Discovery of Orally Active Hydroxyethylamine Based SPPL2a Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00044

SPPL2a (Signal Peptide Peptidase Like 2a) is an intramembrane aspartyl protease engaged in the function of B-cells and dendritic cells. Despite being an attractive target for modulation of the immune system, selective SPPL2a inhibitors are barely describe

Full text of DOI:10.1021/acsmedchemlett.9b00044

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Discovery of Orally Active Hydroxyethylamine Based SPPL2a
Inhibitors
Juraj Velcicky,*,† Casey J. N. Mathison,§ Victor Nikulin,§ Daniel Pflieger,† Robert Epple,§
Mihai Azimioara,§ Christopher Cow,§ Pierre-Yves Michellys,§ Pascal Rigollier,† Daniel R. Beisner,§
Ursula Bodendorf,‡ Danilo Guerini,‡ Bo Liu,§ Ben Wen,§ Samantha Zaharevitz,§ and Trixi Brandl†
‡
†Global Discovery Chemistry, and Autoimmunity Transplantation Inflammation, Novartis Institutes for BioMedical Research,
CH-4002 Basel, Switzerland
§The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, United
States
S
* Supporting Information
ABSTRACT: SPPL2a (Signal Peptide Peptidase Like 2a) is
an intramembrane aspartyl protease engaged in the function
of B-cells and dendritic cells. Despite being an attractive target
for modulation of the immune system, selective SPPL2a
inhibitors are barely described in the literature. Recently, we
have disclosed a selective, small molecular weight agent
SPL‑707 which confirmed that pharmacological inhibition of
SPPL2a leads to the accumulation of its substrate CD74/p8
and as a consequence to a reduction in the number of B-cells
as well as myeloid dendritic cells in mice. In this paper we
describe the discovery of novel hydroxyethylamine based SPPL2a inhibitors. Starting from a rather lipophilic screening hit,
several iterative optimization cycles allowed for its transformation into a highly potent and selective compound 15 (SPL-410)
which inhibited in vivo CD74/p8 fragment processing in mice at 10 mg/kg oral dose.
KEYWORDS: SPPL2a, CD74, hydroxyethylamine, sulfonamide, inhibitor, oral activity
CD74 for its transport from the endoplasmic reticulum to the
he immune system is a powerful host defense mechanism
Tspecializing in antigen recognition. As a consequence, it is late endosome for antigen binding.11 Once arrived in the
endosome, the membrane anchored CD74 undergoes a rapid
cathepsin mediated degradation. Subsequently, the released
MHC II binds endosomaly processed antigens and presents
them on the cell surface whereas the remaining membrane
bound CD74/p8 NTF is cleared from the membrane by
SPPL2a. The B-cell receptor (BCR) is important for the
survival of the developing B-cells.12 It could be shown that
processing of CD74/p8 NTF by SPPL2a is required for
appropriate levels of tonic BCR signaling to promote B cell
maturation.13 In line with the studies using SPPL2a deficient
mice, the SPPL2a low molecular weight inhibitor SPL-707
(Figure 1) demonstrated that pharmacological inhibition of
SPPL2a also prevents CD74/p8 NTF processing and as a
consequence leads to a reduction in the number of B-cells and
myeloid dendritic cells.14 As a part of our continuous effort
toward identification of SPPL2a inhibitors, herein we describe
the discovery of a novel, potentially active site binding scaffold.
Whereas the potent and selective SPPL2a inhibitor
SPL‑70714 could be developed starting from the known
of a vital importance for the immune system to properly learn
to distinguish between foreign and self-antigens in order to
prevent destruction of the body’s own tissue.1 Loss of tolerance
to self-antigens leads to pathological conditions which are
manifested as autoimmune diseases.2 Drugs reducing the
antigen presenting capacity of the immune system, for example
B-cell depleting agents such as rituximab (anti-CD20 anti-
body), are therapeutically beneficial in the treatment of
rheumatoid arthritis, systemic lupus erythematosus, multiple
sclerosis, or graft versus host disease.3 The successful use of B-
cell depleting agents in the control of autoimmune diseases
fueled the search for low molecular weight agents reducing the
number of antigen presenting cells (APCs) and/or their
antigen presenting capacity.4 SPPL2a (Signal Peptide
Peptidase Like 2a) is an intramembrane aspartyl protease5
shown to be engaged in the function of B-cells6−8 and
conventional dendritic cells6−9 in mice and humans. The
mechanism how SPPL2a inhibition leads to reduction in the
number of APCs is not yet fully understood. However, it could
be shown that among several type II transmembrane proteins,
SPPL2a is involved in the intramembrane-cleavage of the
membrane-bound N-terminal p8 fragment (NTF) of CD74.10
Major histocompatibility complex class II (MHC II) requires
Received: February 4, 2019
Accepted: May 16, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00044
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Table 1. Initial Round of Optimization
Figure 1. Structures of the known SPPL2a inhibitors LY-411,575 and
SPL-707, HTS hit 1, and HIV protease inhibitor darunavir.
