Dual Glycolate Oxidase/Lactate Dehydrogenase A Inhibitors for Primary Hyperoxaluria

Article abstract of DOI:10.1021/acsmedchemlett.1c00196

Both glycolate oxidase (GO) and lactate dehydrogenase A (LDHA) influence the endogenous synthesis of oxalate and are clinically validated targets for treatment of primary hyperoxaluria (PH). We investigated whether dual inhibition of GO and LDHA may provide advantage over single agents in treating PH. Utilizing a structure-based drug design (SBDD) approach, we developed a series of novel, potent, dual GO/LDHA inhibitors. X-ray crystal structures of compound 15 bound to individual GO and LDHA proteins validated our SBDD strategy. Dual inhibitor 7 demonstrated an IC50 of 88 nM for oxalate reduction in an Agxt-knockdown mouse hepatocyte assay. Limited by poor liver exposure, this series of dual inhibitors failed to demonstrate significant PD modulation in an in vivo mouse model. This work highlights the challenges in optimizing in vivo liver exposures for diacid containing compounds and limited benefit seen with dual GO/LDHA inhibitors over single agents alone in an in vitro setting.

Full text of DOI:10.1021/acsmedchemlett.1c00196

pubs.acs.org/acsmedchemlett
Letter
Dual Glycolate Oxidase/Lactate Dehydrogenase A Inhibitors for
Primary Hyperoxaluria
Jinyue Ding,* Rajesh Gumpena, Marc-Olivier Boily, Alexandre Caron, Oliver Chong, Jennifer H. Cox,
Valerie Dumais, Samuel Gaudreault, Aaron H. Graff, Andrew King, John Knight, Renata Oballa,
Jayakumar Surendradoss, Tim Tang, Joyce Wu, W. Todd Lowther,* and David A. Powell
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ABSTRACT: Both glycolate oxidase (GO) and lactate dehydrogenase A (LDHA)
influence the endogenous synthesis of oxalate and are clinically validated targets for
treatment of primary hyperoxaluria (PH). We investigated whether dual inhibition of GO
and LDHA may provide advantage over single agents in treating PH. Utilizing a structure-
based drug design (SBDD) approach, we developed a series of novel, potent, dual GO/
LDHA inhibitors. X-ray crystal structures of compound 15 bound to individual GO and
LDHA proteins validated our SBDD strategy. Dual inhibitor 7 demonstrated an IC50 of 88
nM for oxalate reduction in an Agxt-knockdown mouse hepatocyte assay. Limited by poor
liver exposure, this series of dual inhibitors failed to demonstrate significant PD modulation
in an in vivo mouse model. This work highlights the challenges in optimizing in vivo liver
exposures for diacid containing compounds and limited benefit seen with dual GO/LDHA
inhibitors over single agents alone in an in vitro setting.
KEYWORDS: Glycolate oxidase, Lactate dehydrogenase A, Dual inhibitors, Structure-based drug design, Primary hyperoxaluria
rimary hyperoxaluria (PH) is a rare genetic disorder
resulting in oxalate overproduction, formation of calcium
oxalate kidney stones, and in some patients, end stage kidney
failure.1 There are three types of PH, 1, 2, and 3.2 PH1 is
caused by mutations in the gene AGXT, which encodes the
alanine-glyoxylate aminotransferase (AGT) protein (Figure 1).
the kidney can lead to inflammation, interstitial fibrosis, tissue
damage, urinary tract infections and, in severe cases, kidney
failure.3a As kidney function deteriorates, concentrations of
plasma oxalate increase, and calcium oxalate can accumulate
and crystallize in other tissues such as eyes, skin, heart, bones,
and brain. Two inhibition mechanisms have been the focus of
therapeutic development for PH: glycolate oxidase (GO)3 and
LDHA.4
P
GO is encoded by the HAO1 gene and expressed exclusively
in the liver peroxisome in humans. As a flavin mononucleotide
(FMN)-dependent α-hydroxyacid oxidase, GO converts
glycolate into glyoxylate (Figure 1), resulting in increased
production of oxalate via LDHA. PH1 patients, with deficiency
in AGT, have higher levels of glycolate.5 The Salido group
reported that Hao1−/− mice developed high urinary glycolate
levels but were overall healthy.6a Compared to Agxt−/− mice
with a hyperoxaluria phenotype, Agxt−/−-Hao1−/− double-
knockout mice demonstrated reduced urinary oxalate levels.
