENCES
Adams, P. D., Grosse-Kunstleve, R. W., Hung, L.-W., Ioerger, T. R., McCoy, A. J.,
Moriarty, N. W., et al. (2002). PHENIX: Building New Software for Automated
Crystallographic Structure Determination. Acta Crystallogr. D Biol. Cryst. 58,
19481954. doi:10.1107/S0907444902016657
Emsley, P., and Cowtan, K. (2004). Coot: Model-Building Tools for Molecular
Graphics. Acta Crystallogr. D Biol. Cryst. 60, 21262132. doi:10.1107/
S0907444904019158
Fukuoka, S.-I., Ishiguro, K., Yanagihara, K., Tanabe, A., Egashira, Y., Sanada, H.,
et al. (2002). Identication and Expression of a cDNA Encoding Human α-
Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase (ACMSD). J. Biol.
Chem. 277, 3516235167. doi:10.1074/jbc.M200819200
Fukuwatari, T., Ohsaki, S., Fukuoka, S. I., Sasaki, R., and Shibata, K. (2004).
Phthalate Esters Enhance Quinolinate Production by Inhibiting α-Amino-
β-Carboxymuconate-ε-Semialdehyde Decarboxylase (ACMSD), a Key
Enzyme of the Tryptophan Pathway. Toxicol. Sci. 81, 302308. doi:10.
1093/toxsci/kfh204
Garavaglia, S., Perozzi, S., Galeazzi, L., Raffaelli, N., and Rizzi, M. (2009). The
crystal Structure of Human α-amino-β-carboxymuconate-ε-semialdehyde
Decarboxylase in Complex with 1,3-dihydroxyacetonephosphate Suggests a
Regulatory Link between NAD Synthesis and Glycolysis. FEBS J. 276,
66156623. doi:10.1111/j.1742-4658.2009.07372.x
Huo, L., Davis, I., Chen, L., and Liu, A. (2013). The Power of Two. J. Biol. Chem.
288, 3086230871. doi:10.1074/jbc.M113.496869
Huo, L., Fielding, A. J., Chen, Y., Li, T., Iwaki, H., Hosler, J. P., et al. (2012).
Evidence for a Dual Role of an Active Site Histidine in α-Amino-β-
Frontiers in Molecular Biosciences | www.frontiersin.org April 2022 | Volume 9 | Article 8347008
Cianci et al. X-Ray Structure of hACMSD With TES-1025
carboxymuconate-ε-semialdehyde Decarboxylase. Biochemistry 51, 58115821.
doi:10.1021/bi300635b
Huo, L., Liu, F., Iwaki, H., Li, T., Hasegawa, Y., and Liu, A. (2015). Human α-
amino-β-carboxymuconate-ε-semialdehyde Decarboxylase (ACMSD): A
Structural and Mechanistic Unveiling. Proteins 83, 178187. doi:10.1002/
prot.24722
Joosten, R. P., Long, F., Murshudov, G. N., and Perrakis, A. (2014). The
PDB_REDO Server for Macromolecular Structure Model Optimization. Int.
Union Crystallogr. J. 1, 213220. doi:10.1107/S2052252514009324
Kabsch, W. (2010). Xds. Acta Crystallogr. D Biol. Cryst. 66, 125132. doi:10.1107/
S0907444909047337
Katsyuba, E., and Auwerx, J. (2017). Modulating NAD + Metabolism, from Bench
to Bedside. EMBO J. 36, 26702683. doi:10.15252/embj.201797135
Katsyuba, E., Mottis, A., Zietak, M., De Franco, F., van der Velpen, V.,
Gariani, K., et al. (2018). De Novo NAD+ Synthesis Enhances
Mitochondrial Function and Improves Health. Nature 563, 354359.
doi:10.1038/s41586-018-0645-6
Katsyuba, E., Romani, M., Hofer, D., and Auwerx, J. (2020). NAD+ Homeostasis in
Health and Disease. Nat. Metab. 2, 931. doi:10.1038/s42255-019-0161-5
Kellum, J. A., and Prowle, J. R. (2018). Paradigms of Acute Kidney Injury in the
Intensive Care Setting. Nat. Rev. Nephrol. 14, 217230. doi:10.1038/nrneph.
2017.184
Kellum, J. A., van Till, J. W. O., and Mulligan, G. (2020). Targeting Acute Kidney
Injury in COVID-19. Nephrol. Dial. Transpl. 35, 16521662. doi:10.1093/ndt/
gfaa231
Krissinel, E., and Henrick, K. (2007). Inference of Macromolecular
Assemblies from Crystalline State. J. Mol. Biol. 372, 774797. doi:10.
1016/j.jmb.2007.05.022
Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993).
PROCHECK: a Program to Check the Stereochemical Quality of Protein
Structures. J. Appl. Cryst. 26, 283291. doi:10.1107/S0021889892009944
Li, T., Iwaki, H., Fu, R., Hasegawa, Y., Zhang, H., and Liu, A. (2006). α-Amino-β-
carboxymuconic-ε-semialdehyde Decarboxylase (ACMSD) Is a New Member
of the Amidohydrolase Superfamily. Biochemistry 45, 66286634. doi:10.1021/
bi060108c
Liebschner, D., Afonine, P. V., Moriarty, N. W., Poon, B. K., Sobolev, O. V.,
Terwilliger, T. C., et al. (2017). Polder Maps: Improving OMIT Maps by
Excluding Bulk Solvent. Acta Cryst. Sect D Struct. Biol. 73, 148157. doi:10.