γ-secretase inhibitor LY-411,575, compound 1 (Figure 1) was
discovered as a hit in the high throughput screening of the
Novartis compound archive.15 Despite the rather high
lipophilicity of 1, the scaffold appealed to us because of its
structural similarity to the marketed HIV-protease inhibitors
such as, for example, darunavir (Figure 1).16 In addition, the
hydroxyethylamine (HEA) fragment present in 1 is a known
transition state binding motif17 which apart from HIV
protease,16 has successfully been applied in the design of
agents inhibiting other aspartyl proteases such as BACE-1,18
cathepsin D,19 and plasmepsin.20
a
IC50 determined as a mean (n ≥ 3) in HEK293 cells cotransfected
with TNFα-NTF/VP16-GAL4 fusion and human SPPL2a (RGA).
log D7.4 was determined at pH 7.4 by a rapid-throughput octanol-
b
buffer lipophilicity measurement based on 96-well shake flask
equilibrium and LC/MS/MS analysis. Equilibrium solubility
c
determined by saturation shake flask method at pH 6.8 using
DMSO solution.21
Whereas for most of the HEA-containing inhibitors it is
beneficial to keep a substituent at the amine (usually
carbamates such as the bis-THF moiety in darunavir), this
was not the case for SPPL2a since the Cbz group removal led
to a more potent while less lipophilic derivative 2 (Table 1).
Additional truncation (benzyl to phenyl group in 3) further
enhanced the potency and at the same time also solubility.
Interestingly, compound 3 displayed a high selectivity against
other aspartyl proteases such as BACE 1 and 2, cathepsin D
and E, as well as renin (all >100 μM) and even against the
closely related γ-secretase (4.8 μM) and SPP (8.1 μM) (Table
2). Therefore, the next round of optimization started with 3
and focused on derivatization of the sulfonamide N-substituent
(Table 2).
Due to a relatively high intrinsic clearance of compound 3,
as assessed in mouse liver microsomes, addition of polar
substituents at this position was tested for potential reduction
in clearance. While hydroxy-derivative 4 led indeed to an
improved intrinsic clearance, a drop in SPPL2a potency was
observed. On the other hand, the oxo-derivative 5 did
essentially keep the same potency but surprisingly it was
even more rapidly metabolized than 3 (Table 2). While larger
lipophilic substituents had no advantage in terms of potency
and clearance as illustrated by the Bn-analog 6, α-branched
aliphatic and especially cyclic groups appeared to be more
optimal at this position (Table 2). For example, cyclobutyl
analog 7 showed not only improved potency but also reduced
clearance compared to 3. Larger rings such as cycloheptyl in 8
further boosted the potency as well as selectivity but led to
increased lipophilicity (log D7.4 4.0 for 8 vs 2.8 for 7) and
hence also clearance. Adding an oxo-substituent to the
cyclobutyl group resulted in a more polar and metabolically
more stable oxetane analog 9 being again less tolerated by
SPPL2a. In addition, further branching to tertiary groups, as
exemplified by derivative 10, was also not favored by SPPL2a.
Based on the balanced SPPL2a potency and clearance, the
cyclobutyl group was kept for further optimization of the
scaffold.
Exploration of the arylsulfonyl substituent revealed this part
of the molecule to be important for gaining potency against
SPPL2a (Table 3). While bulky lipophilic substituents like
tetramethyltetrahydronaphthalene (11) improved the potency,
polar substituents such as, for example, benzothiazole (12)
were not well accepted by SPPL2a. Interestingly, a single t-Bu
group at the phenyl ring was found to be sufficient to keep high
SPPL2a potency while the clearance of these mono-t-Bu
analogs was dependent on its position, with m-derivative 13
being metabolized quicker than its p-analog 14 (Table 3).
