In Vitro Assessment of Putative PD-1/PD-L1 Inhibitors: Suggestions of an Alternative Mode of Action

Article abstract of DOI:10.1021/acsmedchemlett.9b00221

The programmed cell death protein 1 (PD-1) signaling axis is among the most important therapeutic targets in modern oncology. Aurigene Discovery Technologies Ltd. (Aurigene) has patented a series of peptidomimetic small molecules derived from the PD-1 pro

Full text of DOI:10.1021/acsmedchemlett.9b00221

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
In Vitro Assessment of Putative PD-1/PD-L1 Inhibitors: Suggestions
of an Alternative Mode of Action
Derek J. Blevins,† Ronan Hanley,† Trevor Bolduc,† David A. Powell,‡ Michael Gignac,†
Kayleigh Walker,§ Mark D. Carr,§ Fraser Hof,† and Jeremy E. Wulff*,†
†Department of Chemistry, University of Victoria, PO Box 3065 STN CSC, Victoria, British Columbia V8W 3V6, Canada
‡Inception Sciences Canada, 210-887 Great Northern Way, Vancouver, British Columbia V5T 4T5, Canada
§Leicester Institute of Structural and Chemical Biology, University of Leicester, Leicester, United Kingdom
S
* Supporting Information
ABSTRACT: The programmed cell death protein 1 (PD-1)
signaling axis is among the most important therapeutic targets in
modern oncology. Aurigene Discovery Technologies Ltd.
(Aurigene) has patented a series of peptidomimetic small
molecules derived from the PD-1 protein sequence for use in
targeting the interaction between PD-1 and its ligand, PD-L1.
We evaluated three of Aurigene’s most potent compounds in
SPR binding assays. Our results showed that these compounds
each of which is known to be potently effective in a splenocyte
recovery assaydo not directly inhibit the PD-1/PD-L1
interaction nor do they appear to bind to either of the
constituent proteins, indicating that another mechanism is at
play. As a result of these studies and upon consideration of
structural features within the PD-1/PD-L1 complex, we
hypothesize that the Aurigene molecules may interact with a currently unknown protein capable of regulating the PD-1 axis.
KEYWORDS: PD-1, PD-L1, protein−protein interaction inhibitors, SPR
Table 1. List of Current FDA-Approved Immunotherapeutics
That Block the PD-1/PD-L1 Interaction13−17
rogrammed cell death protein 1 (PD-1) and its ligand (PD-
PL1) are transmembrane immunosuppressive checkpoint
proteins.1 PD-1 is expressed on activated T cells, and PD-L1 is
expressed on somatic tissue and antigen-presenting cells.2,3 The
binding interaction between PD-1 and PD-L1 causes a down-
regulation of T cell proliferative gene expression, thus disrupting
cytotoxic activity. Certain aggressive cancers, such as pancreatic
cancer, breast cancers, and nonsmall lung carcinomas, have
evolved to overexpress PD-L1 as a means of immune evasion.3,4
In these cases, PD-L1 expresses constitutively, allowing the
tumor to masquerade as immune privileged tissue and evade
detection.5−8 Overexpression of PD-L1 is therefore a strong
prognostic biomarker in oncology.2−12
Pharmaceutical companies have sought to develop modu-
lators for the PD-1/PD-L1 interaction to recover lymphocyte
activity. Current treatments are antibody-based therapies
targeting either PD-1 or PD-L1 (Table 1). Despite the
remarkable therapeutic success of these antibodies,13,17−25
there are considerable drawbacks such as poor bioavailability,
immunogenicity, and the high cost of large-scale produc-
tion.13,26,27 Small molecule inhibitors have the potential to
overcome these obstacles, and multiple research groups are
pursuing this objective.28−30 However, there are currently no
FDA-approved small molecule inhibitors capable of blocking the
PD-1/PD-L1 interaction.31,32
name
company
target
FDA approval year
nivolumab
Bristol-Myers Squibb
Merck
Genentech/Roche
Merck
AstraZeneca
Regeneron/Sanofi
PD-1
PD-1
PD-L1
PD-L1
PD-L1
PD-1
2014
2014
2016
2017
2017
2018
pembrolizumab
atezolizumab
avelumab
durvalumab
cemiplimab
Aurigene has an extensive patent portfolio of peptides and
peptidomimetic small molecules that mimic various regions of
the PD-1 protein sequence. The most promising peptidomi-
metic compounds are reported to exhibit nanomolar potency in
a phenotypic cell-based splenocyte recovery assay that Aurigene
uses to scout for PD-1/PD-L1 inhibitors.33−37 However, no
direct protein binding experiments have been reported for this
family of small molecules. We selected three of the most
promising compounds (Figure 1) from three recent pat-
ents35−37 and aimed to characterize them using new surface
Received: May 15, 2019
Accepted: July 2, 2019
Published: July 2, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00221
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ACS Medicinal Chemistry Letters
Letter
concentrations (30 nM) and high concentrations (30 μM). For
titration data associated with 4, see Figures S3 and S4.
