Implementation of the CYP Index for the Design of Selective Tryptophan-2,3-dioxygenase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.0c00004

A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation was demonstrated in vivo, which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the risk of drug-drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor containing a Lewis basic motif capable of binding heme.

Full text of DOI:10.1021/acsmedchemlett.0c00004

pubs.acs.org/acsmedchemlett
Letter
Implementation of the CYP Index for the Design of Selective
Tryptophan-2,3-dioxygenase Inhibitors
Brendan T. Parr,* Richard Pastor, Benjamin D. Sellers, Zhonghua Pei, Firoz A. Jaipuri,
Georgette M. Castanedo, Lewis Gazzard, Sanjeev Kumar, Xiaokai Li, Wen Liu, Rohan Mendonca,
Roheeth K. Pavana, Hima Potturi, Cheng Shao, Venkata Velvadapu, Jesse P. Waldo, Guosheng Wu,
Po-wai Yuen, Zuhui Zhang, Yamin Zhang, Seth F. Harris, Angela J. Oh, Antonio DiPasquale,
Kevin Dement, Hank La, Leanne Goon, Amy Gustafson, Erica C. VanderPorten, Mario R. Mautino,
and Yichin Liu
Cite This: https://dx.doi.org/10.1021/acsmedchemlett.0c00004
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via
structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation
was demonstrated in vivo, which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the
risk of drug−drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein
referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We
anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor
containing a Lewis basic motif capable of binding heme.
KEYWORDS: Tryptophan-2,3-dioxygenase, indoleamine-2,3-dioxygenase, CYP inhibition, property-based drug design
argeting the cellular mechanisms whereby tumors evade
detection and clearance by the host immune response
molecule clinical9−12 and preclinical13,14 entities. In the context
of human tumors, populations of cancer patients are
characterized by highly IDO1 expressing tumors, and increased
translation has been observed as a potential mechanism of
resistance to immunotherapies in the clinic.3,4,15−17
Only within the past decade has the transcriptional regulation
and potential role of TDO in immune evasion by cancer cells
been reported.6,18 TDO is predominantly expressed in the liver
and responsible for regulation of systemic TRP concentrations
and hepatic KYN concentrations. As with IDO1, abnormal
TDO expression profiles have been observed in several cancers
T
comprises an area of intense research effort.1 The kynurenine
pathway, which involves the rate-limiting oxidative metabolism
of tryptophan (TRP) to kynurenine (KYN) via N-formylkynur-
enine (NFK) by the cytosolic enzymes indoleamine-2,3-
dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO),
has been posited as a viable site for small molecule
intervention.2−4 Genetic knockouts of IDO1 and TDO have
implicated complementary roles for these enzymes in
metabolism.5,6 Specifically, IDO1−/− mice exhibit a marked
decrease in KYN concentration with minimal effect on TRP
concentrations. TDO−/− animals, on the other hand, are
characterized by a profound increase systemic TRP.7,8 Since
both tryptophan and kynurenine serve as independent
modulators of immune function, selectively targeting either
IDO1 or TDO is an attractive point of intervention.
Received: January 3, 2020
Accepted: March 4, 2020
Published: March 4, 2020
Selective inhibition of IDO1 has been of particular interest to
the pharmaceutical community, exemplified by numerous small
https://dx.doi.org/10.1021/acsmedchemlett.0c00004
© XXXX American Chemical Society
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
A

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
a
Table 1. Correlation of QM Calculated Partial Negative Charge and Inhibitor Activity of Tricyclic Heme-Binders
a
Calculated partial charge (coulomb) of the iron-binding nitrogen (red) by quantum mechanical calculations. IC50: Half-maximal inhibitory
concentration for recombinant human enzyme in biochemical assay, reported in μM (see the Supporting Information).
and are inversely correlated with overall survival rates.19 One
proposed mechanism for acquired immunosuppression involves
upregulation of TDO within the tumor microenvironment
leading to localized depletion of TRP. The consequent amino
acid deprivation leads to accumulation of uncharged tRNAs,
activating general control non-derepressible 2 (GCN2) kinase,
thereby promoting tumor angiogenesis.20 Direct immune
inactivation is promoted by GCN2 in T cells: blockade of
CD4+ T cell differentiation to T helper cells (TH17) and cell
cycle arrest induced in CD8+ T cells are two reported
mechanisms.21,22
We pursued a medicinal chemistry program aimed at
identifying a potent and selective inhibitor of TDO, which
would be compatible for coadministration in the clinic with an
IDO1-selective inhibitor. Due to the interconnected biological
function of the two enzymes, the substrate TRP, and the
metabolite KYN, it was anticipated that the ability to
independently modulate IDO1 versus TDO enzymatic activity
might be beneficial for both patient safety and efficacy. By
utilizing the IDO1 inhibitor GDC-0919 as a starting point for
chemistry, we envisioned the added benefit of informing any
future efforts to identify an IDO1/TDO dual inhibitor. In
contrast to its structurally diverse counterparts, GDC-0919 is
unique in its relative potency against both IDO1 and TDO. For
example, clinical and preclinical inhibitors INCB24360, BMS-
986205, and PF-06840003 bind IDO1 with low nanomolar
affinity but exhibit minimal inhibitory activity against TDO
(IC50 > 10 μM). By comparison, GDC-0919 shows half-maximal
inhibition of both enzymes at concentrations within one
logarithm (IDO1 EC50 = 0.45 μM; TDO EC50 = 2.0 μM).