Further decoration of 14 by the trifluoromethoxy group at the
para-position of the unsubstituted phenyl ring led to
compound 15 (SPL-410), a highly potent and selective
SPPL2a inhibitor.
The potent inhibition of SPPL2a by compound 15, as
measured in the reporter gene assay (RGA), could also be
confirmed using an orthogonal, imaging based SPPL2a assay
(high content imaging assay; HCA).14,15 Both human and
mouse SPPL2a was inhibited with high potency (IC50 of 0.004
μM and 0.005 μM) in the respective HCA. In addition, the
processing of the endogenous substrate, CD74/p8 NTF, was
inhibited by 15 with an IC50 of 0.15 μM as assessed in the
mouse B cell line A20 by a quantitative Western Blot assay
(Supporting Information Figure S1). Moreover, compound 15
proved to be selective against closely related human aspartyl
proteases such as SPP (0.65 μM) and γ-secretase (1.3 μM)
(Table 3) as well as SPPL2b (0.27 μM in HCA). Cellular data
B
DOI: 10.1021/acsmedchemlett.9b00044
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Table 2. SAR around Sulfonamide N-Substituent
Table 3. Optimization of the Aryl Substituent
a
IC50 determined as a mean (n ≥ 3) in HEK293 cells cotransfected
with TNFα-NTF/VP16-GAL4 fusion and human SPPL2a (RGA);
a
IC50 determined as a mean (n ≥ 3) in HEK293 cells cotransfected
with TNFα-NTF/VP16-GAL4 fusion and human SPPL2a (RGA);
HEK293 cells cotransfected with Notch1-VP16-GAL4 fusion
(RGA); stable U-2 OS cell lines expressing human SPP constitutively
b
HEK293 cells cotransfected with Notch1-VP16-GAL4 fusion
(RGA); stable U-2 OS cell lines expressing human SPP constitutively
b
c
c
and an inducible EGFP-labeled EnvSigSeq-SEAP fusion protein
and an inducible EGFP-labeled EnvSigSeq-SEAP fusion protein
substrate (HCA).14,15 Intrinsic clearance determined by the
d
substrate (HCA).14,15 Intrinsic clearance determined by the
d
disappearance of the parent compound from the reaction media
using mouse liver microsomes.22 Dashed lines represent the
attachment points of substituents to the scaffold.
disappearance of the parent compound from the reaction media
using mouse liver microsomes.22 Dashed lines represent the
attachment points of substituents to the scaffold.
In vivo inhibition of SPPL2a by compound 15 was evaluated
by treating mice with a 10, 30, and 100 mg/kg single p.o. dose
of 15 and measuring the accumulation of CD74/p8 NTF 4 h
later in splenocyte lysates14 (Figure 2B,C and Supporting
Information Figure S2). In this mechanistic model, 15 showed
a dose-linear exposure in plasma and spleen and a nice dose−
response for CD74/p8 NTF accumulation in splenocytes. In
addition, 15 achieved a higher efficacy than our benchmark
compound LY-411,575 at the same dose (Figure 2B).
Although a relatively high exposure of 15 was reached in
spleen at 10 mg/kg dose (Figure 2C), no full efficacy was
observed at this dose. The reason for this might be a poor
activity (4.0 μM) of compound 15 as measured in a mouse
whole blood assay assessing accumulation of the CD74/p8
NTF (Supporting Information Table S1 and Figure S3).
Hence, the 6.0 μM spleen levels reached at 4 h with the 10
mg/kg dose appear rather low for getting the full inhibition of
SPPL2a. The 30-fold drop in potency seen in the whole blood
assay most probably reflects the very high plasma protein
for different assay formats and enzymes are summarized in
Supporting Information Table S1.
Interestingly, despite increased lipophilicity compared to its
analog 14, compound 15 displayed a slightly improved
metabolic stability in mouse liver microsomes (Table 3).
