Introduction
Cancer is among the leading causes of morbidity and mortality in humans. The International Agency for Research on Cancer projects 27.5 million new cancer cases and an alarming mortality rate of 16.3 million by 2040. Cancer treatment requires expensive procedures and follow-ups, necessitating the development of advanced, cost-effective therapies. Current cancer treatments involve various approaches, including radiotherapy, chemotherapy using protein kinase inhibitors and epigenetic modulators, interleukin therapy, and anti-angiogenic therapy targeting vascular endothelial growth factor, which have been found to enhance the body’s anti-tumor immunity. Among these therapies, cancer immunotherapy has gained significant importance, with enormous market growth.1 The 2018 Nobel Prize in Physiology or Medicine was awarded for immunotherapy with immune checkpoint inhibition, as it has revolutionized cancer treatment. The U.S. Food and Drug Administration (FDA) has approved programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) antibodies, such as Dostarlimab, Avelumab, Atezolizumab, and Nivolumab, for renal, prostate, breast, and lung carcinomas. Immune checkpoint inhibitors or modulators, which fall under the category of immunotherapy, have revolutionized oncology.
Various immunoreceptors, such as V-domain Ig suppressor of T cell activation (VISTA), B and T cell attenuator, T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin and mucin-domain containing-3 (TIM3), lymphocyte activation gene-3, Cytotoxic T-lymphocyte antigen 4 (CTLA-4), PD-1, and PD-L1,2 have been employed to inhibit cancer proliferation by acting as immune checkpoints that regulate immune responses. These checkpoints are significant because correcting their aberrant behavior may strengthen the immune system’s ability to combat cancer cell proliferation. Immune cells, such as T cells, are equipped with checkpoints that function as receptors. The interaction between immune checkpoint receptors and complementary receptors on cancer cells protects cancer cells from T cells, allowing the cancer cells to become immortal and evade T cells. Immune checkpoint inhibitors (ICIs) prevent these immune checkpoint receptors from interacting with complementary receptors on cancer cells, helping T cells destroy cancerous cells. Anti-PD-L1 or PD-1 and anti-CTLA4 antibodies are ICIs that have been successfully employed as cancer therapeutics.3 Ipilimumab, which has significantly improved the quality of life in patients with metastatic melanoma, targets CTLA-4, mediating T cell antitumor immune responses.4–7 It was the first FDA-approved immune checkpoint inhibitor. Later, the FDA approved nivolumab for the inhibition of PD-1 in the treatment of advanced melanoma, improving the quality of life.8,9 Following this research, blocking PD-1 or its ligand PD-L1 with monoclonal antibody drugs has gained importance and development potential. However, current ICIs have benefitted only a few patients,10,11 while others develop resistance and adverse side-effects.12,13 Moreover, they can lead to immune-related adverse events (irAEs), causing multiple organ disorders, hyper-progression,14–16 or overactive immune responses.17–19 Monoclonal antibody treatment for inhibiting immune checkpoints has gained momentum as a cancer therapy, but it also suffers from toxicities or irAEs, which are different from the toxicities linked to traditional chemotherapy. These irAEs may affect several organs, including the skin, gastrointestinal tract, liver, lungs, and endocrine systems, due to the nature of the immune system’s response.
The other side effects include neurotoxicity, myocarditis, hematological toxicities, and nephritis.3,20 Identifying these adverse events and managing them is necessary to prevent morbidity in patients undergoing immunotherapy. Thus, it is evident that there is a need for rational design and in-depth clinical and biological phenotyping of ICIs to improve the therapeutic outcomes of next-generation ICIs.
Recently, circular RNAs (circRNAs), which are stable, loop-structured molecules with both coding and non-coding properties, have been reported. They help control cell signaling pathways and regulate tumors.21–25 circRNAs modulate immune checkpoint interactions to influence ICI resistance; however, the other adverse reactions and resistance to ICIs need further exploration in the case of circRNAs. Moreover, the role of circRNA-mediated epigenomics, its control over immune checkpoints, and resistance to cancer immunotherapy are yet to be explained.
Among several checkpoint proteins/receptors, PD-1 and CTLA-4 play important roles in immune function.26–28 Monoclonal antibodies (mAbs) sometimes have low response rates due to poor tissue permeability, inhibition of a single target, long half-life, and inherent immunogenicity, which is responsible for their irAEs. Additionally, they are expensive and involve high costs in clinical applications because of their intravenous or subcutaneous route of administration. These challenges are major constraints to their widespread use in cancer immunotherapy. Thus, an alternative strategy is necessary to improve the clinical efficacy of cancer immunotherapy. To this end, small molecule checkpoint inhibitors are gaining importance, as they target tumor immunity, have good oral bioavailability, short half-life, lower molecular weights, extensive penetration into cells and tissues, lower immunogenicity, and are cost-effective compared to mAbs.29–33 Small molecules also offer easy pharmacokinetic optimization for flexible dosing, which may help avoid mAb-associated irAEs. Furthermore, optimizing checkpoint inhibitory activity, pharmacokinetic parameters, and unwanted toxicity through structural modifications, guided by state-of-the-art computational structural and ligand-based approaches, can be more easily applied to small molecules than to mAbs. These approaches now involve virtual screening to identify new lead drug-like molecules for further clinical trials. The virtual screening protocols include two major approaches: structure-based and ligand-based. The former works on a similarity approach, considering physicochemical properties, chemical functionality, and ligand shape similarity, while the latter is based on the complementarity of the ligand with the target protein’s binding sites. Predictive models developed using these approaches may be useful for virtual screening of large databases or focused libraries for selecting lead molecules for lead optimization.34–38 Hence, the major limitations of mAbs in terms of irAEs may be overcome with chemical entities under/for preclinical studies, and these may also provide insights for optimization and the design of novel chemical entities for cancer immunotherapy. Such small chemical molecules may have the potential to develop more effective immune checkpoint inhibitors, offering a better alternative to mAbs. Therefore, this review is limited to describing small molecules that have shown checkpoint inhibitory activity, including FDA-approved drugs, repurposed drugs, candidate drugs used alone or in combination with mAbs, and novel potential lead molecules under preclinical investigation.
Small molecules immune checkpoint inhibitors
Drugs
There are some FDA-approved drugs that work as immune checkpoint inhibitors, as shown in Figure 1, along with repurposed drugs. FDA-approved drug 1 (Gefitinib) activates the anti-tumor activity of immune cells by inhibiting EGF signaling and destabilizing PD-L1. It has been used to treat metastatic non-small cell lung cancer patients with abnormal epidermal growth factor receptor and no history of medical treatment for cancer.39
Drugs 2 (Fedratinib) and 3 (Ruxolitinib) downregulated PD-L1 expression in non-small cell lung cancer (NSCLC) and breast cancer cells by targeting Janus kinase/signal transducer and activator of transcription pathways.40 These two drugs were approved by the FDA in 2019 and 2022 for myelofibrosis and relapsed/refractory multiple myeloma, respectively.41
The mitogen-activated protein kinase (MEK1/2) inhibitor, drug 4 (Selumetinib), approved by the FDA, inhibited PD-L1 in lung adenocarcinoma cells and is used for the treatment of neurofibromatosis type 1 in children around the age of 2 years with plexiform neurofibromas.42,43
In view of the role of histone acetylation at the PD-L1 promoter region in regulating PD-L1 expression, the histone deacetylase inhibitor drugs 5 (Belinostat), 6 (Panobinostat), 7 (Vorinostat), and 8 (Romidepsin) increased PD-L1 expression and enhanced in vivo proliferation of cancer cells by inducing cell cycle arrest and promoting apoptosis.44–46 FDA-approved drug 5 is used for treating peripheral T-cell lymphoma.47 Drug 6 is an oral deacetylase inhibitor used for treating multiple myeloma.
Injections of drug 8 are employed for the treatment of cutaneous T-cell lymphoma in people with a history of prior treatment with other medications. Drug 8 is a histone deacetylase inhibitor.48 Further, it is in a Phase I pharmacokinetic study in patients with cancer and hepatic dysfunctions.
Drug 9 (Etoposide), also an FDA-approved drug used in cancer chemotherapy, interferes with PD-L1 glycosylation induced by epithelial-to-mesenchymal transition and disrupts surface PD-L1 in tumor cells. The drug, along with metformin, is reported to improve the effectiveness of anti-TIM-3 and anti-CTLA-4 therapies. The well-known natural drug 10 (Curcumin) also destabilizes PD-L1, inhibits the deubiquitylation of the CSN5 protein, and supports the development of anti-CTLA-4 therapy.49
Repurposed drugs
Several drugs used for diseases other than cancer have also been investigated under the novel approach of accelerated drug discovery and development, as it overcomes steps viz. general toxicity, pharmacokinetics, and human tolerance. In this context, the following repurposed drugs (Fig. 1, drugs 11–13) showing potential in checkpoint inhibition are described below:
The antihypertensive FDA-approved drug 11 (Azelnidipine) has been found to inhibit CD47/SIRPα and TIGIT/poliovirus receptor (PVR) through molecular docking studies using Molecular Operating Environment software. It has been demonstrated to inhibit the growth of CT26 tumors in vivo due to enhanced penetration and improved functioning of CD8+ T cells in tumors.50 Drug 12 (Metformin), a well-known anti-diabetic drug, activated T-cells by triggering AMP-activated protein kinase to influence the phosphorylation of PD-L1, preventing PD-L1 glycosylation and promoting antitumor immunity.51
Drug 13 (Rapamycin/Sirolimus), a well-known FDA-approved immunosuppressant (mTOR inhibitor) used after organ transplants, enhances lysosomal protein degradation and decreases protein synthesis.52 Its combination with anti-PD-1 significantly reduced tumor growth in a mouse model of lung cancer, where an increase in CD3+ T cells and a decrease in FoxP3+ Tregs were observed.52
Molecules in clinical trials
The potential drug molecules in various stages of clinical trials are shown in Figure 2. Molecule 14 (INCB086550), a PD-L1 inhibitor,53,54 reduced tumor growth in mice by activating T cells and blocking PD-1/PD-L1 pathways in peripheral blood mononuclear cells, representing the activation of the immune system. It is in phase 2 clinical trials for solid tumors and may provide alternatives to antibody therapies.55
An oral PD-L1 inhibitor, compound 15 (IMMH-010), quickly metabolizes into YPD-29B. It exhibits notable antitumor efficacy in colon cancer xenograft mouse models and melanoma.56 It is in phase 1 clinical trials for malignant neoplasms in advanced malignant solid tumors.57
Compound 16 (MAX-10181) is under phase 1 study, where its safety, tolerability, and pharmacokinetic characteristics are being evaluated in patients with advanced solid tumors. It interferes with the interaction of PD-L1 and PD-1, inhibiting immunosuppressive signals.58
ASC61 (NCT05287399), a small-molecule PD-L1 inhibitor, is under phase 1 clinical study for its safety in treating solid tumors. It is an oral prodrug whose active metabolite, ASC61-A, blocks the PD-1/PD-L1 interaction via PD-L1 internalization and dimerization. It displayed remarkable antitumor efficacy in multiple animal models and humanized mouse models. Preclinical examinations revealed significant safety and pharmacokinetic properties in animal models.59,60
Compound 17, GS-4224, an antiviral drug and PD-L1 inhibitor for treating advanced solid tumors, was under phase 1 studies, but the studies have been terminated for unknown reasons.61
DNA hypomethylating agents have also been explored for their checkpoint inhibition. Among these, compounds 18 (Azacytidine, NCT02447666) and 19 (Decitabine) upregulated PD-L1 expression and potentiated anti-PD-L1 antibody efficacy in different cancer mouse models for colorectal cancer, gastric cancer, and NSCLC.62,63 They are in phase 2 clinical trials for myelodysplastic syndrome and leukemia.64