Encouraged by these observations, a combined structure- and
property-based design approach was initiated to identify a TDO
selective small molecule inhibitor.
heme-binders, a putative minimal pharmacophore, were then
synthesized to cover a range of predicted partial negative
character (Table 1). The correlation was validated by
comparison to biochemical inhibition. Of the analogues for
which a partial charge was calculated, the imidazoindazole and
III analogues 1 and 2 were predicted the most Lewis basic (QM
charge = −0.526 and −0.527 coulomb, respectively). The near
equal partial charges translated into equipotent TDO inhibitory
activity. Transposition of the nonbinding nitrogen to afford
imidazoindole 3, with its lone pair of electrons in immediate
conjugation with the benzenoid ring, resulted in a subtle
decrease in partial negative character and a corresponding loss of
binding affinity. Similarly, more significant reductions of partial
charge, as with isothiazoloindene 4 and 1,2,4-triazolobenzimi-
dazole 5, were correlated with substantial losses of inhibitory
activity (IC50 = 7.0 and 17 μM, respectively). Still further
attenuation of the heterocycle Lewis basicity in the form of 1,2,3-
triazoloindole 6 resulted in a loss of measurable inhibition. Thus,
we pursued optimization of the ancillary portion of the ligand
with the imidazoisoindole core for the combination of potency
and relative ease of chemical synthesis.
Despite the functional homology of IDO1 and TDO, turning
our attention to X-ray structures alluded to subtle differences
that we anticipated were exploitable handles for selectivity
(Figure 1). While a few amino acid residue differences present
We recognized that engaging the dioxygenase catalytic heme
imparts significant inhibitory activity to GDC-0919. Unlike
IDO1, which exclusively contains ferrous iron as the catalytic
engine, the metal center in TDO exists in equilibrium between
ferrous and ferric states. Thus, we considered whether the
imidazoisoindole (III) heme-binder would prove optimal for
inhibition of TDO. Wishing to maintain the general features of
the III motif, which provided intrinsic TDO inhibitory activity,
we explored isosteric replacements of the Lewis basic imidazole,
tuning the electronics of the iron-coordinating nitrogen atom, to
determine if enhancements in binding could be identified.
Designs were triaged by estimating the propensity of the core to
coordinate to the heme iron using quantum mechanical
calculations of the partial charge of the iron-binding nitrogen
(see the Supporting Information). Atoms with more negative
partial charges might coordinate more strongly with the iron
leading to increased affinity as has been investigated previously
with IDO1 inhibitors. A number of 5-ethoxy substituted tricyclic
Figure 1. Overlay of the cocrystal structure of GDC-0919 in IDO1
(cyan) with TDO (navy).
B
https://dx.doi.org/10.1021/acsmedchemlett.0c00004
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
along the inner sphere of the active site adjacent the tricyclic
core, we focused attention on the extraneous portion of the
ligand, where affinity might be modulated independent of heme-
binding potential. Alignment of the cocrystal structure of GDC-
0919 bound to IDO1 with a structure of TDO about the ferrous
center suggested that the van der Waals surface of TDO along
the hydrophobic channel could accommodate a larger ligand
than IDO1. Despite conserved inner sphere residues, we
targeted the spacious lipophilic pocket available adjacent to
TDO F140 (IDO1 F226) to improve TDO affinity. Introducing
a methyl group at C(10) of 7, a simplified GDC-0919 analogue,
resulted in concomitant gain in TDO potency, loss in IDO1
activity, and net ∼20× improvement in selectivity (cf. Table 2,
Upon comparison of TDO potency and selectivity for IDO1
across a range of ligands of varied ring size, there appeared a
modest preference of TDO for four-membered alcohols (Figure
2). By contrast, five-to-seven membered alcohols exhibited
Table 2. Structure-Guided Design of C(10)-Cyclic Inhibitors
Figure 2. TDO vs IDO cellular potency of cyclic III inhibitors.
trivial distinctions in cellular activity. All subseries of inhibitors,
however, displayed a propensity for reversible inhibition of the
cytochrome P450 subfamily member 3A4. In that regard, the
four-membered cyclic alcohols also differentiated in their
exquisitely potent CYP3A4 inhibition. We envisioned remedy-
ing reversible CYP inhibition by reducing lipophilicity via
introduction of heteroatom functionality into the ancillary
portion of the ligand. Due to the limited vectors available for
modifying the four-membered ring inhibitors, our attention
turned to the six-membered cyclic alcohols, offering a balance
between lipophilicity and vectors for property-based optimiza-
tion to improve selectivity and metabolic stability.
