Discovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor of Soluble Adenylyl Cyclase (sAC, ADCY10)

Article abstract of DOI:10.1021/acsmedchemlett.1c00273

Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.

Full text of DOI:10.1021/acsmedchemlett.1c00273

pubs.acs.org/acsmedchemlett
Letter
Discovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor
of Soluble Adenylyl Cyclase (sAC, ADCY10)
Makoto Fushimi, Hannes Buck, Melanie Balbach, Anna Gorovyy, Jacob Ferreira, Thomas Rossetti,
Navpreet Kaur, Lonny R. Levin, Jochen Buck, Jonathan Quast, Joop van den Heuvel, Clemens Steegborn,
Efrat Finkin-Groner, Stacia Kargman, Mayako Michino, Michael A. Foley, Michael Miller,
,
‡
‡
Nigel J. Liverton, David J. Huggins,* and Peter T. Meinke
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sı Supporting Information
ABSTRACT: Soluble adenylyl cyclase (sAC) has gained attention
as a potential therapeutic target given the role of this enzyme in
intracellular signaling. We describe successful efforts to design
improved sAC inhibitors amenable for in vivo interrogation of sAC
inhibition to assess its potential therapeutic applications. This work
culminated in the identification of TDI-10229 (12), which displays
nanomolar inhibition of sAC in both biochemical and cellular
assays and exhibits mouse pharmacokinetic properties sufficient to
warrant its use as an in vivo tool compound.
KEYWORDS: sAC, Soluble adenylyl cyclase, ADCY10, Inhibitor, Structure-based drug design
yclic AMP (cAMP), a critical second messenger, is
involved in a broad range of physiological processes.
cAMP is produced from ATP by adenylyl cyclases (ACs) and
degraded by catabolizing phosphodiesterases (PDEs). Pres-
ently, there are two known distinct classes of adenylyl cyclases
in mammals: bicarbonate-regulated soluble adenylyl cyclase
therapeutic intervention in multiple disease states, including
for hypotony or as an on-demand, nonhormonal contraceptive.
While tool compounds, including LRE1 (1) and KH7 (1b),
C
2
8
are known (Figure 1), their properties (weak potencies, low
(
sAC, ADCY10) and G protein-regulated transmembrane
adenylyl cyclases (tmACs; ADCY1−9). While PDEs are
established drug targets, at this juncture, there exist no
therapeutic agents targeting sAC.
In contrast to tmACs, which respond to signals originating
in other surrounding cells, sAC functions as an environmental
sensor: it is directly regulated by bicarbonate and serves as a
−
1
Figure 1. Structures of LRE1 and KH7.
physiological CO /HCO /pH sensor. sAC is localized in
2
3
i
intracellular microdomains and is found distributed through
the cytoplasm and in cellular organelles, including inside the
nucleus and the mitochondrial matrix. It regulates a diverse
selectivities, poor pharmacokinetic profiles, etc.) render them
2
unsuitable for careful scrutiny of sAC driven pharmacology in
array of biological functions, including sperm activation and
motility, intraocular pressure, ciliary beat frequency in airways,
luminal pH in the epididymis, the mitochondrial electron
transport chain, and glucose-stimulated insulin release from β
9
cellular or animal models. While additional sAC inhibitors
have been described by pharma (primarily via patent
10,11
disclosures),
limited data exists regarding their profiles.
This work describes our efforts to identify compounds,
typified by TDI-10229, that combine significantly improved
1,3
cells of the pancreas.
While sAC is ubiquitously expressed, two independently
derived sAC knockout (KO) mouse strains show male-specific
sterility with only mild other phenotypes (e.g., elevated
Received: May 12, 2021
Accepted: July 8, 2021
3
−6
intraocular pressure).
Importantly, in humans, loss of sAC
function appears similarly benign: two adult males bearing an
7
ADCY10 frameshift variant were sterile but otherwise healthy.
Based on its apparent functions, potent and selective
inhibitors of sAC potentially provide a mechanism for
©
XXXX American Chemical Society
https://doi.org/10.1021/acsmedchemlett.1c00273
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Letter
intrinsic potency with the ability to sustain systemic exposure
above cell-based IC50 values to enable investigations of sAC-
mediated biology in preclinical species.