When compounds 1−3 were tested in this assay, we observed
no change in binding between the two proteins (Figure 2).
Moreover, in the absence of PD-1 protein (data shown in hashed
columns) the Aurigene compounds 1−3 also elicited little signal
variance between 30 nM and 30 μM, suggesting that there is no
directly observable binding to PD-L1. While a very small signal
was likewise observed for 4 in the absence of PD-1, this is
consistent with the known 2:1 binding mode between PD-L1
and compound 4.38 Such behavior would effectively double the
molecular weight of the receptor (since 4 binds to a PD-L1
homodimer, rather than to monomeric PD-L1), while reducing
the density of receptor on the surface of the chip (since not all
surface-bound proteins would be able to homodimerize). The
result would be very weak signal since SPR response is
proportional to both the ratios of the molecular weights of the
two interacting species and to the immobilization density.
We repeated the inhibition experiment, this time attaching
biotinylated PD-1 to the gold surface and flowing recombinant
PD-L1 protein (corresponding to residues A18-T239, chosen to
represent both extracellular domains of the native protein)
across the chip along with various concentrations of 1−4 (Figure
3). This type of reciprocal binding assay serves as an important
control for binding artifacts in SPR experiments. Once again, the
positive control molecule (4) was observed to inhibit the
interaction (data shown in yellow), although in this case higher
concentrations of 4 were required because of the high
concentration of PD-L1 required in the experiment. These
data provide additional support for the earlier study determining
PD-L1 as the biological target for compound 4.39
Figure 1. Compounds used for the present study. Compounds 1−3 are
patented immunomodulators from Aurigene, hypothesized to disrupt
the PD-1/PD-L1 interaction. Compound 4 is a known inhibitor
developed by Bristol-Myers Squibb, employed here as a positive
control.
plasmon resonance (SPR)-based methods. Each test compound
was chosen with an eye toward maximizing both potency and
drug-like properties, while optimally representing the com-
pounds claimed within each patent.
Using SPR, we developed inhibition assays to detect how
binding between the extracellular domain of PD-1 and the
extracellular domain of PD-L1 is affected in the presence of the
Aurigene compounds (refer to Figures S1 and S2 in the
Supporting Information for sensorgrams showing PD-1/PD-L1
binding). In our first inhibition assay (Figure 2), biotinylated
PD-L1 was adsorbed on a streptavidin-coated gold chip (SA
chip). Varied concentrations (ranging from 30 nM to 30 μM) of
Aurigene compounds 1−3 were titrated over a flow cell in the
presence of recombinant soluble PD-1 (corresponding to
residues P34−E150 of the native protein).
The binding responses were normalized to PD-1 binding to
PD-L1 (control). Compounds acting as inhibitors of the PD-1/
PD-L1 interaction would decrease the binding response relative
to the control. To validate the assay, we used a known PD-1/PD-
L1 inhibitor from Bristol-Myers Squibb as a positive control
(compound 4, data shown in yellow).29 Previous studies have
shown that compound 4 induces a dimerization of the PD-L1
proteins, preventing PD-1 from binding.38−40 As expected,
compound 4 showed effective in vitro inhibition at both low
As in the previous assay, however, the Aurigene compounds
1−3 showed no inhibition of the PD-1/PD-L1 interaction
(Figure 3) nor did they appear to elicit any statistically
significant binding to adsorbed PD-1. At very high concen-
trations (300 μM), Aurigene compounds 1 and 2 gave an
increase in SPR response (i.e., negative inhibition), but this is
likely due either to molecules nonspecifically adsorbing to the
attached protein or else to compound precipitation. The same
results were observed in our assay for soluble PD-1 flowed over
another adsorbed binding partner, PD-L2 (Figure S6).