We decided to investigate the structure−activity relationships
of tetrahydropyranols and piperidinols as readily accessible
scaffolds for reducing the lipophilicity. While 3-tetrahydropyr-
anol 13 was tolerated by TDO and improved IDO1 selectivity
relative to 9, the compound−as with its cycloalkyl counter-
parts−was plagued by submicromolar CYP3A4 inhibition
(Table 3, entry 1). The phenylalanine (F72, F140) and leucine
(L46, L147) lined pocket of TDO presents a very lipophilic
surface, and attempts to introduce polarity in that vector as with
4-tetrahydropyranol were less compatible (Table 3, entry 2, 14,
TDO EC50 = 0.21 μM). Based on the cyclic ether structure−
activity relationship (SAR), we targeted 3-piperidinols. Due to
the proximity of the nitrogen atom to Arg144, it was not
surprising to find the basic N-methyl-4-piperinol 15 was not
accommodated. By reducing basicity, however, we were pleased
to find that an N-methanesulfonylpiperidine analogue 16
provided comparable TDO potency and IDO1 selectivity to
its carbocyclic counterpart. Consistent with the proposal of
increasing polarity to mitigate CYP inhibition, 50% maximal
inhibition of CYP3A4 was not observed at 10 μM of the
compound. Nonetheless, we continued our efforts to identify an
inhibitor with improved balance of IDO1 and CYP3A4
selectivity. Unfortunately, N-acetylpiperidine was not accom-
modated (entry 5, 17), potentially due to steric clash between
the low energy conformer of the amide and the protein.
a
Half-maximal efficacious concentration in SW48 and A172 cellular
b
assays, reported in μM (see the Supporting Information). F.S. = Fold
c
selectivity. Clearance in human, rat, and mouse liver microsomes,
reported in mL × min−1 × kg−1.
entries 1 and 2). By truncating the ligand into a cyclohexanol
ring (entry 3, 9), still further improvements in IDO1 selectivity
were observed. Due to apparent subtle changes in the rotameric
disposition of the heme propionate, we then considered
optimizing the presumed hydrogen bonding interaction. The
apparent mobility of that motif led us to question its significance
for potency or selectivity. While eliminating the interaction by
removing the alcohol was tolerated for both potency and
selectivity (entry 4, 10), the increased lipophilicity was
accompanied by reduced in vitro stability as measured by
turnover in liver microsomes. Modulating the vector by
introducing a cyclopentanol or cyclobutanol ring (entries 5
and 6, 11 and 12, respectively) improved both the IDO1
selectivity and in vitro stability relative to 9.
From a survey of the inhibitors profiled, we recognized that
reversible CYP inhibition presented a significant challenge.
Turning to the literature for guidance to mitigate CYP inhibition
C
https://dx.doi.org/10.1021/acsmedchemlett.0c00004
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Table 3. C(10)-Heterocyclic Inhibitor SAR
a
Half-maximal efficacious concentration in SW48 and A172 cellular
b
assays, reported in μM (see the Supporting Information). F.S. = Fold
selectivity.
in the presence of a heme-binding motif, we identified two
Figure 3. Property-based correlations with CYP3A4 inhibition. (a)
CYP3A4 IC50/TDO EC50 fold selectivity vs lipophilicity; red = <3×,
orange = 3−10×, yellow = 10−30×, green = >30×. CYP3A4 IC50 vs (b)
rotatable bond count, (c) ring count, and (d) CYP Index; red = <0.3
μM, orange = 0.3−1.0 μM, yellow = 1.0−3.0 μM, green = >3.0 μM.
strategies: reduce lipophilicity (decrease cLogP) and introduce
conformational rigidity (decrease rotatable bonds).23
A
property-based analysis of III inhibitors, however, suggested
that relying on traditional remedies would not suffice.
Specifically, a comparison of TDO versus CYP3A4 inhibition
as a function of lipophilicity indicated a lack of correlation
between LogD and selectivity (Figure 3a). Although reduced
rotatable bond count, as a surrogate for increased conforma-
tional rigidity, did track with improved chances of decreased
CYP activity, the odds of achieving an IC50 > 3 μM were still very
low for molecules with a single rotatable bond (Figure 3b).
Further, we considered ring count, under the assumption that
increased rings would correlate with increased conformational
rigidity and, perhaps, improved CYP margins. Much to our
dismay, ring count did not trend in a meaningful way with CYP
inhibition (Figure 3c). Moreover, many promising leads were
already well optimized with respect to conventional property-
based refinements, such as 13 (LogD = 1.3, rotatable bonds = 1).
We considered whether an intuitive, multiparameter approach
might offer a solution to the challenge of promiscuous heme-
binding. We hypothesized that simple ring count did not
accurately predict CYP inhibition due to the propensity to also
trend with increasing lipophilicity. Therefore, by introducing a
lipophilicity correction, the relationship between rigidity and
CYP binding might be realized. Accordingly, we defined the
design parameter CYP Index as the quotient of ring count and
cLogP (Figure 3; Figure 4).
