Research paperDevelopment of small molecule inhibitors targeting TGF-β ligand and receptor: Structures, mechanism, preclinical studies and clinical usage
Graphical abstract
Introduction
Transforming growth factor-β (TGF-β) was first discovered from murine sarcoma virus-transformed fibroblasts in 1978 by Joseph De Larco and George Todaro [1,2], from the National Cancer Institute, Bethesda, Maryland in the United States. More than 33 TGF-β members have been identified so far that form a large superfamily and are divided into different subfamilies including TGF-β, activin, bone morphogenetic protein (BMP), growth and differentiation factors (GDFs), Nodal and Anti-Müllerian Hormone (AMH) [3]. TGF-β superfamily ligands trigger their intracellular signals by binding to two types of transmembrane receptors, type I (TβR-I; also known as activin receptor-like kinase, ALK) and type II (TβR-II) receptors with a serine/threonine kinase domain. Currently, seven ALKs (ALK1∼ALK7) and five TβR-IIs have been identified, which may form specific heterocomplexes with each TGF-β superfamily member. Each ligand can bind multiple receptors, but with different affinities. For example, TGF-β binds to ALK1 and ALK5 with high affinity; activin binds to ALK2 and ALK4 with high affinity but with moderate affinity to ALK7; and BMP binds to ALK1, ALK3 and ALK6 with high affinity but with moderate affinity to ALK2. The activated ALKs then phosphorylate intracellular SMAD/non-SMAD proteins and mediate signal transduction [4].
As the TGF-β subfamily consists of cytokines with pleiotropic functions, TGF-βs play a critical role in numerous cellular processes such as proliferation, differentiation, migration, embryonic development, extracellular matrix (ECM) expression and immune responses [5]. To date, five different isoforms of TGF-β (TGF-β1∼5) have been described. Among them, TGF-β1, TGF-β2 and TGF-β3 are mainly expressed in mammals, of which TGF-β1 accounts for more than 90% [6]. There are 3 types of receptors for the TGF-βs, among which, TβR-I (ALK5) and TβR-II, contain extracellular binding domains for protein-protein interactions with the ligands and intracellular kinase domains, while the type III receptor (also termed betaglycan) does not contain kinase active regions, and mainly regulates the binding of TGF-β to the type II receptor [7].
TGF-β signal transduction is initiated by the binding of two TGF-β ligands to two TβR-II extracellular domains, followed by the binding of two TβR-Is to the complex. After the formation of the TGF-β-TβR-II-TβR-I heterohexameric complex, the intracellular domain of TβR-I is phosphorylated and downstream signaling is activated. Intracellular signals activated by TGF-β are then mediated by SMAD and non-SMAD associated proteins. In the TGF-β/SMAD signaling pathway (canonical pathway), receptor-specific (R) SMADs (SMAD2 and SMAD3) are phosphorylated and form oligomers with SMAD4 and this complex then translocates to the nucleus to regulate the transcription of various target genes [8,9]. TGF-β also activates SMAD-independent signaling pathways (non-canonical pathways), such as the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase and protein kinase B (PI3K/Akt), and Rho-Rock pathways. Notably, the downstream effectors of the MAPK pathway include extracellular signal-regulated protein kinase 1/2 (ERK1/2), c-jun amino terminal kinase (JNK), and p38 MAPK signaling pathways [10]. In the nucleus, SMAD and/or non-SMAD signaling regulatory proteins bind to specific transcription cofactors and/or accessory proteins to regulate the transcription of downstream target genes. Collectively, transcriptional control of target genes regulates a range of biological responses and deregulation of TGF-β signaling is associated with a variety of diseases, such as cancer, atherosclerosis, myeloproliferative syndromes and organ fibrosis in a context- and tissue-specific manner [[11], [12], [13]].
Epithelial–mesenchymal transition (EMT) is a well-recognized trans-differentiation process, by which epithelial cells lose their intercellular adhesion and gain the migratory and invasive characteristics of mesenchymal cells [14,15]. Physiologically, EMT is responsible for the development of various tissues during embryonic growth as well as tissue regeneration and wound healing in adulthood. However, dysregulation of EMT contributes to a number of disease pathologies, such as carcinogenesis, chronic cardiovascular disease (i.e. atherosclerosis, myeloproliferative syndromes), and the development of fibrosis in organ systems (skin, heart, liver, kidney, and lung); inhibition or reversal of pathological EMT is expected to be beneficial for the treatment of these diseases [16,17]. TGF-β plays a key role in the EMT process due to its functions in activating EMT-related intracellular signal transduction, promoting the expression of mesenchymal markers (such as fibronectin, alpha smooth muscle actin (α-SMA) and vimentin), down-regulating epithelial and/or endothelial markers (such as epithelial or vascular endothelial adhesion protein (E-cadherin/VE-cadherin)), and enhancement of cell motility [18].
TGF-β plays a critical and dual role in the progression of human cancer. In early or primary stages of cancer, TGF-β can act as a tumor suppressor by inducing cell-cycle arrest and promoting apoptosis. Substantial evidence suggests that TGF-β causes G1 cycle arrest and induces apoptosis by up-regulating the expression of cyclin-dependent kinases (CDKs) inhibitors such as p21 and apoptotic regulators such as Bcl-2-like protein 11 (BIM) in the early stages of malignancy. However, in late or advanced stages of cancer, TGF-β can function as a tumor promoter. Tumor cells in the more advanced stages of the disease often lose susceptibility to the inhibitory effects of TGF-β as a result of mutations in some key components of the TGF-β pathway. For example, high rates of mutation and/or deletion of smad4 are reported in patients with pancreatic cancer and inactivating mutations in smad2 have been widely identified in patients with hepatocellular carcinoma, colorectal cancer or lung cancer. When cancer cells become resistant to the growth suppressive effects of TGF-β but maintain their responsiveness to TGF-β, the ligand favors tumor progression, and EMT, invasion and metastasis are promoted [19,20].
TGF-β can regulate the functions of stroma cells involved in fibrotic tissues and the tumor microenvironment by promoting the differentiation of fibroblasts to myofibroblasts, inducing the recruitment of immune cells, driving tissue angiogenesis and suppressing adaptive immune cell responses. One of the hallmark events during the development of organ fibrosis and cancer is TGF-β-mediated myofibroblast differentiation and the activation of resident/infiltrating fibroblasts, which result from the secretion of pro-fibrotic/pro-tumorigenic cytokines and chemokines, and the expression of mesenchymal/cancer-associated biomarkers and cause excessive accumulation of ECM, providing a hotbed for the infiltration of immune cells and a precondition conducive to the cell (i.e. tumor cells, fibroblasts, vascular endothelial cells). The infiltration of immune cells into the tissue microenvironment further enhances the fibrotic or tumorigenic responses by secreting cytokines and chemokines responsible for the myofibroblast differentiation, the stimulation of ECM deposition and the further recruitment of immune cells [19,21]. TGF-β also displays an immunosuppressive effect on most immune cells such as dendritic cells, natural killer cells, macrophages and cytotoxic T lymphocytes, and thereby, the activation of TGF-β signaling in the tumor microenvironment has been associated with the poor prognosis. Furthermore, recent immune evasion studies show that a transcriptional signature of TGF-β signaling in cancer-associated fibroblasts contributes to T cell exclusion, which results in a poor response to immune checkpoint blockade of the programmed cell death-1/programmed death ligand-1 (PD-1/PD-L1) pathway induced by the immunotherapy drug atezolizumab [[21], [22], [23]]. Notably, the lack of response to the immunotherapy drug was associated with the activation of TGF-β signaling in fibroblasts.
In summary, many function roles for aberrant TGF-β signaling in the pathogenesis of many diseases have been described over the past decades and, therefore, targeting TGF-β signaling modulators and/or downstream effectors may lead to entirely new drugs for specific diseases. Different strategies have been used in the past to antagonize the actions of TGF-β. Antibodies to TGF-β [24,25], engineered mutant TGF-β ligands [26], anti-sense oligonucleotides targeting specific TGF-β signaling components [27], decorin [28,29], recombinant soluble betaglycan [30,31] and a soluble TβR-II: Fc fusion protein [[32], [33], [34], [35]] all have been developed to inhibit TGF-β activity or signaling. The soluble TβR-II: Fc fusion protein is particularly interesting because it inhibits mammary tumor metastases [36] without the development of long-term adverse side effects that might be predicted from the phenotypes of TGF-β null mice [37]. The work of Yang et al. [36], therefore, provides “proof of principle” that the long-term inhibition of TGF-β activity can inhibit the metastatic dissemination of breast cancer cells. The use of proteins/peptides in a therapeutic capacity, however, is limited because of poor bioavailability due to the conformational instability and susceptibility to proteolytic degradation, poor membrane penetration, induction of host immune responses and unfavorable pharmacokinetics.
These limitations have prompted the development of small molecule inhibitors, and currently there are two potential ways to inhibit the TGF-β signaling pathway, either by down-regulating the expression of TGF-β itself or by inhibiting the kinase activities of the TGF-β receptors. This review will focus on the chemical structures, binding poses and biological functions of the TGF-β inhibitors.
Section snippets
Molecules that reduce TGF-β expression
Idiopathic pulmonary fibrosis (IPF) is a serious chronic disease that affects the tissue surrounding the air sacs, or alveoli, which can cause permanent scarring in the lung (i.e. fibrosis), which makes it progressively more difficult for patients to breathe. There is substantial evidence to indicate that the pathogenesis of IPF is associated with EMT of injured respiratory epithelial cells, excessive differentiation of fibroblasts to myofibroblasts and desmoplastic deposition of ECM [18,38].
TGF-β receptor kinase inhibitors
Most of the small molecule inhibitors under development for the inhibition of TGF-β signaling target the kinase domain of TβR-I. Such TβR-I kinase inhibitors have been shown to affect various hallmarks of diseases such as invasion/metastasis, angiogenesis, ECM production and immune responses. With a greater understanding of the structure and function of TGF-β receptors, especially the crystal structures of the kinase domain, we can reasonably expect that more potent kinase domain inhibitors
Concluding remarks
Encouraged by the success of pirfenidone, the design and development of new potent small molecule TGF-β inhibitors with low toxicity is currently an area of intense research. In this review, we have discussed those TGF-β inhibitors that have either been approved, are in clinical trials or are in pre-clinical development. As members of the TGF-β superfamily play critical roles in the regulation of cell growth, differentiation and development, the use of inhibitors that lack specificity is likely
Declaration of competing interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Acknowledgments
This work was supported by National Natural Science Foundation of China (81660588, 81960623, 81773582, 81973383, 8132100, 81621064), the Key R&D Program of Ningxia (2018BFG02004), the Program for Leading Talents of Ningxia Province (KJT2018004), the National Mega-project for Innovative Drugs (2019ZX09721001), the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program”, China (2019ZX09201001-003-007), CAMS Initiative Fund for Innovative Medicine (
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2021, Advances in ImmunologyCitation Excerpt :A corresponding family of membrane TGF-β receptors (TβRs) with cytoplasmic protein serine/threonine kinase domains, are widely expressed and transduce signals through the highly conserved Smad pathway (Batlle & Massagué, 2019; Derynck & Budi, 2019). Genetic deficiencies in gene-targeted transgenic mice result in lethal inflammatory disorders within weeks of birth (Kulkarni et al., 1993; Shull et al., 1992), which are even recapitulated when T cells alone cannot express TGF-β (Li, Sanjabi, & Flavell, 2006; Marie, Liggitt, & Rudensky, 2006); consequently experimental manipulations employ more refined constructs such as mutated receptors with a dominant negative kinase domain (Gorelik & Flavell, 2000), conditional deletions targeted to specific lineages (Lewis, Wehrens, Labarta-Bajo, Streeck, & Zuniga, 2016), or pharmacological kinase inhibitors with preferential activity against individual TβRs (Inman et al., 2002; Wang, Chen, et al., 2020). A further invaluable experimental tool is the monoclonal antibody 1D11 which neutralizes all three isoforms of mammalian TGF-β (Dasch, Pace, Waegell, Inenaga, & Ellingsworth, 1989).