Table 1. 4-Substituted Pyrazole Analogs
First generation sAC inhibitors, such as KH7, suffered from
general cell toxicity and potential non-sAC mediated effects on
ATP/cAMP levels that are likely to confound their utility as
2
2
tool molecules. The more recently discovered LRE1 binds in
the bicarbonate binding site of sAC, preventing its enzymatic
function. LRE1 represented a significant improvement in
overall profile relative to KH7 and permitted more extensive
interrogation of sAC biology in vitro. However, its relatively
modest potency in biochemical and cell-based assays suggests
that high micromolar levels would be required in vivo to elicit
sAC-driven pharmacology in translational experiments. LRE1
also showed low oral bioavailability (<5%) in mice, a liability
exacerbated by rapid mouse and human microsomal clearance
(
>768 and 360 μL/(min/mg), respectively), further limiting in
vivo options.
More potent sAC inhibitors with appropriate systemic PK to
afford sustained sAC inhibition in vivo would represent a
significant advance in enabling interrogation of sAC
pharmacology in vivo. As a low molecular weight (280.8 g/
mol) lead, LRE1 represented an auspicious entry point for
medicinal chemistry efforts focused on discovering superior
sAC inhibitors. Along with improved potency, microsomal
stability and pharmacokinetics, we also wished to evolve the
LRE1 lead toward more drug-like molecules, lacking the
cyclopropylamine and monosubstituted thiophene moieties,
12,13
which represent potential metabolic liabilities.
We leveraged significant structural biology data throughout
this project. In the first instance, utilization of free-energy
14
perturbation (FEP) calculations with FEP+ based on the X-
2
ray crystal structure of LRE1 bound to sAC led to the design
of 2 through scaffold hopping (Table 1). Compound 2 bears a
methylpyrazole in lieu of the less desirable cyclopropylamino
moiety and yields a 10-fold improvement in potency over
LRE1. Medicinal chemistry efforts next focused on mod-
ifications around the conserved aminochloropyrimidine head-
group. Unfortunately, this headgroup proved to have extremely
narrow SAR, as predicted by FEP+, with very small changes,
such as chloro (6) to methyl (10), leading to a dramatic loss in
activity. We then moved on to replacement of the undesirable
thiophene ring. It was noted that various aromatic and
heteroaromatic moieties were well tolerated on the 4-position
methylene of the pyrazole (3−8), whereas direct attachment of
the thiophene ring to the pyrazole (9) was not. Of particular
note is that substituted phenyl rings, such as the fluorophenyl
in compound 6, yielded similar potency to the thiophene
compound 2.
a
Compounds were tested at minimum in triplicate.
2
Furthermore, substitution of the R methyl group with hydroxy
(21) or dimethylamino (22) resulted in reduced potency
versus methyl (12). To analyze the binding details of our
improved inhibitors and to aid in further lead optimization, we
then obtained crystallographic data with 12 bound to sAC (see
Figure 2; Supplementary Table 1; Supplementary Figure 1).
Figure 2 shows the conserved overlap of the two
aminochloropyrimidine headgroups. Importantly, the key
hydrogen-bonding contacts made by the aminochloropyrimi-
dine headgroup of LRE1 are maintained in 12. Compared to
LRE1, a very subtle rotation of the headgroup of 12 allows it to
position its central pyrazole group in the area occupied by
LRE1’s central amine and additional cyclopropane substituent.
The larger pyrazole moiety fills this area more completely and
its additional interactions likely contribute to the higher
affinities of 2 and 12. The phenyl group of 12 is also oriented
similarly to LRE1’s thiophene, albeit with a slightly rotated ring
plane. The rotation and larger ring of 12 are accommodated by
a minor rearrangement of the interacting Arg176, which
Addition of a methyl group at the 5-position of the pyrazole
was also noted to lead to a small improvement in potency as
shown in Table 2 (IC50 of 140 nM in 2 vs IC50 of 74 nM in
11). Gratifyingly, the equivalent methyl substitution on the
phenyl compound 7 also improved potency and yielded
compound 12 with an IC of 195 nM. Alternative ring systems
50
were also found to retain potency in this context (13−15),
while others abrogated affinity (16 and 17). Next, we
investigated the effect of substitution at the 5-position of the
2
pyrazole ring (Table 2). Substitution at R on the central
pyrazole was found to be sensitive to functionality. For
example, the methyl ester substituted analog 18 exhibited
higher potency, while other closely related analogs displayed
reduced potency (e.g., acid 19 or methyl amide 20).
2
previously was observed to adopt multiple different rotamers.
B
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Table 2. 5-Substituted Pyrazole Analogs
a
Compounds were tested at minimum in triplicate unless otherwise noted.
permeability (parallel artificial membrane permeability assay,
PAMPA), it was gratifying to see much improved inhibition of
sAC in a cellular assay (Table 3).