Figure 4. Design strategy for increasing CYP index of III inhibitors.
interaction with the lipophilic CYP binding pocket. An analysis
of reversible CYP3A4 inhibition as a function of the CYP Index
provided a reasonable correlation, and guided future design
space. Specifically, we noted that designs would need to meet the
criteria of CYP Index > 2 for improved odds of mitigating
submicromolar CYP3A4 binding.
For design compliance with the new property-based
parameter, the team recognized that even for relatively
nonlipophilic inhibitors (e.g., cLogP = 2.5), a minimum of five
rings would be necessary to meet the CYP Index > 2 threshold.
Not wishing to increase molecular complexity with additional
contiguous stereocenters, we envisioned fusing an arene to the
cycloalkanol inhibitor to both introduce an element of rigidity
Thus, increased rigidity without a proportionate increase in
cLogP could improve the odds of introducing an offending
D
https://dx.doi.org/10.1021/acsmedchemlett.0c00004
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Table 4. Tetrahydroarenol Inhibitor SAR
a
b
Half-maximal efficacious concentration in SW48 and A172 cellular assays, reported in μM (see the Supporting Information). F.S. = Fold
c
selectivity. Clearance in human, rat, and mouse liver microsomes, reported in mL × min−1 × kg−1.
and a handle for modulating lipophilicity. From our seminal
investigations, we observed that aryl alcohols (e.g., Figure 4, 18)
were potent TDO inhibitors; though, from the outset they were
less desirable due to increased lipophilicity compared to their
aliphatic alcohol counterparts. Nonetheless, replacement of the
solvent-exposed phenyl ring with a 2-oxazole analogue 19
demonstrated the ability to attenuate lipophilicity while
maintaining TDO activity. Only by subsequent introduction
E
https://dx.doi.org/10.1021/acsmedchemlett.0c00004
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
of additional rigidity at comparable LogD (Figure 4, 20) did we
observe appreciable CYP margins. Implementing a strategy of
prioritizing molecules with increased CYP Index, the team
devised to synthesize a series of fused tetrahydroarene analogues
covering a range of lipophilicity in the hope of identifying an
inhibitor with suitable margins over both CYP3A4 and IDO1
(Table 4). In addition to excellent TDO potency and an
acceptable CYP3A4 margin, tetrahydroisoquinolin-8-ol ana-
logue 20 exhibited desirable IDO1 selectivity and moderate liver
microsome stability. As expected, increased lipophilicity and
reduced CYP Index with tetrahydronaphthalene 21 was
detrimental to CYP3A4 selectivity. Despite a comparable CYP
Index, regioisomeric tetrahydroisoquinolin-5-ol 22 suffered a
dramatic reduction in the CYP3A4 margin. We hypothesized
that the slight decrease in steric encumbrance of the pyridyl
nitrogen relative to 20 may be sufficient to increase its aptitude
as a second heme-binding moiety. Substitutions of the
tetrahydronaphthalene core with aim of reducing lipophilicity
to improve IDO1 and CYP selectivity were considered.
Both 7- and 6-(methylsulfonyl)tetrahydronaphthalene-1-ol
(23 and 24, respectively) were potent TDO inhibitors; however,
the former regiosiomer exhibited a modest benefit with respect
to both CYP3A4 margins and liver microsome stability.
Although the dimethylphosphineoxide-substituted tetrahydro-
naphthalene 25 was promising due to excellent IDO1 selectivity
and in vitro stability, poor CYP selectivity, due in part to reduced
TDO affinity, impeded further advancement. The nitrile-
substituted analogue 26 was surprisingly potent and selective
over CYP3A4; however, the increased lipophilicity translated to
rapid turnover in liver microsome incubations. Increasing
polarity, as with the primary carboxamide-substituted com-
pound 27, was well accommodated by TDO and restored
CYP3A4 selectivity, as predicted by the increased CYP index,
with a concomitant improvement in IDO1 selectivity. Due to
increased hydrogen bond donor count and TPSA, however, the
molecule suffered poor passive permeability in MDCK cell lines
(Papp A:B = 1.7 × 10−6 cm × s−1). We shifted focus to
introducing polarity via five-membered heteroaromatic rings in
the solvent-exposed vector. Unfortunately, substantial decreases
in lipophilicity with tetrahydrotriazolopyridin-8-ol 28 resulted in
dramatic loss of TDO inhibitor activity. Nonetheless, we found
that increased polarity beyond the tetrahydroisoquinolinol
derivatives could be accommodated as with the tetrahydro-
benzooxadiazol-4-ol 29. Moreover, the modest increase in CYP
Index furnished improvements in CYP3A4 selectivity, and
similarly, the reduced lipophilicity correlated with improved in
vitro stability across species. Though the TDO potency was
diminished with N-fused tetrahydropyrazolopyridin-4-ol 30, the
increased CYP Index once again translated to improved
CYP3A4 margins. Looking to restore some TDO potency to
24 by slight modulation of lipophilicity, N-methyl tetrahy-
droindazol-4-ol 31 was investigated. Despite generating a
reasonable docking pose, the substitution pattern was not well
tolerated by TDO, leading to further loss in potency.