Notably, such a positive effect of the trifluoromethoxy group
on clearance seems to be rather specific for this compound
since it usually led to an opposite effect within this series, it
means to a lower metabolic stability of the corresponding
trifluoromethoxy analogs. In order to determine the in vivo
clearance as well as other pharmacokinetic parameters of this
promising candidate, mice were dosed with 15 at 5 mg/kg i.v.
and 20 mg/kg p.o. (Figure 2A). Whereas the compound
showed moderate in vivo clearance (41 mL/min/kg), a rather
long t1/2 of 5.0 h was observed being driven by its high volume
of distribution (12.3 L/kg). The oral bioavalability (35%) and
oral exposure (282 h*nM AUC dose normalized) were
moderate but suitable for oral dosing.
C
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a
Scheme 1. Synthesis of Compound 15 and Its Analogs
a
Reagents and conditions: (a) DIPEA, CH2Cl2, 23 °C, 16 h (85%); (b)
DBU, i-PrOH, 85 °C, 16 h (69%); (c) Me3P, THF/H2O, 23 °C, 3 h
(71%).
Figure 2. (A) Pharmacokinetic profile of compound 15 determined as
a mean plasma concentration of three animals (Balb\c mice) after
dosing the compound using a PEG300/D5W (3:1) formulation for
i.v. and p.o. application. (B) Inhibition of CD74/p8 NTF processing
in mice after oral treatment with 15. Mice received single oral doses of
100 mg/kg LY-411,575 or 10, 30, and 100 mg/kg 15 using PEG300/
D5W (3:1) formulation. Four h after dosing, inhibition of SPPL2a in
vivo was assessed by measuring CD74/p8 NTF accumulation in
splenocyte lysates by Western blot analysis with an antimouse CD74
antibody. Relative quantification of the p8 bands is shown, and the
reference sample with LY-411,575 was set arbitrarily as 100%. (C)
In summary, in this paper we have described our discovery
of a novel HEA-containing SPPL2a scaffold derived from an
HTS campaign. Without any available structural data it
remains uncertain how these inhibitors bind into SPPL2a.
However, thanks to the present HEA, a known aspartyl
protease transition state binding motif, it may be assumed that
the described SPPL2a inhibitors bind into the active site of
SPPL2a. The hit 1 could be optimized into a selective and
highly potent SPPL2a inhibitor 15 (SPL-410) with decent oral
exposure in mice. In the mechanistic mouse model, this
compound displayed a significant inhibition of CD74/p8 NTF
processing even at 10 mg/kg oral dose. The main drawback of
this series is the loss of potency of CD74/p8 NTF processing
in the presence of whole blood which may be a result of the
high PPB being typically >99% across species. This parameter
is most probably driven by the high lipophilicity of this scaffold
which, however, seems to be dictated by the SPPL2a binding
site. Therefore, we believe that more polar analogs may help to
address this issue and potentially allow for improved potency
in the whole blood assay and in vivo inhibition of SPPL2a at
lower doses.
Total plasma and spleen concentrations (means
SEM) of 15 at
termination of the study 4 h after dosing. Statistics are one-way
ANOVA followed by Dunnett’s test compared to matched vehicle
group: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
binding (PPB, >99% across species) determined for 15, being
the case also for other compounds within the series. Therefore,
further work focusing on lowering the PPB will be necessary
for this scaffold for improvement of its potency in whole blood
and hence, in vivo.
The described compounds were prepared as illustrated by
synthesis of 15 (Scheme 1). The required trifluoromethoxy
building block 18 was obtained in five steps starting from the
known allylic alcohol 1723 in analogy to the procedure
described for preparation of the unsubstituted azidoepoxide
20.24 Base catalyzed epoxide opening25 in 18 was achieved
using sulfonamide 16 that was prepared in a single step from
the corresponding sulfonyl chloride and cyclobutylamine.
Reduction of the azide group in 19 provided compound 15
in good overall yield. The hydroxylamine analog 5 was
assembled in a similar manner but starting from the Boc-
protected aminoepoxide 21,26 and the Bn-analog 2 was
prepared from 2227 (Scheme 1). Attachment of the Cbz-
group to the primary amine in 2 provided compound 1.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00044.