Table 3. Cell Activity and Selected Parameters
a
cell IC50
(nM)
PAMPA
(nm/s)
HPLC LogD
(pH = 7.4)
kinetic solubility
compd
(μg/mL)
2
3
1
1
196
225
102
92
258
257
280
274
2.5
2.8
2.7
2.9
6.3
7.5
0.38
1.3
1
2
a
Human 4-4 cell data: compounds were tested at minimum in
triplicate.
Of the new analogs generated, TDI-10229 (12) was deemed
Figure 2. Overlay of crystal structures of sAC complexes with
compound 12 and LRE1. The crystal structure of sAC in complex
with 12 is displayed as a gray cartoon with gray wires and overlaid
with the ligand from the crystal structure of sAC in complex with
LRE1. Compound 12 is displayed as atom-colored balls and sticks
with green carbons. LRE1 is displayed as atom-colored balls and sticks
with magenta carbons.
most promising overall in terms of activity coupled with drug-
like qualities and was therefore selected for additional
characterization. Mouse pharmacokinetic studies of TDI-
10229 indicated that this compound, with its 59% bioavail-
ability, is well suited for interrogation of sAC inhibition in an in
vivo context at reasonable dosages with coverage up to 50-fold
over the cell IC based on free drug concentrations (mouse fu
5
0
The protein conformations are otherwise very similar between
the two structures.
With sAC inhibitors displaying improved intrinsic potency
relative to LRE1 in hand, their activity was evaluated in a
cellular context. Given that many compounds exhibited good
= 0.1, Table 4). Furthermore, TDI-10229 was not cytotoxic at
20 μM (200-fold above its IC50 for sAC in cells), it had a low
propensity to form reactive intermediates (glutathione trapping
study), and it did not show appreciable activity against a
panel of 310 kinases and 46 other well-known drug targets
16
C
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Table 4. Mouse Pharmacokinetic Properties of TDI-10229
Lonny R. Levin − Department of Pharmacology, Weill Cornell
Medicine, New York, New York 10021, United States
Jochen Buck − Department of Pharmacology, Weill Cornell
Medicine, New York, New York 10021, United States
Jonathan Quast − Department of Biochemistry, University of
Bayreuth, 95440 Bayreuth, Germany
(12)
a
dosage
AUC (μM·h)
Cmax (μM)
MRT (h)
2
2
0 mg/kg (po)
0 mg/kg (ip)
94
72
15.5
45
3.95
2.82
a
Mean residence time.
Joop van den Heuvel − Helmholtz-Zentrum fur
̈
Infektionsforschung, 38124 Braunschweig, Germany
Clemens Steegborn − Department of Biochemistry, University
of Bayreuth, 95440 Bayreuth, Germany; orcid.org/0000-
(
GPCRs, ion channels, and nuclear receptors) [Supporting
0002-0913-1467
Information S1]. TDI-10229 also displayed high selectivity for
sAC versus the closely related tmAC subtypes.
Efrat Finkin-Groner − Tri-Institutional Therapeutics
Discovery Institute, New York, New York 10021, United
States
15
To summarize, in order to identify an inhibitor suitable for
exploration of sAC-driven pharmacology in vivo, a set of sAC
inhibitors derived from LRE1 were designed and synthesized,
efforts facilitated by structural biology input coupled to
computational drug design tools. From the novel analogs
synthesized, TDI-10229 displayed suitable potency, selectivity,
and drug-like properties to facilitate in vivo studies of sAC
pharmacology in preclinical animal models of disease,
including its utility for hypotony or as an on-demand,
Stacia Kargman − Tri-Institutional Therapeutics Discovery
Institute, New York, New York 10021, United States
Mayako Michino − Tri-Institutional Therapeutics Discovery
Institute, New York, New York 10021, United States
Michael A. Foley − Tri-Institutional Therapeutics Discovery
Institute, New York, New York 10021, United States
Michael Miller − Tri-Institutional Therapeutics Discovery
Institute, New York, New York 10021, United States
Nigel J. Liverton − Tri-Institutional Therapeutics Discovery
Institute, New York, New York 10021, United States
Peter T. Meinke − Tri-Institutional Therapeutics Discovery
Institute, New York, New York 10021, United States;
Department of Pharmacology, Weill Cornell Medicine, New
York, New York 10021, United States
3
,9,15
nonhormonal contraceptive.
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00273.
■
*
Synthesis and characterization for compounds 2−22,
biology assay methods, ADMET assay methods, mouse
pharmacokinetic studies, crystal structure determination,
diffraction and refinement statistics, crystal structure of
hsAC-cat in complex with TDI-10229 (12), and
supplementary references (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00273
Author Contributions
‡
D.J.H. and P.T.M. contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Accession Codes
Coordinates and structure factors for the sAC/TDI-10229
crystal structure have been deposited with the worldwide PDB
Funding
(
www.pdb.org) under accession ID 7OVD.