moderately stable by hepatocyctes (CLhep MHep = 30−70%
LBF), whereas the opposite was true for compound 29,
exhibiting increased lability in hepatocytes relative to micro-
somes (Table 5). Only compound 30 binned as moderate
Table 5. Mouse In Vitro and In Vivo Pharmacokinetics of
Compounds 20, 29, and 30
20
71
33
11
139
29
29
45
73
23
165
63
30
46
56
13
149
28
CLhep MLM (mL × min−1 × kg−1
)
)
)
CLhep MHep (mL × min−1 × kg−1
MDCK Papp (A:B) (10−6 cm × s−1
kinetic solubility (μM)
CLp (mL × min−1 × kg−1
)
PPB fub
7
410
27
9
310
CLp,ub (mL × min−1 × kg−1
)
230
1.1
0.21
<5
V
ss (L × kg−1
T1/2 (h)
)
1.4
0.62
68
0.34
78
1.1
0.78
140
0.81
70
%F cassette (1 mg × kg−1
AUC∞ (μM × h)
)
)
a
BLQ
%F discrete (5 mg × kg−1
a
BLQ = Below limit of quantification.
stability in both liver microsomes and hepatocytes. All three
inhibitors, however, categorically fell into high solubility (kinetic
solubility > 100 μM) and high permeability (MDCK Papp A:B >
10 × 10−6 cm × s−1) space ideal for an orally bioavailable
molecule. Upon discrete intravenous dosing in mice, both 20
and 30 exhibited moderate plasma clearance (CLp = 29 and 28
mL × min−1 × kg−1, respectively), whereas compound 29 was
rapidly cleared (CLp = 63 mL × min−1 × kg−1). Correcting for
differences in plasma protein binding, however, indicated the
unbound clearances were all comparable. When dosed in oral
cassette PK studies, furazan compound 29 was not detected (F
below limit of quantification), while both pyridine 20 and
pyrazole 30 showed good exposure, which was confirmed in
subsequent discrete oral studies.
Due to the indistinguishable murine pharmacokinetic profiles
of compounds 20 and 30, the former was advanced into a PK/
PD study on the basis of superior TDO potency. As a
consequence of poor mouse-to-human TDO homology,
human inhibitors typically suffer one logarithm loss in potency
against the murine isoform; thus, conducting pharmacodynamic
studies with the more potent molecule was critical for proof-of-
concept in vivo target engagement. Treatment with 20 resulted
in a dose-dependent increase in blood−tryptophan concen-
trations (Figure 5). Above a 30 mg × kg−1 dose of 20, minimal
increase in TRP Cmax was observed; however, a prolonged
increase in TRP concentration resulted in a 2-fold increase in
AUC0−6h upon dose escalation to 100 mg × kg−1 (Table 6).
Moreover, in the maximum dose cohort, a nearly 4-fold increase
in TRP AUC was observed relative to vehicle.
In conclusion, utilizing structure-based drug design, we
designed a series of TDO-selective inhibitors from the scaffold
of the IDO-selective inhibitor GDC-0919. A novel and intuitive
design parameter, coined the CYP Index, was conceived and
implemented to facilitate the design of heme-binding inhibitors
with reduced CYP3A4 inhibition. Moreover, we believe the CYP
Index may be utilized in any discovery program to optimize away
from CYP inhibition due to presence of a heme-binding motif. A
tool compound was identified suitable for demonstrating dose-
dependent increases in blood tryptophan concentrations as a
pharmacodynamic indicator of TDO inhibition in vivo. We
Based on the combination of TDO potency, CYP3A4 and
IDO1 selectivity and in vitro stability, we advanced compounds
20, 29, and 30 on to mouse in vivo pharmacokinetic studies to
identify a suitable candidate for subsequent PK/PD studies. The
three compounds exhibited complementary mouse liver micro-
some and hepatocyte predicted stability profiles, which we
anticipated would be helpful for understanding higher species in
vitro−in vivo correlations. Compound 20 was predicted to be
labile by liver microsomes (CLhep MLM > 70% LBF) and
F
https://dx.doi.org/10.1021/acsmedchemlett.0c00004
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Benjamin D. Sellers − Genentech, South San Francisco,
California 94080, United States; orcid.org/0000-0002-
4194-1240
Zhonghua Pei − Genentech, South San Francisco, California
94080, United States
Firoz A. Jaipuri − NewLink Genetics, Ames, Iowa 50010, United
States
Georgette M. Castanedo − Genentech, South San Francisco,
California 94080, United States; orcid.org/0000-0003-
4584-6715
Lewis Gazzard − Genentech, South San Francisco, California
94080, United States
Sanjeev Kumar − NewLink Genetics, Ames, Iowa 50010, United
States
Xiaokai Li − NewLink Genetics, Ames, Iowa 50010, United States
Wen Liu − Genentech, South San Francisco, California 94080,
United States
Rohan Mendonca − Genentech, South San Francisco, California
94080, United States
Figure 5. Mouse pharmacodynamics of compound 20
Roheeth K. Pavana − NewLink Genetics, Ames, Iowa 50010,
United States
Hima Potturi − NewLink Genetics, Ames, Iowa 50010, United
States
Cheng Shao − Pharmaron, Beijing 100176, People’s Republic of
China
Venkata Velvadapu − NewLink Genetics, Ames, Iowa 50010,
United States
Jesse P. Waldo − NewLink Genetics, Ames, Iowa 50010, United
States
Guosheng Wu − Pharmaron, Beijing 100176, People’s Republic
of China
Table 6. Dose−Response Summary for Mouse PK/PD Study
of Compound 20
20
TRP
max (μM) AUC0−6h (μM × h)
570
a
dose
AUC∞ (μM × h)
C
Cmax (μM)
0
10
30
340
420
560
540
5.0
35
210
4.1
21.2
70
680
1,120
1,980
100
a
mg × kg−1 compound 20.