Description of in vitro assays, pharmacokinetic measure-
ments, synthesis procedures and characterization data
for all compounds, and UPLC and NMR charts for
compound 15 (PDF)
D
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Hanin, F.; Arlehamn, C. S. L.; Rao, G.; Picard, C.; Lasseau, T.;
Latorre, D.; Hambleton, S.; Deswarte, C.; Itan, Y.; Abarca, K.;
Moraes-Vasconcelos, D.; Ailal, F.; Ikinciogullari, A.; Dogu, F.;
AUTHOR INFORMATION
Corresponding Author
*Juraj Velcicky, e-mail: juraj.velcicky@novartis.com.
ORCID
Juraj Velcicky: 0000-0001-6564-1448
Present Address
M.A.: Plexium, Inc., 4204 Sorrento Valley Boulevard, Suite A,
San Diego, CA 92121, USA. D.R.B.: Vividion Therapeutics,
5820 Nancy Ridge Dr., San Diego, CA 92121, USA.
■
̈
Benhsaien, I.; Sette, A.; Abel, L.; Boisson-Dupuis, S.; Schroder, B.;
Nussenzweig, M. C.; Liu, K.; Geissmann, F.; Tangye, S. G.; Gros, P.;
Sallusto, F.; Bustamante, J.; Casanova, J.-L. Disruption of an
antimycobacterial circuit between dendritic and helper T cells in
human SPPL2a deficiency. Nat. Immunol. 2018, 19, 973−985.
̈
(10) Voss, M.; Schroder, B.; Fluhrer, R. Mechanism, specificity, and
physiology of signal peptide peptidase (SPP) and SPP-like proteases.
Biochim. Biophys. Acta, Biomembr. 2013, 1828, 2828−2839.
Notes
̈
(11) Schroder, B. The multifaceted roles of the invariant chain
The authors declare no competing financial interest.
CD74 - more than just a chaperone. Biochim. Biophys. Acta, Mol. Cell
Res. 2016, 1863, 1269−1281.
(12) Niiro, H.; Clark, E. A. Regulation of B-cell fate by antigen-
receptor signals. Nat. Rev. Immunol. 2002, 2, 945−956.
ACKNOWLEDGMENTS
■
The authors would like to thank Stephane Rodde and Damien
Hubert for the physicochemical measurements and Corinne
Marx for HRMS measurements.
(13) Huttl, S.; Klasener, K.; Schweizer, M.; Schneppenheim, J.;
̈
̈
̈
Oberg, H.-H.; Kabelitz, D.; Reth, M.; Saftig, P.; Schroder, B.
Processing of CD74 by the intramembrane protease SPPL2a is critical
for B cell receptor signaling in transitional B cells. J. Immunol. 2015,
195, 1548−1563.
ABBREVIATIONS
■
BACE, β-secretase; DBU, 1,8-diazabicyclo[5.4.0]undec-7-en;
DIPEA, ethyldiisopropyl amine; D5W, 5% dextrose in water;
EGFP, green fluorescent protein; EnvSigSeq, hepatitis C virus
envelope protein signal peptide; GAL4, regulatory protein
galactose 4; HCA, high content imaging assay; RGA, reporter
gene assay; SEAP, secreted embryonic alkaline phosphatase;
SPP, signal peptide peptidase; TNFα, tumor necrosis factor
alpha; VP16, virus protein 16.
(14) Velcicky, J.; Bodendorf, U.; Rigollier, P.; Epple, R.; Beisner, D.
R.; Guerini, D.; Smith, P.; Liu, B.; Feifel, R.; Wipfli, P.; Aichholz, R.;
Couttet, P.; Dix, I.; Widmer, T.; Wen, B.; Brandl, T. Discovery of the
first potent, selective, and orally bioavailable Signal Peptide Peptidase-
Like 2a (SPPL2a) inhibitor displaying pronounced immunomodula-
tory effects in vivo. J. Med. Chem. 2018, 61, 865−880.
̈
(15) Zhang, X.; Gotte, M.; Ibig-Rehm, Y.; Schuffenhauer, A.;
Kamke, M.; Beisner, D.; Guerini, D.; Siebert, D.; Bonamy, G. M. C.;
Gabriel, D.; Bodendorf, U. Identification of SPPL2a inhibitors by
multiparametric analysis of a high-content ultra-high-throughput
screen. SLAS Discov 2017, 22, 1106−1119.
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