Compound 4 was also shown to not inhibit the PD-1/PD-L2
Figure 2. Aurigene compounds (1−3) show no inhibition between 30 nM and 30 μM for PD-1 flowing over PD-L1. Soluble PD-1 was flowed across
surface-bound PD-L1 with and without test compounds at varying concentrations. The response is normalized to the control protein interaction (PD-1
only). Responses were measured in triplicate, and error bars represent standard deviation.
B
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Figure 3. Aurigene compounds (1−3) show no inhibition between 300 nM and 300 μM for PD-L1 flowing over PD-1. Soluble PD-L1 was flowed
across surface-bound PD-1 with and without test compounds at varying concentrations. The response is normalized to the control protein interaction
(PD-L1 only). Responses were measured in triplicate, and error bars represent standard deviation.
Figure 4. Binding interface between PD-1 and PD-L1 (from PDB: 4ZQK), together with an illustration of the discovery process leading to Aurigene’s
lead compounds. PD-1 and PD-L1 are shown in cartoon representation (orange) and surface representation (blue), respectively. Residues highlighted
in sticks are the residues the patent compounds are derived from. (A) Left: Residues (57−63, 95−99, 127−136) of the PD-1 protein. Right: Patented
peptide derived as a structural analogue to the region. (B) Left: Residues (57−63) of the PD-1 protein. Right: Patented macrocyclic compound derived
as a structural analogue to the region. (C) Left: Residues (57−59) of the PD-1 protein. Right: Small molecule peptidomimetic derived as a structural
analogue to the region.
C
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ACS Medicinal Chemistry Letters
Letter
interaction and showed no sign of binding directly to PD-L2,
highlighting the specificity of the BMS compound.
disclosed, but this lead candidate apparently emerged from a
focused library at Aurigene that was designed to exploit hotspots
within the PD-1/PD-L1 complex.49 In a recent review by two of
the inventors of the Aurigene molecules 1−3, it is stated that
CA-170 was designed to target one or more conserved pockets
found in PD-L1 and VISTA, a nonredundant immunosuppress-
ing protein of the B7-superfamily.28 These authors are careful to
not indicate whether CA-170 is structurally related to
compounds 1−3, but two subsequent reviews (from two
different groups) both speculate that CA-170 is related to
Aurigene’s earlier disclosed compounds.50,51 One of these recent
reviews asserts that CA-170 is an oxadiazole (like compounds 2
and 3),51 while the other describes CA-170 as a molecule
capable of disrupting the PD-1/PD-L1 complex.50 The existence
of a clinical candidate that is likely related to compounds 1−3
and that is thought to function through direct blockade of PD-1/
PD-L1 binding provides motivation to better understand the
function for this series of small molecules. In this study, we
performed surface-plasmon resonance assays to test the
hypothesis of direct protein binding inhibition and found that
none of the compounds tested can disrupt the interaction
between soluble PD-1 and adhered PD-L1 or between soluble
PD-L1 and adhered PD-1. Preliminary testing also did not reveal
binding of 1−3 to surface-bound VISTA (Figure S7). Based on
these data and an analysis of structural features of the PD-1/PD-
L1 interaction (and particularly the region of the PD-1 BC-loop
from which Aurigene’s lead compounds were derived), we
hypothesize that this family of small molecules may regulate the
function of some other PD-1 binding partner.52
Seeking confirmation of these results in an orthogonal assay
type, we also briefly explored the function of compound 1 in a
commercial ELISA assay for PD-1/PD-L1 inhibitors (Figure
S8). Once again, however, we saw no significant inhibitory
activity. Positive controls (including compound 4 and two
related inhibitors from Bristol-Myers-Squibb, as well as a known
antibody inhibitor) all worked as expected, once again validating
the assay.
Aurigene characterized the efficacy of their lead compounds
using a phenotypic cell-based assay built around splenocyte
recovery as a proxy for immune activation. Compounds 1−3
triggered high recovery in this experiment (68%, 93%, and 92%,
respectively, relative to an uninhibited positive control) at 100
nM.35−37 The limited experimental data in Aurigene’s patents
(including controls with PD-L1 in the absence of small
molecule) suggest that the phenotype was PD-L1 specific. But
as we have shown above, the results from Aurigene’s cell-based
assays cannot be attributed to direct inhibition of PD-1/PD-L1
binding. Instead, it would appear that an alternative mechanism
is responsible for the observed phenotypic effect.