The combination of decitabine and the poly (ADP-ribose) polymerase inhibitor Talazoparib is in a phase 1 clinical trial for therapy in relapsed/refractory acute myeloid leukemia.65
An orally available small molecule 20 (CA170), containing L-threonine, D-asparagine, and L-serine partially linked to urea and diacylhydrazine linker moieties, targets both VISTA and PD-L1.66 A single daily dose suppressed metastasis and tumor growth in mouse models of melanoma cells (B16F10) and colorectal cells (MC38).67 It rescued interferon gamma release from human peripheral blood mononuclear cells blocked by recombinant VISTA, PD-L1, and PD-L2. In a phase 1 study, it was found to be well-tolerated at an oral dose of 50–1,200 mg, with a plasma half-life of 4–9.5 h depending on the dosage.68 In phase 2 studies, it showed remarkable clinical activity, with a 30% overall response rate in Hodgkin lymphoma and a more than 85% clinical benefit rate at 400 mg daily, with progression-free survival of up to 19.6 weeks in advanced non-squamous NSCLC.69 It is also currently in a phase 1 trial for advanced solid tumors or lymphomas.70
Additionally, several other PD-L1 inhibitors, such as BPI-371153, are progressing through phase 1 clinical trials for advanced solid tumors or relapsed/refractory lymphoma71; compound 21 (Tomivosertib) is in phase 2 clinical trials for solid tumors72; compound 22 (Navtemadlin) is in phase 1 clinical trials for Merkel cell carcinoma73; compound 23 (Abemaciclib) is in phase 2 clinical trials for head and neck neoplasms74; compound 24 (Ciforadenant) is in phase 1 clinical trials for the treatment of renal cell cancers75; and recently developed small molecules like ABSK043 are in phase 1 clinical trials for neoplasms and advanced solid tumors.76
Small molecules have been explored for cancer treatment to overcome the limitations of mAbs. The following molecules, PD-1 and PD-L1 checkpoint inhibitors, are in various stages of clinical trials and have been investigated in combination with different mAbs, as shown in Table 1 and Figure 3.77–85 Small molecules are used in combination with mAbs to address issues such as lack of oral bioavailability and immune-related side effects.
Table 1Small molecules in clinical trials in combination with mAbs
| S.N | Small molecule | Synergistic mAbs | Cancer | The phase of clinical trials | Reference |
|---|
| 1 | 25 (Pexidartinib) (MW = 417.09) | Sirolimus | Unresectable sarcoma and malignant peripheral nerve tumors | Phase 1 | 77 |
| 2 | 26 (6-Thio-2′-deoxyguanosine) (MW = 283.309) | Cemiplimab | Advanced NSCLC | Phase 2 | 78 |
| 3 | 27 (Vismodegib) (MW = 420.01) | Atezolizumab | Platinum-resistant ovarian, fallopian tube, and primary peritoneal cancer | Phase 2 | 79 |
| 4 | 28 (Cabozantinib) (MW = 501.50) | Nivolumab and Ipilimumab | Renal-cell carcinoma | Phase 3 | 80 |
| 5 | 29 (Itacitinib) (MW = 552.52) | Pembrolizumab | Metastatic NSCLC | Phase 3 | 81 |
| 6 | 30 (SF1126) (MW = 865.86) | Nivolumab | Advanced hepatocellular carcinoma | Phase 1 | 82 |
| 7 | 31 (Sitravatinib) (MW = 629.19) | Nivolumab | Nonsquamous NSCLC progressing | Phase 2 | 83 |
| 8 | 32 (Talazoparib) (MW = 380.35) | Avelumab | Repair proficient endometrial cancer | Phase 1 | 84 |
| 9 | 33 (Apatinib) (MW = 397.19) | Camrelizumab | First-line platinum-resistant or PD-1 inhibitor resistant recurrent/metastatic nasopharyngeal carcinoma | Phase 2 | 85 |
Lead molecules
Apart from the drugs, repurposed drugs, and checkpoint inhibitors under different stages of clinical trials discussed above, novel lead molecules with the potential to serve as checkpoint inhibitors are explored in this section and shown in Figure 4.
Virtual screening using Molecular Operating Environment software led to the development of PVR binder compound 34 (Liothyronine), which interferes with interactions between the ITIM domain (TIGIT) and PVR, as well as T cell immunoglobulin. Its in vivo administration increased CD8+ T cell infiltration and immune responses in tumor-bearing mice, leading to tumor growth arrest.86 Homology modeling of VISTA, coupled with virtual screening, identified compound 35 as a VISTA binder with potent immunomodulatory activity in coculture cellular assays.87
Compound 36, incorporating a benzimidazole moiety with substituted phenyls, is a VISTA inhibitor with submicromolar binding affinity. It promoted VISTA degradation and enhanced the expression of lipidated MAP1LC3, an autophagosome membrane marker in HepG2 cancer cells.88 It also exhibited promising activity in the CT26 mouse model by suppressing tumor growth.89
Compound 37 [2-Fluoro-L-fucose (2F-Fuc)] reduced checkpoint glycosylation (fucosylation) and PD-1 levels on the surface of activated T-cells.90 It increased T-cell activity by inhibiting Fut8 and reducing PD-1 expression. Its combination with anti-PD-L1 ameliorated B7-H3 expression and enhanced efficacy in triple-negative breast cancer.
In addition to the molecules discussed above, other small molecules reported as ICIs interacting with PD-1 and PD-L1 include substituted biphenyls (38–41),30 compound 42 (2-bromopalmitate),49,91 compound 43 (BMS1166) (in preclinical phase),92 and compound 44 (NGI-1) (in preclinical phase).93 NGI-1 inhibited the activity of STT3A and STT3B, with higher specificity to the latter.94,95 The migration, invasion, and proliferation of lung adenocarcinoma cells were well inhibited by the compound.96 NGI-1 was also found to inhibit triple-negative breast tumors, which tend to be “cold” with low T-cell infiltration.93
Conclusions
It is evident from the above discussion that ICIs are substantially used clinically for various cancers. ICIs appear to be revolutionizing cancer therapeutics as an alternative to traditional chemotherapeutics for treating diverse malignancies. Despite several recent advancements in this field, it may still be considered in its infancy. Hence, more research is needed to overcome major shortcomings such as irAEs and heterogeneity. It is well known that each patient’s response to ICIs and the onset of irAEs can differ, so a careful understanding of these factors may help in devising strategies for personalized treatment.
Compared to widely used mAbs, small molecules offer advantages such as improved drug penetration into tumors or organs, enhanced stability, and better movement across cell membranes. Although the use of mAbs has been optimistic, they have not shown significant clinical improvements in patients with different types of cancer at various stages in several clinical trials. The efficacy of immunomodulators depends on the cancer type and its microenvironment. The therapeutic efficacy of these molecules is greatly influenced by mutations and specific molecular changes. Thus, identifying the relevant proteins or genes and understanding their molecular mechanisms may help enhance the efficacy of current immunomodulators. Another major drawback to consider is off-target toxicity or “cytokine storm”, which needs to be addressed for successful treatment with ICIs. Given these limitations, small molecules as immuno-oncology drugs have been explored as potential immune checkpoint inhibitors. However, these developments are still in the early stages and require further exploration to improve efficacy, reduce toxicity, increase bioavailability, and ensure specific action for their selection as candidate molecules for clinical development. Despite some disadvantages, small molecule ICIs have shown potential, and some are in various stages of clinical trials as immuno-oncology drugs. In this review, we summarized FDA-approved drugs, repurposed drugs, candidate drugs alone or in combination with mAbs, and novel potential lead molecules under preclinical investigation. The review described ten FDA-approved drugs that target PD-L1/PD-1 signaling, CTLA-4/CD28, TIGIT/PVR, and CD47/SIRPα pathways, as well as three repurposed drugs, eleven candidate drugs alone, and nine in combination with mAbs that are in different phases of clinical trials. Additionally, eleven novel chemical entities under pre-clinical studies may provide insights for optimization and the design of novel chemical entities for cancer immunotherapy.
Declarations
Funding
No funding was received for this research.
Conflict of interest
One of the authors, AKS, has been an editorial board member of the Oncology Advances journal since May 2021. The authors have no other conflicts of interest to declare.
Authors’ contributions
Literature search and compilation (NS), topic conception and design (AS), intellectual input, manuscript writing, and critical revision of the manuscript (AKS). All authors have made significant contributions to this study and have approved the final manuscript.