This work was supported by NIH Grants P50 HD100549 and
R01 HD088571 (to J.B. and L.R.L.), DFG grant STE1701/11
(C.S.), F31 AG069501 (T.R.), T32 GM073546-13 (J.F.), and
Male Contraceptive Initiative (M.B.).
AUTHOR INFORMATION
■
Corresponding Author
David J. Huggins − Tri-Institutional Therapeutics Discovery
Institute, New York, New York 10021, United States;
Department of Physiology and Biophysics, Weill Cornell
Medicine, New York, New York 10021, United States;
orcid.org/0000-0003-1579-2496; Phone: +1-646-962-
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
6
133; Email: dhuggins@tritdi.org; www.tritdi.org
We thank the BESSY beamline staff for excellent technical
1
3
Authors
support and Dr. Tanweer Khan for generating C NMR data.
The authors gratefully acknowledge the support to the project
generously provided by the Tri-Institutional Therapeutics
Discovery Institute (TDI), a 501(c)(3) organization. TDI
receives financial support from Takeda Pharmaceutical
Company, TDI’s parent institutes (Memorial Sloan Kettering
Cancer Center, The Rockefeller University, and Weill Cornell
Medicine) and from a generous contribution from Mr. Lewis
Sanders and other philanthropic sources.
Makoto Fushimi − Tri-Institutional Therapeutics Discovery
Institute, New York, New York 10021, United States
Hannes Buck − Department of Pharmacology, Weill Cornell
Medicine, New York, New York 10021, United States
Melanie Balbach − Department of Pharmacology, Weill
Cornell Medicine, New York, New York 10021, United States
Anna Gorovyy − Department of Pharmacology, Weill Cornell
Medicine, New York, New York 10021, United States
Jacob Ferreira − Department of Pharmacology, Weill Cornell
Medicine, New York, New York 10021, United States
Thomas Rossetti − Department of Pharmacology, Weill
Cornell Medicine, New York, New York 10021, United States
Navpreet Kaur − Department of Pharmacology, Weill Cornell
Medicine, New York, New York 10021, United States
ABBREVIATIONS
■
AC, adenylyl cyclase; sAC, soluble adenylyl cyclase; tmAC,
transmembrane adenylyl cyclase; po, per os; ip, intraperitoneal
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15) Balbach, M.; Ghanem, L.; Rossetti, T.; Kaur, N.; Ritagliati, C.;
REFERENCES
■
Ferreira, J.; Krapf, D.; Molina, L. P.; Santi, C. M.; Hansen, J. N.;
Wachten, D.; Fushimi, M.; Meinke, P. T.; Buck, J.; Levin, L. R.
Soluble adenylyl cyclase inhibition prevents human sperm functions
essential for fertilization. Mol. Hum. Reprod. 2021, submitted for
publication. https://www.biorxiv.org/content/10.1101/2021.04.27.
(
1) Rossetti, T.; Jackvony, S.; Buck, J.; Levin, L. R. Bicarbonate,
carbon dioxide and pH sensing via mammalian bicarbonate-regulated
soluble adenylyl cyclase. Interface Focus 2021, 11, 20200034.
2) Ramos-Espiritu, L.; Kleinboelting, S.; Navarrete, F. A.; Alvau, A.;
Visconti, P. E.; Valsecchi, F.; Starkov, A.; Manfredi, G.; Buck, H.;
Adura, C.; Zippin, J. H.; van den Heuvel, J.; Glickman, J. F.;
Steegborn, C.; Levin, L. R.; Buck, J. Discovery of LRE1 as a specific
and allosteric inhibitor of soluble adenylyl cyclase. Nat. Chem. Biol.
016, 12, 838−844.
3) Wiggins, S. V.; Steegborn, C.; Levin, L. R.; Buck, J.
(
441671v1.article-info.
(
16) Sameshima, T.; Miyahisa, I.; Yamasaki, S.; Gotou, M.;
Kobayashi, T.; Sakamoto, J. High-Throughput Quantitative Intrinsic
Thiol Reactivity Evaluation Using a Fluorescence-Based Competitive
Endpoint Assay. SLAS Discov 2017, 22, 1168−1174.
2
(
Pharmacological modulation of the CO2/HCO3-/pH-, calcium-,
and ATP-sensing soluble adenylyl cyclase. Pharmacol. Ther. 2018,
NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published ASAP on July 14, 2021, with errors
in the Supporting Information. The corrected version was
reposted on July 20, 2021.
1
(
90, 173−186.