Po-wai Yuen − Pharmaron, Beijing 100176, People’s Republic of
China
Zuhui Zhang − NewLink Genetics, Ames, Iowa 50010, United
States
Yamin Zhang − Pharmaron, Beijing 100176, People’s Republic of
China
Seth F. Harris − Genentech, South San Francisco, California
94080, United States
believe that these tool molecules will be valuable in assessing
TDO as a viable target for cancer immunotherapy as single agent
or in combination with IDO inhibitors.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00004.
Angela J. Oh − Genentech, South San Francisco, California
94080, United States
Antonio DiPasquale − Genentech, South San Francisco,
Assay conditions, experimental procedures, and crystal-
lography data (PDF)
X-ray coordinates for 7 (CIF)
X-ray coordinates for 8 (CIF)
X-ray coordinates for 9 (CIF)
X-ray coordinates for 20 (CIF)
X-ray coordinates for ent-12 (CIF)
X-ray coordinates for ent-13 (CIF)
X-ray coordinates for ent-14 (CIF)
X-ray coordinates for ent-21 (CIF)
X-ray coordinates for ent-22 (CIF)
X-ray coordinates for ent-30 (CIF)
California 94080, United States
Kevin Dement − Genentech, South San Francisco, California
94080, United States
Hank La − Genentech, South San Francisco, California 94080,
United States
Leanne Goon − Genentech, South San Francisco, California
94080, United States
Amy Gustafson − Genentech, South San Francisco, California
94080, United States
Erica C. VanderPorten − Genentech, South San Francisco,
California 94080, United States
Mario R. Mautino − NewLink Genetics, Ames, Iowa 50010,
United States; orcid.org/0000-0003-2729-4996
Yichin Liu − Genentech, South San Francisco, California 94080,
United States
AUTHOR INFORMATION
Corresponding Author
Brendan T. Parr − Genentech, South San Francisco, California
94080, United States; orcid.org/0000-0001-5774-8122;
Email: parr.brendan@gene.com
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00004
Authors
Richard Pastor − Genentech, South San Francisco, California
94080, United States
G
https://dx.doi.org/10.1021/acsmedchemlett.0c00004
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
(7) Suzuki, Y.; Suda, T.; Furuhashi, K.; Suzuki, M.; Fujie, M.;
Hahimoto, D.; Nakamura, Y.; Inui, N.; Nakamura, H.; Chida, K.
Increased serum kynurenine/tryptophan ratio correlates with disease
progression in lung cancer. Lung Cancer 2010, 67, 361−365.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(8) de Jong, R. A.; Nijman, H. W.; Boezen, H. M.; Vomer, M.; ten
Hoor, K. A.; Krijnen, J.; van der Zee, A. G. J.; Hollema, H.; Kema, I. P.
Serum tryptophan and kynurenine concentrations as parameters for
indoleamine 2,3-dioxygenase activity in patients with endometrial,
ovarian, and vulvar cancer. Int. J. Gynecol. Cancer 2011, 21, 1320−1327.
(9) Yue, E. W.; Sparks, R.; Polam, P.; Modi, D.; Douty, B.; Wayland,
B.; Glass, B.; Takvorian, A.; Glenn, J.; Zhu, W.; Bower, M.; Liu, X.;
Leffet, L.; Wang, Q.; Bowman, K. J.; Hansbury, M. J.; Wei, M.; Li, Y.;
Wynn, R.; Burn, T. C.; Koblish, H. K.; Fridman, J. S.; Emm, T.; Scherle,
P. A.; Metcalf, B.; Combs, A. P. INCB24360 (Epacadostat), a highly
potent and selective indoleamine-2,3-dioxygenase 1 (IDO1) inhibitor
for immuno-oncology. ACS Med. Chem. Lett. 2017, 8, 486−491.
(10) Crosignani, S.; Bingham, P.; Bottemanne, P.; Cannelle, H.;
Cauwenbergs, S.; Cordonnier, M.; Dalvie, D.; Deroose, F.; Feng, J. L.;
Notes
The authors declare no competing financial interest.