Additionally, CCPST (4-carboxy-5-[(4-chlorophenyl)-
sulfanyl]-1,2,3-thiadiazole), a modestly potent GO inhibitor,
Figure 1. Metabolic pathways of oxalate synthesis.
PH2 and PH3 are caused by mutations in the genes which
produce glyoxylate reductase (GR) and 4-hydroxy-2-oxogluta-
rate aldolase (HOGA) proteins, respectively. These mutations
result in an autosomal recessive disorder of glyoxylate
metabolism, impairing hepatic detoxification of glyoxylate
and corresponding excessive oxalate production in the liver via
the action of lactate dehydrogenase A (LDHA). Oxalate
produced in the liver is excreted via the kidneys, but when
present at high concentrations, it complexes with calcium to
form calcium oxalate salt. Deposits of calcium oxalate within
Received: April 7, 2021
Accepted: May 18, 2021
Published: May 20, 2021
https://doi.org/10.1021/acsmedchemlett.1c00196
© 2021 American Chemical Society
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caused oxalate reductions in a cellular Agxt−/− mouse
hepatocyte assay. When administered orally to Agxt−/− mice,
CCPST reduced urinary oxalate excretion by 30−50%.6a
Clinically, lumasiran, a GalNAc-conjugated GO-siRNA ther-
apeutic developed by Alnylam, achieved robust urinary oxalate
reduction in PH1 patients.3
In contrast to the indirect influence of GO on oxalate
production, the LDH enzymes, encoded by LDH A−C genes,
not only catalyze the reversible conversion of pyruvate and
NADH to lactate and NAD+ but also convert glyoxylate to
oxalate (Figure 1). Thus, inhibition of liver LDHA represents a
viable approach for blocking oxalate production and, being the
terminal and committed step in oxalate synthesis, may be
effective in treating all three types of PH. Individuals with
LDHB deficiency often do not have adverse symptoms,7
although LDHA-deficient individuals can experience muscle-
related adverse events which can lead to myoglobinuria and
kidney damage.8 Importantly, liver LDHA has been selectively
targeted using the GalNAc−siRNA conjugate nedosiran from
Dicerna. This approach is currently in phase 3 trials and has
shown efficacy in reducing urinary oxalate levels in PH1 and
PH2 patients.4,9 This suggests that LDHA-inhibition in the
liver is sufficient for reducing urinary oxalate in PH patients.
Because GO and LDHA inhibition have been validated
clinically via GalNAc-conjugated siRNA therapeutics, we
sought to investigate whether additional benefit may be
obtained from a dual GO/LDHA small molecule inhibitor.10
This would represent a novel and clinically derisked
therapeutic approach for all three forms of PH. With
multispecific drugs emerging as a powerful drug targeting
approach in modern biopharmaceutical industry,11 we also
envisioned that a class of bispecific small molecules would
further illustrate the effectiveness of target-based therapeutic
molecules. To evaluate the possibility of dual GO/LDHA
inhibition by a single small molecule agent, we utilized
published structural data of small molecule inhibitors bound to
GO and LDHA to inform our approach.
Figure 2. Representative examples of GO and LDHA inhibitors.
Several potent, small molecule GO inhibitors have been
reported, which are defined structurally by a nonpolar tail and
polar acidic warhead. The latter resembles the GO substrate,
glycolate, and forms multiple key interactions with the enzyme.
Examples of well-studied acidic warheads include 3-hydroxy-
1H-pyrrole-2,5-dione,12 1,2,3-thiadiazole-4-carboxylic acid,6
and 1,2,3-triazole-4-carboxylic acid13 (Figure 2). The crystal
structure of GO in complex with CDST (4-carboxy-5-
dodecylsulfanyl-1,2,3-triazole; Ki 15 nM) containing a triazole
carboxylic acid warhead was studied by Murray et al.13a
A
Figure 3. (A) Key interactions of GO with CDST (PDB 2RDT)13a
and (B) LDHA with compound 63 (PDB 5W8L).19a
hybrid of CDST and CCPST, compound A, was synthesized
and determined as a potent GO inhibitor (Figure 2). Several
patented examples of potent GO inhibitors featuring a triazole
acid warhead have also been described.14 The numerous
interactions between CDST and the GO active site are detailed
in Figure 3A. Residues His260 and Tyr132 interact with the
triazole ring, while Arg167 and Arg263 coordinate the acid
moiety. The phenol moiety of Tyr26 has a long-range
interaction with the sulfur atom of the thioether. We
considered that a triazole warhead of a dual inhibitor could
be utilized to engage the active site and the solvent exposed,
hydrophobic tail could potentially be substituted with LDHA-
inhibitor.
have been developed by pharmaceutical companies, including
AstraZeneca (compound 33),16 GlaxoSmithKline (GSK
2837808A),17 and Genentech (GNE-140)18 (Figure 2).
Upon examining the SAR and the crystal structures of these
compounds bound to LDHA, we were intrigued by the class of
pyrazole-based LDHA inhibitors and their unique binding
conformation.19 The nanomolar inhibitor compound 63
(Figure 3B) highlights three key interactions: (1) thiazole
acid warhead with residue Arg168 and Thr247, (2) cyclo-
propyl π−π interaction with Tyr238 (not shown for clarity),
and (3) sulfonamide with Asp140, Glu191, and Ile141. More
recently, the same group reported the beneficial impact of a 2-
fluorine substituent on the benzenesulfonamide ring.19b Of
Given the central role of LDHA in glycolysis, LDHA
inhibitors have been studied as potential therapeutics in
oncology.15 Several scaffolds with potent LDHA inhibition
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a
note, the terminal biphenyl tail points toward solvent-exposed
area and does not form any key interactions with the LDHA
enzyme.
Scheme 1. Synthesis of Analogue 7
On the basis of structural analysis of GO-CDST and LDHA-
compound 63 complexes described above, both scaffolds
contain a rather large solvent-exposed terminal moiety.
Therefore, we hypothesized that upon merging the two
scaffolds at the solvent-exposed fronts, a series of diacid-
containing compounds may engage the required protein
residues to potently inhibit both GO and LDHA (Figure 4).
We sought to identify shorter linkers to minimize the overall
molecular weight of the dual inhibitors and ideally maintain
drug-like properties.
a
Reagents and conditions: (a) LiOH, THF, H2O, 23 °C, 4 h, 66%;
(b) (Boc)2O, DMAP, iPr2NEt, CH2Cl2, 23 °C, 3 h, 83%; (c) K2CO3,
DMF, 130 °C, 2 h, 87%; (d) 1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-
cyclopropylethan-1-one, MgBr2·Et2O, iPr2NEt, CH2Cl2, 23 °C, 2 h,
51%; (e) 4-(bromomethyl)-2-fluorobenzenesulfonamide, Cs2CO3, KI,
DMSO, 23 °C, 2 h, 78%; (f) NH2NH2·H2O, EtOH, 70 °C, 2 h, 95%;
(g) sodium tert-pentoxide, DMF, 0 °C, 1 h, 21%; (h) TFA, Et3SiH, 40
°C, 18 h, 18%; (i) 1 M aqueous LiOH, THF, MeOH, 23 °C, 2 h,
45%.
a
Scheme 2. Synthesis of Analogue 11
Figure 4. Strategy for the generation of dual GO/LDHA inhibitors.
On the basis of the above hypothesis, a series of compounds
containing both the GO warhead (triazole acid) and LDHA
warhead (thiazole carboxylic acid) were synthesized (Scheme
1). The GO warhead precursor, N-PMB protected triazole
carboxylate 1, was assembled following a literature method.20
The corresponding tert-butyl ester 2 was obtained upon
hydrolysis of ethyl ester 1 followed by treatment with di-tert-
butyl dicarbonate. After an SNAr reaction with 1-(3-
sulfanylphenyl)ethanone, the sulfur-linked triazole carboxylate
3, was synthesized. The acetyl group of 3 allowed further
construction of the compound 63-like scaffold, following
literature procedures.19 The acetophenone 3 underwent a
magnesium bromide-catalyzed soft enolization in the presence
of Hu
̈
nig’s base,21 yielding 1,3-diketone 4 in 51% yield, which
was then alkylated with 4-(bromomethyl)-2-fluorobenzenesul-
fonamide. This was followed by cyclization with hydrazine,
yielding the desired pyrazole intermediate 5 in a quantitative
yield. When treated with sodium tert-pentoxide at 0 °C,
pyrazole 5 underwent a regioselective arylation reaction with
ethyl 2-fluorothiazole-4-carboxylate, favoring the desired
regioisomer 6. Use of other bases and the chloro or bromo
thiazole ester resulted in reduced selectivity for the desired
arylated pyrazole isomer. Both tert-butyl and PMB protecting
groups in compound 6 were removed in one step by reaction
with trifluoroacetic acid and triethylsilane. Further hydrolysis
of the resulting ethyl ester resulted in diacid 7.
a
Reagents and conditions: (a) Pd(dppf)Cl2, B2pin2, KOAc, dioxane,
100 °C, 18 h, 46%; (b) urea hydrogen peroxide, MeOH, 23 °C, 18 h,
82%; (c) 2, K2CO3, DMF, 130 °C, 2 h, 19%; (d) TFA, Et3SiH, 40 °C,
18 h, 37%; (e) 1 M aqueous LiOH, THF, MeOH, 23 °C, 2 h, 53%.
The bromo intermediate 8 underwent a Miyaura borylation
reaction, yielding the corresponding pinacol boronate 9, which
was further oxidized to the desired phenol intermediate 10.
After SNAr reaction with intermediate 2, deprotection of PMB
and tert-butyl ester, and saponification of ethyl carboxylate, the
oxygen linker analogue 11 was accessed.
The corresponding oxygen linker analogue 11 (Scheme 2)
was synthesized from the bromo intermediate 8, which can be
accessed in the same manner as described in Scheme 1 (steps
d−g), starting from 1-(3-bromo-4-fluorophenyl)ethan-1-one.
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Both sulfur and oxygen-linker analogues were further
evaluated in a series of in vitro assays. Gratifyingly, both
compounds exhibited good potency against both GO and
LDHA enzymes, with IC50 values ranging from 0.3−11 nM
(Table 1). Both compounds were further examined in a mouse
Scheme 3. Synthesis of Second-Generation Dual GO/LDHA
Inhibitors
a
Table 1. In Vitro Profile of First-Generation Dual GO/
a
LDHA Inhibitors 7 and 11
enzyme IC50 (nM)
primary mouse hepatocyte
compd GO LDHA mLDHA
IC50 (nM)
clogD7.4
7
11
4.7
11
0.5
0.4
0.7
0.3
136
732
−0.3
−1.1
a
See Supporting Information for assay conditions; clogD7.4 values
were calculated using ACD laboratories (ACD/Percepta 2014
Release). All IC50 values are mean values determined from more
than two replicates.
primary hepatocyte assay, where hepatocytes were freshly
isolated from wild-type mice (CD1 from Charles River
Laboratories) and incubated with tested compound in the
presence of pyruvate. Compound potency was determined by
measuring inhibition of lactate production in the freshly
isolated hepatocytes. Measurement of oxalate production
requires ion chromatography mass spectrometry (ICMS)22
for enhanced sensitivity. Interestingly, compound 7 was about
6-fold more potent in the mouse primary hepatocyte assay
than compound 11, while both compounds showed similar
potency against mouse LDHA (mLDHA) and the human
enzyme. Because this primary hepatocyte assay measures
intracellular LDHA inhibition, their minor potency difference
against GO would have little impact in this discrepancy.
Compound 7 exhibits a 0.8 unit higher clogD7.4 value than that
of compound 11 (Table 1) and may possess higher cellular
permeability, which could explain the cellular potency
differences. Additionally, structural changes from compounds
7 to 11 might have an impact on LDHA binding kinetics.19b
Given the improved cellular LDHA inhibition of the less
hydrophilic analogue 7, we next explored the synthesis of
analogues with more lipophilic moieties in the linker region
(Scheme 3) with the goal of further improving the mouse
hepatocyte cellular potency via improved permeability. The
more lipophilic oxygen-linker analogue 14 was accessed in
three steps, starting from a SNAr reaction between 3-
bromophenol and triazole intermediate 1 (Scheme 3). The
resulting bromide 12 underwent a Pd-catalyzed Suzuki−
Miyaura coupling reaction with pinacol boronate intermediate
9, yielding the diethyl ester 13. After PMB deprotection and
ester hydrolysis, 14 was obtained in 15% yield over two steps.
Three more analogues, 15, 16, and 17, were synthesized in a
similar manner, starting from 4-bromophenol, 4-bromothio-
phenol, and tert-butyl 5-(4-iodophenyl)-1H-1,2,3-triazole-4-
carboxylate, respectively.
a
Reagents and conditions: (a) 1, K2CO3, DMF, 130 °C, 9 h, 93%; (b)
9, Pd(dppf)Cl2, 2 M aqueous K3PO4, dioxane, 130 °C, 1 h, 60%; (c)
TFA, Et3SiH, 40 °C, 18 h, 59%; (d) 1 M aqueous LiOH, THF,
MeOH, 23 °C, 18 h, 25%.
Table 2. In Vitro Profile of Second-Generation Dual GO/
LDHA Inhibitors
a
enzyme IC50 (nM)
primary mouse hepatocyte
compd GO LDHA mLDHA
IC50 (nM)
clogD7.4
14
15
16
17
0.9
1.2
1.1
3.6
0.1
0.07
0.3
0.2
0.4
0.2
0.4
143
198
230
170
0.6
0.6
1.2
0.7
0.3
a
See Supporting Information for enzyme and hepatocyte assay
conditions; clogD7.4 values were calculated using ACD laboratories
(ACD/Percepta 2014 Release). All IC50 values are mean values
determined from more than two replicates.
assay, further strengthening our hypothesis between lip-
ophilicity and cell activity for the current structural series.
Our strategy of utilizing a structure-based drug design
(SBDD)23 led to a series of highly potent dual GO/LDHA
inhibitors. To validate the mode of binding for these novel
dicarboxylic acid series, we determined the crystal structure of
compound 15 in complex with GO and LDHA at 2.07 and
2.50 Å resolution (Supporting Information, Table S1),
respectively. In the GO-compound 15 complex, the triazole
acid warhead preserves all key interactions with residues
Arg167/263, Tyr26, Trp110, Tyr132, and His260 (Figure 5A),
previously observed in the GO-CDST complex (Figure 3A).
The remainder of the compound, similar to the lipid-like tail of
CDST, was accommodated into the solvent exposed area by
loop 4 movements.13a
In contrast to GO, compound 15 binds to LDHA in two
slightly different but well-defined conformations (Figure 5B).
In both conformations, the LDHA binding regions of
compound 15 were well aligned, highlighting all three binding
features observed in the LDHA-compound 63 complex (Figure
3B), including thiazole acid warhead with Arg168/Thr247,
These less hydrophilic compounds, with clogD7.4 values in
the 0.6−1.2 range, exhibited subnanomolar IC50 values against
both GO and LDHA enzymes. The direct-linked analogue 17
had an IC50 of 3.6 nM against GO (Table 2). Compared to the
oxygen or sulfur-linker analogues, the moderately reduced
potency observed in the direct-linker analogue 17 could be due
to the polar hydroxyl group of Tyr26 in the GO active site
(Figure 3A). Nonetheless, all four analogues demonstrated
good potency (EC50 143−230 nM) in a mouse hepatocyte
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a
Table 3. Wild-Type and Agxt-KD Hepatocyte Assays
mouse primary
mGO mLDHA
hepatocyte
AgxtKD mouse primary
IC50
IC50
(pyruvate to lactate) hepatocyte (glycolate
compd (nM)
(nM)
IC50 (nM)
to oxalate) IC50 (nM)
7
7.7
0.8
136
88
A
63
14.6
149
181
b
0.1
a
See Supporting Information for assay conditions. All IC50 values are
mean values determined from more than two replicates unless
b
otherwise specified. IC50 value from n = 1 experiment.
Encouraged by the robust inhibition in the PH1 mouse
hepatocyte assay demonstrated by compound 7, we sought to
further assess the in vivo pharmacodynamic (PD) effect of this
series of dual inhibitors. It is challenging to measure changes in
urinary oxalate in vivo in wild-type mice, where their basal
urinary oxalate is low. Measurements of urinary oxalate in
Agxt-KD mice can be achieved but require 5+ days of
compound treatment.6a Thus, we sought to measure plasma
glycolate in wild-type mice, a convenient and noninvasive PD
marker for GO inhibition, as a more acute measurement of
compound PD.25 Compound 7 and the second-generation
analogue compound 14, which showed superior potency in a
mouse primary hepatocyte assay, were further evaluated in the
mouse plasma glycolate-PD study. Compounds were orally
administered at 100 mg/kg to C57BL/6N mice (n = 3 per
group), and plasma glycolate levels were measured at 6 h after
compound administration.26 Neither of the dual inhibitors
demonstrated a significant PD effect for plasma glycolate
elevation (Figure 6). In comparison, compound A produced a
4−5-fold elevation of plasma glycolate 2 h post oral dosing.
Figure 5. X-ray crystal structures of compound 15 in complex with
(A) GO (PDB 7M2O) and (B) LDHA (PDB 7M2N).
cyclopropyl moiety with Tyr238 (not shown for clarity), and
the sulfonamide with Asp140/Glu191/Ile141, all of which are
critical for a potent LDHA inhibition. Interestingly, the
terminal triazole acid GO warhead group extended toward
solvent, with a ∼90° rotation between conformers, demon-
strating the considerable conformational freedom afforded by
this region. The helix associated with Arg105 also moved
outward to facilitate binding.
While the structural and chemical hypotheses proposed
herein were validated with the new dual inhibitors, the
question of whether the dual compound scaffold exhibits
efficacy in cellular assays and animals remained. Therefore, we
examined the profile of the current series of dual GO/LDHA
inhibitors in a cellular model of PH1. In this assay, compound-
mediated inhibition of glycolate to oxalate conversion was
assessed in freshly isolated hepatocytes from mice deficient in
AGT through the hepatic knockdown of the Agxt gene via an
Agxt-siRNA encapsulated in a lipid nanoparticle (LNP) as
reported in the literature.24 Conversion of glycolate to oxalate
was measured using ICMS.22 The advantage of this lower
throughput assay is the direct measurement of reduction of
oxalate production from glycolate, which encompasses both
GO and LDHA inhibitory pathways simultaneously (Figure 1).
Seven days following a single intravenous administration of
Agxt-siRNA LNP to CD1 mice, hepatocytes were isolated and
freshly incubated with glycolate and inhibitor at various
concentrations (Table 3). In this assay, compound 7
demonstrated robust inhibition of oxalate production with an
IC50 of 88 nM, further validating the role of these dual GO/
LDHA inhibitors in inhibiting oxalate synthesis, the etiological
driver in PH. In the same oxalate reduction assay, compound 7
was only moderately more potent compared to individual GO
inhibitor A or LDHA inhibitor 63, while their corresponding
intrinsic mouse GO or LDHA potency are comparable,
suggesting dual GO/LDHA inhibition might not be additive
or synergistic compared to individual inhibition.
Figure 6. Mouse PK/PD of compounds 7, 14 (6 h post oral dosing),
and A (2 h post oral dosing).
We investigated the hepatic exposure of both compounds to
understand the relationship between pharmacokinetic (PK)
and the modest GO-driven PD effect. As a result, mouse liver
samples were collected at the end of the PD experiment. Both
dual inhibitors showed low μM liver exposures, which were
significantly below liver exposures seen with compound A at
the same dose. Poor liver exposure may be attributed to low
oral absorption of the highly polar dicarboxylic acid series.
Recognizing the limitations of this dicarboxylic acid series is
important for further optimization and acid isosteres27 or
prodrug approaches28 are possible strategies to improve the
oral bioavailability and liver exposures.
In summary, we developed a series of potent dual GO/
LDHA inhibitors utilizing a structure-based drug design
strategy. The binding mode of these novel inhibitors was
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confirmed by the X-ray crystal structures of compound 15 in
complex with GO and LDHA. Careful tuning of clogD7.4 led to
a series of second-generation dual GO/LDHA inhibitors,
which showed improved potency in a mouse primary
hepatocyte assay, presumably via improved passive cellular
permeability. Compound 7 demonstrated potent cellular
inhibition of oxalate production in an PH1 mouse cellular
model, the Agxt-knockdown primary hepatocyte assay. Further
mouse PD studies with hepatic PK analysis revealed a
limitation of the current series of dual inhibitors in reaching
sufficient liver exposure to engage the hepatic GO enzyme.
Finally, we were disappointed that serial inhibition of oxalate
synthesis via dual GO/LDHA inhibition did not appear to be
additive.29 Individual novel GO or LDHA small-molecule
inhibitors demonstrated similar potencies as dual inhibition in
the Agxt-knockdown primary hepatocyte assay (50−200 nM
range). Our efforts to further develop GO and LDHA small-
molecule inhibitors for primary hyperoxaluria will be presented
in future publications.
John Knight − Department of Urology, University of Alabama
at Birmingham, Birmingham, Alabama 35294, United States
Renata Oballa − Chinook Therapeutics, Seattle, Washington
98102, United States
Jayakumar Surendradoss − Chinook Therapeutics, Seattle,
Washington 98102, United States
Tim Tang − Chinook Therapeutics, Seattle, Washington
98102, United States; orcid.org/0000-0002-7055-440X
Joyce Wu − Chinook Therapeutics, Seattle, Washington
98102, United States
David A. Powell − Chinook Therapeutics, Seattle, Washington
98102, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00196
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
ASSOCIATED CONTENT
■
The authors declare the following competing financial
interest(s): Some authors are current or former employees of
Chinook Therapeutics and potentially own and/or hold stocks
in the company.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00196.
1
Detailed description of chemical synthesis, H NMR
ACKNOWLEDGMENTS
data, methods of protein expression and purification, X-
ray crystallography, enzyme and hepatocyte (both wild-
type and Agxt-knock down) assays, in vivo pharmacoki-
netic, pharmacodynamic studies, and analysis (PDF)
■
We thank Chinook leadership team in supporting this project,
Wake Forest School of Medicine (under the supervision of
W.T.L.) for providing crystallization and X-ray diffraction
facilities (Wake Forest Baptist Comprehensive Cancer Center
P30 CA012197), and University of Alabama at Birmingham
(under the supervision of J.K.) for ICMS facilities. We also
thank Terrence L. Smalley for many helpful discussions.
AUTHOR INFORMATION
■
Corresponding Authors
W. Todd Lowther − Center for Structural Biology,
Department of Biochemistry, Wake Forest School of Medicine,
Winston-Salem, North Carolina 27157, United States;
Email: tlowther@wakehealth.edu
Jinyue Ding − Chinook Therapeutics, Seattle, Washington
98102, United States; orcid.org/0000-0003-3883-2470;
Email: jding@chinooktx.com
ABBREVIATIONS
■
siRNA, small interfering RNA; GalNAc, N-acetylgalactos-
amine; NAD, nicotinamide adenine dinucleotide; PMB, p-
methoxybenzyl; SNAr, nucleophilic aromatic substitution;
DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; TFA, trifluoroacetic acid; LNP,
lipid nanoparticle.
Authors
Rajesh Gumpena − Center for Structural Biology, Department
of Biochemistry, Wake Forest School of Medicine, Winston-
Salem, North Carolina 27157, United States
Marc-Olivier Boily − Chinook Therapeutics, Seattle,
Washington 98102, United States
Alexandre Caron − Chinook Therapeutics, Seattle,
Washington 98102, United States
Oliver Chong − Chinook Therapeutics, Seattle, Washington
98102, United States
Jennifer H. Cox − Chinook Therapeutics, Seattle, Washington
98102, United States
REFERENCES
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Valerie Dumais − Chinook Therapeutics, Seattle, Washington
98102, United States
Samuel Gaudreault − Chinook Therapeutics, Seattle,
Washington 98102, United States
Aaron H. Graff − Center for Structural Biology, Department of
Biochemistry, Wake Forest School of Medicine, Winston-
Salem, North Carolina 27157, United States
Andrew King − Chinook Therapeutics, Seattle, Washington
98102, United States
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