Despite the recent appearance of a number of papers related
to small molecule modulation of the PD-1/PDL-1 interac-
tion,14,28,31,41−44 there is little explanation of how the Aurigene
compounds exert their function. However, a close reading of
Aurigene’s patent portfolio provides some insight into the
development of this class of small molecules. Aurigene’s early
patent filings claimed large peptides that mimic a significant
portion of the PD-1 extracellular domain (Figure 4A).33 This
includes much of the PD-L1 binding interface, and so it is likely
that these large peptides would be competitive inhibitors of the
PD-1/PD-L1 interaction. Attempts to achieve more drug-like
properties, however, led to subsequent filings describing the
development of macrocyclic peptides that mimic the 7-residue
BC-loop (Figure 4B).34 The most recent filings further refine the
structure (and, presumably, improve the pharmacokinetic
properties) by developing small tripeptides and peptidomimetic
analogues that mimic the central serine-asparagine-threonine
tripeptide of this loop (Figure 4C).35−37 This is the strategy that
ultimately led to small molecules 1−3.
Critically, however, the BC-loop (and particularly the region
mimicked by 1−3) points away from the PD-L1 binding
interface (Figure 4B), and so it is unclear why these molecules
would be expected to be direct binding inhibitors, notwithstand-
ing their apparent potency in cell-based assays and the fact that
they are referred to (with little supporting data) in Aurigene’s
patents35−37 and subsequent reviews28,42,45 as inhibitors of the
PD-1/PD-L1 interaction.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00221.
Supplementary Figures S1−S8, together with experimen-
tal details for protein expression, analytical methods, and
compound synthesis, as well as NMR spectra for
synthesized compounds 1−4 (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: wulff@uvic.ca.
■
ORCID
Fraser Hof: 0000-0003-4658-9132
Jeremy E. Wulff: 0000-0001-9670-160X
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
While it is possible that there is some key difference (e.g.,
glycosylation state) between our in vitro system and the “real-
life” PD-1 and PD-L1 proteins expressed on the surface of T cells
and antigen presenting cells, a more likely scenario is that the
compounds do mimic the PD-1 surface as intended, but that this
serves not to directly disrupt binding with the PD-L1 protein as
has been assumed, but perhaps to modulate the function of some
other PD-1 binding partner. Both PD-1 and PD-L1 are thought
to participate in regulatory binding interactions with other
proteins,46,47 and we hypothesize that one or more of these
might be the true biological target of 1−3.
Funding
This work was supported by funding from the Canadian Cancer
Society Research Institute (grant # 701684), as well as an
NSERC CREATE grant (# 497311−2017) and an NSERC
Discovery grant to J.W. (# 4283). Additional support came from
the Michael Smith Foundation for Health Research, the Canada
Research Chairs program, and the University of Victoria.
Notes
In 2015, Curis Inc. initiated clinical trials with a small
molecule called CA-170, which had been developed at
Aurigene.48 The exact structure of CA-170 has yet to be
The authors declare the following competing financial
interest(s): D.A.P. is an employee of Inception Sciences Canada.
The remaining authors declare no competing financial interest.
D
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ACKNOWLEDGMENTS
■
D.J.B. thanks the Polymer Nanoparticles for Drug Delivery
(POND) CREATE program for a stipend and the University of
Victoria for partial operating support that was used to offset the
cost of this research. F.H. and J.W. thank the Canada Research
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ABBREVIATIONS
■
PD-1, programmed cell death protein 1 (CD279);; PD-L1,
programmed death-ligand 1 (CD274 or B7−H1); VISTA, V-
domain Ig suppressor of T cell activation; SPR, surface plasmon
resonance
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Drugs 2018, 78 (17), 1841−1846.
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(32) Konstantinidou, M.; Zarganes-Tzitzikas, T.; Magiera-Mularz, K.;
(52) Following the submission of this manuscript, a study on the in
vitro function of a likely CA-170 candidate (structurally related to 1−3)
was disclosed. This study likewise finds that the candidate is inactive in a
variety of protein-binding experiments and concludes that an
alternative mechanism may be responsible for its biological activity.
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DOI: 10.1021/acsmedchemlett.9b00221
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