Absolute stereochemical configuration of compounds 7, 8, 9,
ent-12, ent-13, ent-14, 20, ent-21, ent-22, and ent-30 were
determined by single crystal X-ray crystallographic analysis.
Structure factors and coordinates are deposited with the CSD
and PDB under accession code 6VBN. The configuration of all
other molecules was tentatively assigned by analogy to the most
TDO-potent isomer of the stereotetrad or stereo-octet.
ACKNOWLEDGMENTS
■
Crystallographic data were collected at the Stanford Synchrto-
tron Radiation Lightsource beamline 12-2. Use of the SSRL,
SLAC National Accelerator Laboratory, is supported by the U.S.
Department of Energy, Office of Science, Office of Basic Energy
Sciences under Contract No. DE-AC02-76SF00515. The SSRL
Structural Molecular Biology Program is supported by the DOE
Office of Biological and Environmental Research, and by the
National Institutes of Health, National Institute of General
Medical Sciences (P41GM103393). The contents of this
publication are solely the responsibility of the authors and do
not necessarily represent the official views of NIGMS or NIH.
́
Gomes, B.; Greasley, S.; Kaiser, S. E.; Kraus, M.; Negrerie, M.; Maegley,
K.; Miller, N.; Murray, B. W.; Schneider, M.; Soloweij, J.; Stewart, A. E.;
Tumang, J.; Torti, V. R.; Van Den Eynde, B.; Wythes, M. Discovery of a
novel and selective indoleamine 2,3-dioxygenase (IDO-1) inhibitor 3-
(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (EOS200271/PF-
06840003) and its characterization as a potential clinical candidate. J.
Med. Chem. 2017, 60, 9617−9629.
(11) Kumar, S.; Waldo, J. P.; Jaipuri, F. A.; Marcinowicz, A.; Van Allen,
C.; Adams, J.; Kesharwani, T.; Zhang, X.; Metz, R.; Oh, A. J.; Harris, S.
F.; Mautino, M. R. Discovery of clinical candidate (1R,4r)-4-((R)-2-
((S)-6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)-
cyclohexan-1-ol (Navoximod), a potent and selective inhibitor of
indoleamine 2,3-dioxygenase 1. J. Med. Chem. 2019, 62, 6705−6733.
(12) Prendergast, G. C.; Malachowski, W. P.; DuHadaway, J. B.;
Muller, A. J. Discovery of IDO1 inhibitors: from bench to bedside.
Cancer Res. 2017, 77, 6795−6811.
(13) Yang, S.; Li, X.; Hu, F.; Li, Y.; Yang, Y.; Yan, J.; Kuang, C.; Yang,
Q. Discovery of tryptanthrin derivatives as potent inhibitors of
indoleamine 2,3-dioxygenase with therapeutic activity in lewis lung
cancer (LLC) tumor-bearing mice. J. Med. Chem. 2013, 56, 8321−
8331.
(14) Zhang, H.; Liu, K.; Pu, Q.; Achab, A.; Ardolino, M. J.; Cheng, M.;
Deng, Y.; Doty, A. C.; Ferguson, H.; Fradera, X.; Knemeyer, I.;
Kurukulasuriya, R.; Lam, Y.-h.; Lesburg, C. A.; Martinot, T. A.;
McGowan, M. A.; Miller, J. R.; Otte, K.; Biju, P. J.; Sciammetta, N.;
Solban, N.; Yu, W.; Zhou, H.; Wang, X.; Bennett, D. J.; Han, Y.
Discovery of amino-cyclobutarene-derived indoleamine-2,3-dioxyge-
nase 1 (IDO1) inhibitors for cancer immunotherapy. ACS Med. Chem.
Lett. 2019, 10, 1530.
ABBREVIATIONS
■
AUC, area under the curve; CLhep, predictive hepatic clearance;
CLp, plasma clearance; cLogP, calculated water−octanol
partition coefficient; fub, unbound fraction; LogD, water−
octanol partition coefficient at pH = 7.4; LBF, liver blood flow;
MDCK, Madin-Darby canine kidney cell line; MHep, mouse
hepatocytes; MLM, mouse liver microsomes; Papp (A:B),
apparent permeability form the apical to basolateral chamber;
PPB, plasma protein binding; QM, quantum mechanics; T1/2,
plasma half-life; Vss, steady-state volume of distribution; %F,
percent fraction absorbed
REFERENCES
■
(1) Mahoney, K. M.; Rennert, P. D.; Freeman, G. J. Combination
cancer immunotherapy and new immunomodulatory targets. Nat. Rev.
Drug Discovery 2015, 14, 561−584.
(15) Munn, D. H.; Mellor, A. L. Indoleamine 2,3-dioxygenase and
metabolic control of immune responses. Trends Immunol. 2013, 34,
137−143.
́
(2) Uyttenhove, C.; Pilotte, L.; Theate, I.; Stroobant, C.; Colau, D.;
Parmentier, N.; Boon, T.; Van den Eynde, B. J. Evidence for a tumoral
immune resistance mechanism based on tryptophan degradation by
indoleamine-2,3-dioxygenase. Nat. Med. 2003, 9, 1269−1274.
