CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 – a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.
We describe a new
class of potent PD-L1/PD-1 inhibitors based on
a terphenyl scaffold that is derived from the rigidified biphenyl-inspired
structure. Using
in silico
docking, we designed and
then experimentally demonstrated the effectiveness of the terphenyl-based
scaffolds in inhibiting PD-1/PD-L1 complex formation using various
biophysical and biochemical techniques. We also present a high-resolution
structure of the complex of PD-L1 with one of our most potent inhibitors
to identify key PD-L1/inhibitor interactions at the molecular level.
In addition, we show the efficacy of our most potent inhibitors in
activating the antitumor response using primary human immune cells
from healthy donors.
In the development of PD-L1-blocking therapeutics, it is essential to transfer initial in vitro findings into proper in vivo animal models. Classical immunocompetent mice are attractive due to high accessibility and low experimental costs. However, it is unknown whether inter-species differences in PD-L1 sequence and structure would allow for human-mouse cross applications. Here, we disclose the first structure of the mouse (m) PD-L1 and analyze its similarity to the human (h) PD-L1. We show that mPD-L1 interacts with hPD-1 and provides a negative signal toward activated Jurkat T cells. We also show major differences in druggability between the hPD-L1 and mPD-L1 using therapeutic antibodies, a macrocyclic peptide, and small molecules. Our study indicates that while the amino acid sequence is well conserved between the hPD-L1 and mPD-L1 and overall structures are almost identical, crucial differences determine the interaction with anti-PD-L1 agents, that cannot be easily predicted in silico.
Immune checkpoint blockade is one
of the most promising strategies
of cancer immunotherapy. However, unlike classical targeted therapies,
it is currently solely based on expensive monoclonal antibodies, which
often inflict immune-related adverse events. Herein, we propose a
novel small-molecule inhibitor targeted at the most clinically relevant
immune checkpoint, PD-1/PD-L1. The compound is capable of disrupting
the PD-1/PD-L1 complex by antagonizing PD-L1 and, therefore, restores
activation of T cells similarly to the antibodies, while being cheap
in production and possibly nonimmunogenic. The final compound is significantly
smaller than others reported in the literature while being nontoxic
to cells even at high concentrations. The scaffold was designed using
a structure–activity relationship screening cascade based on
a new antagonist-induced dissociation NMR assay, called the weak-AIDA-NMR.
Weak-AIDA-NMR finds true inhibitors, as opposed to only binders to
the target protein, in early steps of lead compound development, and
this process makes it less time and cost consuming.
The PD-1/PD-L1 axis has proven to be a highly efficacious target for cancer immune checkpoint therapy with several approved antibodies. Also, small molecules based on a biphenyl core can antagonize PD-1/PD-L1, leading to the in vitro formation of PD-L1 dimers. However, their development remains challenging, as we do not yet fully understand their mode of action. In this work, we designed a new scaffold based on our previously solved high-resolution structures of low-molecular-weight inhibitors bound to PD-L1. A small compound library was synthesized using the Groebke−Blackburn− Bienaymémulticomponent reaction (GBB-3CR), resulting in the structure−activity relationship of imidazo[1,2-a]pyridine-based inhibitors. These inhibitors were tested for their biological activity using various biophysical assays giving potent candidates with low-micromolar PD-L1 affinities. An obtained PD-L1 cocrystal structure reveals the binding to PD-L1. Our results open the door to an interesting bioactive scaffold that could lead to a new class of PD-L1 antagonists.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.