Beyond inhibition against the PD-1/PD-L1 pathway: development of PD-L1 inhibitors targeting internalization and degradation of PD-L1.

Tumor cells hijack the programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway to suppress the immune response through overexpressing PD-L1 to interact with PD-1 of T cells. With in-depth ongoing research, tumor-intrinsic PD-L1 is found to play important roles in tumor progression without interaction with PD-1 expressed on T cells, which provides an additional important target and therapeutic approach for development of PD-L1 inhibitors. Existing monoclonal antibody (mAb) drugs against the PD-1/PD-L1 pathway generally behave by conformationally blocking the interactions of PD-1 with PD-L1 on the cell surface. Beyond general inhibition of the protein-protein interaction (PPI), inhibitors targeting PD-L1 currently focus on the functional inhibition of the interaction between PD-1/PD-L1 and degradation of tumor-intrinsic PD-L1. This perspective will clarify the evolution of PD-L1 inhibitors and provide insights into the current development of PD-L1 inhibitors, especially targeting internalization and degradation of PD-L1.

Discovery of Novel Phenoxyaryl Pyridones as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with High Selectivity for the Second Bromodomain (BD2) to Potentially Treat Acute Myeloid Leukemia.

Bromodomain-selective BET inhibition has emerged as a promising strategy to improve the safety profiles of pan-BET inhibitors. Herein, we report the discovery of potent phenoxyaryl pyridones as highly BD2-selective BET inhibitors. Compound 23 (IC50 = 2.9 nM) exhibited a comparable BRD4 BD2 inhibitory activity relative to 10 (IC50 = 1.0 nM) and remarkably improved selectivity over BRD4 BD1 (23: 2583-fold; 10: 344-fold). This lead compound significantly inhibited the proliferation of acute myeloid leukemia (AML) cell lines through induction of G0/G1 arrest and apoptosis in vitro. Excellent in vivo antitumor efficacy with 23 was achieved in an MV;411 mouse xenograft model. Pleasingly, compound 23 (hERG IC50 > 30 µM) mitigated the inhibition of the human ether-à-go-go-related gene (hERG) ion channel compared with 10 (hERG IC50 = 2.8 µM). This work provides a promising BD2-selective lead for the development of more effective and safe BET inhibitors as anticancer agents.

Rising Star in Immunotherapy: Development and Therapeutic Potential of Small-Molecule Inhibitors Targeting Casitas B Cell Lymphoma-b (Cbl-b).

Casitas B cell lymphoma-b (Cbl-b) is a vital negative regulator of TCR and BCR signaling pathways, playing a significant role in setting an appropriate threshold for the activation of T cells and controlling the tolerance of peripheral T cells via a variety of mechanisms. Overexpression of Cbl-b leads to immune hyporesponsiveness of T cells. Conversely, the deficiency of Cbl-b in T cells results in markedly increased production of IL-2, even in the lack of CD28 costimulation in vitro. And Cbl-b-/- mice spontaneously reject multifarious cancers. Therefore, Cbl-b may be associated with immune-mediated diseases, and blocking Cbl-b could be considered as a new antitumor immunotherapy strategy. In this review, the possible regulatory mechanisms and biological potential of Cbl-b for antitumor immunotherapy are summarized. Besides, the potential roles of Cbl-b in immune-mediated diseases are comprehensively discussed, with emphasis on Cbl-b immune-oncology agents in the preclinical stage and clinical trials.

Discovery of Novel PD-L1 Inhibitors That Induce the Dimerization, Internalization, and Degradation of PD-L1 Based on the Fragment Coupling Strategy.

Tumor cells can evade immune surveillance through overexpressing programmed cell death-ligand 1 (PD-L1) to interact with programmed cell death-1 (PD-1). Besides, tumor-intrinsic PD-L1 is involved in tumor progression without interaction with PD-1, which provides more challenges for the discovery of PD-L1 inhibitors. Herein, we report the discovery of novel PD-L1 inhibitors using the fragment coupling strategy. Among them, B9 was found to inhibit the PD-1/PD-L1 interaction with the best IC50 value of 1.8 ± 0.7 nM. Beyond the blockade of the PD-1/PD-L1 axis, B9 promotes the dimerization, internalization, and degradation of PD-L1. Furthermore, B9 displayed high in vivo antitumor efficacy in the CT26 mouse model and activated the immune microenvironment and induced PD-L1 degradation of PD-L1 in the tumor. These results show that B9 is a promising lead PD-L1 inhibitor through the blockade of PD-1/PD-L1 interaction and functional inhibition of the PD-L1 signal pathway.