(3) Godin-Ethier, J.; Hanafi, L.-A.; Piccirillo, C. A.; Lapointe, R.
Indoleamine-2,3-dioxygenase expression in human cancers: clinical and
immunologic perspectives. Clin. Cancer Res. 2011, 17, 6985−6991.
(4) Platten, M.; von Knebel Doeberitz, N.; Oezen, I.; Wick, W.; Ochs,
K. Cancer immunotherapy by targetining IDO1/TDO and their
downstream effectors. Front. Immunol. 2015, 5, 673.
(5) Koblish, H. K.; Hansbury, M. J.; Bowman, K. J.; Yang, G.; Neilan,
C. L.; Haley, P. J.; Burn, T. C.; Waeltz, P.; Sparks, R. B.; Yue, E. W.;
Combs, A. P.; Scherle, P. A.; Vaddi, K.; Fridman, J. S. Hydroxyamidine
inhibitors of indoleamine-2,3-dioxygenase potently suppress systemic
tryptophan catabolism and growth of IDO-expressing tumors. Mol.
Cancer Ther. 2010, 9, 489−498.
(16) Pallotta, M. T.; Orabona, C.; Volpi, C.; Vacca, C.; Belladonna, M.
L.; Bianchi, R.; Servillo, G.; Brunacci, C.; Calvitti, M.; Bicciato, S.;
Mazza, E. M. C.; Boon, L.; Grassi, F.; Fioretti, M. C.; Fallarino, F.;
Puccetti, P.; Grohmann, U. Indoleamine 2,3-dioxygenase is a signaling
protein in long-term tolerance by dendritic cells. Nat. Immunol. 2011,
12, 870−880.
(17) Sharma, M. D.; Hou, D.-Y.; Baban, B.; Koni, P. K.; He, Y.;
Chandler, P. R.; Blazar, B. R.; Mellor, A. L.; Munn, D. H.
Reprogrammed Foxp3+ regulatory T cells provide essential help to
̈
support cross-presentation and CDK8+ T cell priming in naive mice.
Immunity 2010, 33, 942−954.
̌
́
(18) Pilotte, L.; Larrieu, P.; Stroobant, V.; Colau, D.; Dolusic, E.;
́
́
Frederick, R.; De Plaen, E.; Uyttenhove, C.; Wouters, J.; Masereel, B.;
Van den Eynde, B. J. Reversal of tumoral immune resistance by
inhibition of tryptophan 2,3-dioxygenase. Proc. Natl. Acad. Sci. U. S. A.
2012, 109, 2497−2502.
(6) D’Amato, N. C.; Rogers, T. J.; Gordon, M. A.; Greene, L. I.;
Cochrane, D. R.; Spoelstra, N. S.; Nemkov, T. G.; D’Alessandro, A.;
Hansen, K. C.; Richer, J. K. A TDO2-AhR signaling axis facilitates
anoikis resistance and metastasis in triple-negative breast cancer. Cancer
Res. 2015, 75, 4651−4664.
(19) Opitz, C. A.; Litzenburger, U. M.; Sahm, F.; Ott, M.; Tritschler,
I.; Trump, S.; Schumacher, T.; Jestaedt, L.; Schrenk, D.; Weller, M.;
Jugold, M.; Guillemin, G. J.; Miller, C. L.; Lutz, C.; Radlwimmer, B.;
H
https://dx.doi.org/10.1021/acsmedchemlett.0c00004
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Lehmann, I.; von Deimling, A.; Wick, W.; Platten, M. An endogenous
tumour-promoting ligand of the human aryl hydrocarbon receptor.
Nature 2011, 478, 197−203.
̈
(20) Wang, Y.; Ning, Y.; Alam, G. N.; Jankowski, B. M.; Dong, Z.; Nor,
J. E.; Polverini, P. J. Amino acid deprivation promotes tumor
angiogenesis through the GCN2/ATF4 pathway. Neoplasia 2013, 15,
989−997.
(21) Munn, D. H.; Sharma, M. D.; Baban, B.; Harding, H. P.; Zhang,
Y.; Ron, D.; Mellor, A. L. GCN2 kinase in T cells mediates proliferative
arrest and anergy induction in response to indoleamine 2,3-
dioxygenase. Immunity 2005, 22, 633−642.
(22) Eleftheriadis, T.; Pissas, G.; Antoniadi, G.; Liakopoulos, V.;
Tsogka, K.; Sounidaki, M.; Stefanidis, I. Differential effects of the two
amino acid sensing systems, the GCN2 kinase and the mTOR complex
1, on primary human alloreactive CDK4+ T-cells. Int. J. Mol. Med. 2016,
37, 1412−1420.
(23) Kumar, S.; Sharma, R.; Roychowdhury, A. Modulation of
cytochrome P450 inhibition (CYP) in drug discovery: a medicinal
chemistry perspective. Curr. Med. Chem. 2012, 19, 3605−3621.
I
https://dx.doi.org/10.1021/acsmedchemlett.0c00004
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX