The Development of Small-molecule Inhibitors Targeting CD47

April 18, 2022

Abstract

Immunotherapy has become an indispensable part of cancer treatment. A pivotal phagocytosis checkpoint, named cluster of differentiation 47 (CD47), which functions as a ‘don’t eat me’ signal to protect cells from phagocytosis upon interaction with signal regulatory protein alpha (SIRPα) on macrophages, has recently attracted much attention. Numerous antibodies targeting the CD47/SIRPα axis have shown encouraging efficacy in clinical trials. Meanwhile, studies on small-molecule inhibitors that interfere with CD47/SIRPα interaction or regulate CD47 expression are also in full swing. In this review, researchers summarize the small-molecule inhibitors interrupting the binding of CD47/SIRPα and regulating CD47 at the transcriptional, translational, and post-translational modification (PTM) levels. They provide perspectives and strategies for targeting the CD47/SIRPα phagocytosis checkpoint.

The Mechanism of Action of CD47

Immune checkpoints that target the adaptive immune system, such as cytotoxic T lymphocyte-associated protein (CTLA-4), programmed death (PD)-1 and their ligand PD-L1, have achieved breakthrough success in patients with advanced cancer. However, low objective response rates (15-30%) and the emergence of drug resistance remain challenges for these immune checkpoint antibodies. Targeting immune checkpoints in the innate immune system may provide a new solution. The accumulation of tumor-associated macrophages (TAMs) around the tumor tissue and the secretion of interleukin (IL-10), transforming growth factor (TGF)-β and other cytokines form an immunosuppressive tumor microenvironment (TME) that prevents the invasion of CD8+T cells into the tumor tissue. These data suggest that targeting TAMs can unlock immunosuppressive TME and stimulate phagocytosis.

So far, CD47 is one of the most promising targets for regulating macrophage phagocytosis. Structurally, the five-fold transmembrane protein CD47 contains a larger extracellular N-terminal domain than the short intracytoplasmic C-terminal tail. The extracellular IGV-like domain on CD47 binds to SIRPα on myeloid cells, including macrophages, to initiate phosphorylation of two tyrosine residues of immune receptor tyrosine inhibitory motifs (ITIMs) in the intracellular domain of SIRPα.

When CD47 located on the surface of tumor cells interacts with SIRPα on the surface of macrophages, it phosphorylates the intracellular portion of SIRPα, which then activates SHP-1 and SHP2, thereby inhibiting myosin IIA and preventing macrophage phagocytosis. When CD47/SIRPα is blocked or interfered with antibodies (Abs) or small molecule inhibitors (SMIs), the disruption of CD47/SIRPα results in the activation of macrophage phagocytosis.

Phosphorylation of ITIMs re-recruits and activates the phosphatase SHP1/2, resulting in the inhibition of the downstream protein in this cascade, non-muscle myosin IIA, and ultimately inhibits phagocytosis. CD47 is expressed at different levels in different cell types. Its high expression on erythrocytes shows its protective function. Furthermore, its expression is critical for controlling the fate of circulating hematopoietic stem cells. What’s more, CD47 is often overexpressed in a variety of cancer cells to help them evade immune surveillance. Blocking CD47/SIRPα binding results in a failure in the recruitment and activation of SHP1/2, thereby promoting phagocytosis. During this process, cross-priming of antigen-presenting cells (APCs) is also initiated, leading to activation of the adaptive immune system. In vitro and in vivo studies showed that CD47/SIRPα blocking antibody significantly enhanced macrophage phagocytosis, dendritic cell (DC) cross-presentation, and neutrophil-mediated killing of cancer cells.

Small-molecule Inhibitors Targeting CD47

Although some antibodies against CD47 have shown acceptable therapeutic effects, adverse reactions such as anemia are unavoidable. This is because CD47 is a ubiquitously expressed cell surface protein that is also expressed on red blood cells. In addition, due to the longer half-life of antibodies in the human body, adverse reactions are prolonged. In contrast, small-molecule compounds have controllable side effects in some respects due to their short metabolic half-lives and flexible in vivo drug exposure. In addition, small molecule inhibitors are more likely to enter tumor cells due to their small molecular weight. Reported small molecule inhibitors of the CD47 axis include blockers that disrupt the CD47/SIRPA interaction at the level of transcriptional, translational and post-translational modifications and modulators of CD47.

1. Small molecule inhibitor that directly blocks CD47 binding to SIRPα

NCGC00138783, PEP-20 and D4-2 are three reported inhibitors that directly block the interaction of CD47 and SIRPα. Their structures and sequences are shown in Table 1. Both NCGC00138783 and PEP-20 have been reported to bind to CD47 to exert its blocking function; D4-2 interferes with the CD47/SIRPα interaction by binding to SIRPα.

2. Small molecule inhibitors that inhibit CD47 expression at the transcriptional and translational levels

RRx-001, metformin, 4-methylumbelliferone (4-Mu), JQ1, and gefitinib have been reported to inhibit CD47 expression at the transcriptional and translational levels. Among them, RRx-001 has entered Phase III clinical trials.

3. Small molecule inhibitors against CD47/SIRPPα

At the level of post-translational modification, it refers to the enzymatic regulation of protein biosynthesis that matures the protein and enables it to perform its function. So far, Glutaminyl-Peptide Cyclotransferase Like (QPCTL) inhibitors that regulate CD47 pyroglutamate formation, such as SEN177 and PQ912, have been reported to affect CD47-SIRPα interaction.

While antibodies to CD47 immune checkpoints, such as Magrolimab, have shown promising anti-tumor effects and some have entered phase III clinical trials, it is equally important to develop small molecule inhibitors because of their advantages over antibodies. First, small molecule inhibitors are not immunogenic and have a shorter half-life than antibodies, making their side effects manageable. Secondly, some CD47 antibodies can cause agglutination because the antibody binds to high-density erythrocyte membrane determinants, such as glucagon A, which can be avoided by small molecule inhibitors. In addition, small molecule inhibitors have the potential for oral administration to improve patient compliance. In addition to focusing on CD47, small molecule inhibitors targeting SIRPα are also feasible. Compared with antibodies, small molecule inhibitors will penetrate cells more easily and inhibit downstream signaling pathways, preventing downstream signaling activation to re-stimulate phagocytosis.

Table 1: Inhibitors of the CD47/SIRPα signaling pathway

Compound	Target	Research stage	Cancer type
NCGC00138783	Binding of CD47 to SIRPα	Preclinical	Leukemia
NCGC00538419	Binding of CD47 to SIRPα	Preclinical	Leukemia
NCGC00538430	Binding of CD47 to SIRPα	Preclinical	Leukemia
Pep-20	Binding of CD47 to SIRPα	Preclinical	Colon carcinoma
D4-2	Binding of CD47 to SIRPα	Preclinical	Lymphoma and melanoma
RRx-001	Expression of CD47 and SIRPα	Phrase 3	Small cell lung cancer
Metformin	Expression of CD47	Preclinical	Breast cancer
4Mu	Expression of CD47	Preclinical	HCC
JQ1	Expression of CD47	Preclinical	Lymphoma
Gefitinib	Expression of CD47	Preclinical	NSCLC
PQ912	Modification of CD47 protein	Preclinical	Melanoma
SEN177	Modification of CD47 protein	Preclinical	Melanoma