4) Esposito, G.; Jaiswal, B. S.; Xie, F.; Krajnc-Franken, M. A. M.;
Robben, T. J. A. A.; Strik, A. M.; Kuil, C.; Philipsen, R. L. A.; van
Duin, M.; Conti, M.; Gossen, J. A. Mice deficient for soluble adenylyl
cyclase are infertile because of a severe sperm-motility defect. Proc.
Natl. Acad. Sci. U. S. A. 2004, 101, 2993−2998.
(5) Hess, K. C.; Jones, B. H.; Marquez, B.; Chen, Y.; Ord, T. S.;
Kamenetsky, M.; Miyamoto, C.; Zippin, J. H.; Kopf, G. S.; Suarez, S.
S.; Levin, L. R.; Williams, C. J.; Buck, J.; Moss, S. B. The ″soluble″
adenylyl cyclase in sperm mediates multiple signaling events required
for fertilization. Dev. Cell 2005, 9, 249−259.
(6) Xie, F.; Garcia, M. A.; Carlson, A. E.; Schuh, S. M.; Babcock, D.
F.; Jaiswal, B. S.; Gossen, J. A.; Esposito, G.; van Duin, M.; Conti, M.
Soluble adenylyl cyclase (sAC) is indispensable for sperm function
and fertilization. Dev. Biol. 2006, 296, 353−362.
(7) Akbari, A.; Pipitone, G. B.; Anvar, Z.; Jaafarinia, M.; Ferrari, M.;
Carrera, P.; Totonchi, M. ADCY10 frameshift variant leading to
severe recessive asthenozoospermia and segregating with absorptive
hypercalciuria. Hum. Reprod. 2019, 34, 1155−1164.
(8) Steegborn, C. Structure, mechanism, and regulation of soluble
adenylyl cyclases - similarities and differences to transmembrane
adenylyl cyclases. Biochim. Biophys. Acta, Mol. Basis Dis. 2014, 1842,
2
(
535−2547.
9) Balbach, M.; Fushimi, M.; Huggins, D. J.; Steegborn, C.; Meinke,
P. T.; Levin, L. R.; Buck, J. Optimization of lead compounds into on-
demand, nonhormonal contraceptives: leveraging a public-private
drug discovery institute collaboration. Biol. Reprod. 2020, 103, 176−
1
(
82.
10) Saalau-Bethell, S. M.; Berdini, V.; Cleasby, A.; Congreve, M.;
Coyle, J. E.; Lock, V.; Murray, C. W.; O’Brien, M. A.; Rich, S. J.;
Sambrook, T.; Vinkovic, M.; Yon, J. R.; Jhoti, H. Crystal structure of
human soluble adenylate cyclase reveals a distinct, highly flexible
allosteric bicarbonate binding pocket. ChemMedChem 2014, 9, 823−
3
(
2.
11) Buchmann, B.; Harter, M.; Cancho-Grande, Y.; Kosemund, D.;
Schirok, H.; Nguyen, D.; Fritsch, M. Azaindoles as Inhibitors of
Soluble Adenylate Cyclase. WO 2009/030725 A2, 2009.
(12) Macmillan, D. The use of Derek Nexus to facilitate decision-
making in chemical safety assessment. Application of non-animal
approaches for decision-making in chemical safety assessment,
December 10−11, 2018, NC3Rs, London, UK. https://www.
lhasalimited.org/publications/the-use-of-derek-nexus-to-facilitate-
decision-making-in-chemical-safety-assessment/5183.
(13) Ellison, C. M.; Madden, J. C.; Judson, P.; Cronin, M. T. D.
Using In Silico Tools in a Weight of Evidence Approach to Aid
Toxicological Assessment. Mol. Inf. 2010, 29, 97−110.
(14) Wang, L.; Wu, Y.; Deng, Y.; Kim, B.; Pierce, L.; Krilov, G.;
Lupyan, D.; Robinson, S.; Dahlgren, M. K.; Greenwood, J.; Romero,
D. L.; Masse, C.; Knight, J. L.; Steinbrecher, T.; Beuming, T.; Damm,
W.; Harder, E.; Sherman, W.; Brewer, M.; Wester, R.; Murcko, M.;
Frye, L.; Farid, R.; Lin, T.; Mobley, D. L.; Jorgensen, W. L.; Berne, B.
J.; Friesner, R. A.; Abel, R. Accurate and Reliable Prediction of
Relative Ligand Binding Potency in Prospective Drug Discovery by
Way of a Modern Free-Energy Calculation Protocol and Force Field.
J. Am. Chem. Soc. 2015, 137, 2695−2703.
E
https://doi.org/10.1021/acsmedchemlett.1c00273
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