1107/S2059798316018210
Manrique-Caballero, C. L., Kellum, J. A., Gómez, H., De Franco, F., Giacchè, N.,
and Pellicciari, R. (2021). Innovations and Emerging Therapies to Combat
Renal Cell Damage: NAD+ as a Drug Target. Antioxid. Redox Signaling 35,
14491466. doi:10.1089/ars.2020.8066
Martynowski, D., Eyobo, Y., Li, T., Yang, K., Liu, A., and Zhang, H. (2006). Crystal
Structure of α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase:
Insight into the Active Site and Catalytic Mechanism of a Novel
Decarboxylation Reaction,. Biochemistry 45, 1041210421. doi:10.1021/
bi060903q
McNicholas, S., Potterton, E., Wilson, K. S., and Noble, M. E. M. (2011). Presenting
Your Structures: the CCP 4 mg Molecular-Graphics Software. Acta Crystallogr.
D Biol. Cryst. 67, 386394. doi:10.1107/S0907444911007281
Pellicciari, R., Liscio, P., Giacchè, N., De Franco, F., Carotti, A., Robertson, J., et al.
(2018). α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase
(ACMSD) Inhibitors as Novel Modulators of De Novo Nicotinamide
Adenine Dinucleotide (NAD+) Biosynthesis. J. Med. Chem. 61, 745759.
doi:10.1021/acs.jmedchem.7b01254
Poyan Mehr, A., Tran, M. T., Ralto, K. M., Leaf, D. E., Washco, V., Messmer, J.,
et al. (2018). De Novo NAD+ Biosynthetic Impairment in Acute Kidney Injury
in Humans. Nat. Med. 24, 13511359. doi:10.1038/s41591-018-0138-z
Pucci, L., Perozzi, S., Cimadamore, F., Orsomando, G., and Raffaelli, N. (2007).
Tissue Expression and Biochemical Characterization of Human 2-amino 3-
carboxymuconate 6-semialdehyde Decarboxylase, a Key Enzyme in
Tryptophan Catabolism. FEBS J. 274, 827840. doi:10.1111/j.1742-4658.
2007.05635.x
Saito, K., Fujigaki, S., Heyes, M. P., Shibata, K., Takemura, M., Fujii, H., et al.
(2000). Mechanism of Increases in L-Kynurenine and Quinolinic Acid in Renal
Insufciency. Am. J. Physiology-Renal Physiol. 279, F565F572. doi:10.1152/
ajprenal.2000.279.3.F565
Segel, I. H. (1993). Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium
and Steady-State Enzyme Systems. John Wiley & Sons.
von Stetten, D., Carpentier, P., Flot, D., Beteva, A., Caserotto, H., Dobias, F., et al.
(2020). ID30A-3 (MASSIF-3) - a Beamline for Macromolecular
Crystallography at the ESRF with a Small Intense Beam. J. Synchrotron
Radiat. 27, 844851. doi:10.1107/S1600577520004002
Weiss, M. S. (2001). Global Indicators of X-ray Data Quality. J. Appl. Cryst. 34,
130135. doi:10.1107/S0021889800018227
Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R.,
et al. (2011). Overview of the CCP4 Suite and Current Developments. Acta
Crystallogr. D Biol. Cryst. 67, 235242. doi:10.1107/S0907444910045749
Yang, Y., Borel, T., De Azambuja, F., Johnson, D., Sorrentino, J. P., Udokwu, C.,
et al. (2021). Diunisal Derivatives as Modulators of ACMS Decarboxylase
Targeting the Tryptophan-Kynurenine Pathway. J. Med. Chem. 64, 797811.
doi:10.1021/acs.jmedchem.0c01762
Yang, Y., Davis, I., Matsui, T., Rubalcava, I., and Liu, A. (2019). Quaternary
Structure of α-amino-β-carboxymuconate-ε-semialdehyde Decarboxylase
(ACMSD) Controls its Activity. J. Biol. Chem. 294, 1160911621. doi:10.
1074/jbc.RA119.009035
Yoshino, J. (2019). ACMSD: A Novel Target for Modulating NAD+ Homeostasis.
Trends Endocrinol. Metab. 30, 229232. doi:10.1016/j.tem.2019.02.002
Zhou, C.-C., Yang, X., Hua, X., Liu, J., Fan, M.-B., Li, G.-Q., et al. (2016). Hepatic
NAD+deciency as a Therapeutic Target for Non-alcoholic Fatty Liver Disease
in Ageing. Br. J. Pharmacol. 173, 23522368. doi:10.1111/bph.13513
Conict of Interest: NG, PL, ER, FD, and JR are employees of TES Pharma S.r.l.;
RP is President and CEO of TES Pharma S.r.l.; AC is a consultant of TES Pharma
S.r.l.; MC, LC, MG, and NR have a research collaboration contract with TES
Pharma S.r.1.
Publishers Note: All claims expressed in this article are solely those of the authors
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the publisher, the editors, and the reviewers. Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Copyright © 2022 Cianci, Giacchè, Cialabrini, Carotti, Liscio, Rosatelli, De Franco,
Gasparrini, Robertson, Amici, Raffaelli and Pellicciari. This is an open-access article
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