Novel Benzimidazoles as Potent Small-Molecule Inhibitors and Degraders of V-Domain Ig Suppressor of T-Cell Activation (VISTA).

The V-domain Ig suppressor of T-cell activation (VISTA) is a promising negative immune checkpoint and plays a critical role in the regulation of the quiescence of naïve T lymphocytes. Most patients however do not experience durable disease control from current immune checkpoint inhibitors and discovery of inhibitors targeting novel immune checkpoints is necessary. Herein, we report our discovery and optimization of benzimidazoles as the bifunctional inhibitors of VISTA. Compound 1 is identified as a bifunctional inhibitor targeting VISTA, which shows good binding affinity to VISTA and induces VISTA degradation in HepG2 cells through an autophagic mechanism. Compound 1 rescues VISTA-mediated immunosuppression effectively and enhances antitumor activity of immune cells. 1 activates the antitumor immunity in vivo and suppresses tumor growth in a CT26 mouse model significantly. Our results show that compound 1 is a promising VISTA inhibitor and degrader and offers novel approach for cancer immunotherapy through VISTA degradation.

Recent advances of targeting nicotinamide phosphoribosyltransferase (NAMPT) for cancer drug discovery.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for the biosynthesis of NAD+ in the salvage pathway. NAMPT is overexpressed in various cancers, associating with a poor prognosis and tumor progression. Beyond cancer metabolism, recent evidence unravels additional roles of NAMPT in cancer biology, including DNA repair machinery, crosstalk with oncogenic signaling pathways, cancer cell stemness, and immune responses. NAMPT is a promising therapeutic target for cancer. However, first-generation NAMPT inhibitors exhibited limited efficacy and dose-limiting toxicities in clinical trials. Multiple strategies are being exploited to improve their efficacy and minimize toxic-side effects. This review discusses the biomarkers predictive of response to NAMPT inhibitors, and summarizes the most significant advances in the evolution of structurally distinct NAMPT inhibitors, the manipulation of targeted delivery technologies via antibody-drug conjugates (ADCs), PhotoActivated ChemoTherapy (PACT) and the intratumoral delivery system, as well as the development and pharmacological outcomes of NAMPT degraders. Finally, a discussion of future perspectives and challenges in this area is also included.

Discovery of diaminotriazine carboxamides as potent inhibitors of hematopoetic progenitor kinase 1.

Hematopoietic progenitor kinase 1 (HPK1), a member of mitogen-activated protein kinase kinase kinase kinase (MAP4K) family of Ste20 serine/threonine kinases, is a negative regulator of T-cell receptor (TCR) signaling. Inactivating HPK1 kinase has been reported to be sufficient to elicit antitumor immune response. Therefore, HPK1 has attracted much attention as a promising target for tumor immunotherapy. A few of HPK1 inhibitors have been reported, and none of them have been approved for clinical applications. Hence, more effective HPK1 inhibitors are needed. Herein, a series of structurally novel diaminotriazine carboxamides were rationally designed, synthesized and evaluated for their inhibitory activity against HPK1 kinase. Most of them exhibited potent inhibitory potency against HPK1 kinase. In particular, compound 15b showed more robust HPK1 inhibitory activity than that of 11d developed by Merck in kinase activity assay (IC50 = 3.1 and 8.2 nM, respectively). The significant inhibitory potency against SLP76 phosphorylation in Jurkat T cells further confirmed the efficacy of compound 15b. In human peripheral blood mononuclear cell (PBMC) functional assays, compound 15b more significantly induced the production of interleukin 2 (IL-2) and interferon γ (IFN-γ) relative to 11d. Furthermore, 15b alone or in combination with anti-PD-1 antibodies showed potent in vivo antitumor efficacy in MC38 tumor-bearing mice. Compound 15b represents a promising lead for the development of effective HPK1 small-molecule inhibitors.

Design, synthesis, and evaluation of PD-1/PD-L1 small-molecule inhibitors bearing a rigid indane scaffold.

Discovery of small-molecule inhibitors against programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) axis provides a promising alternative to overcome the inevitable defects of PD-1/PD-L1 monoclonal antibodies (mAbs). Here, we report a series of indanes as novel small-molecule inhibitors of PD-1/PD-L1 interaction. Thirty-one indanes were synthesized and the structure-activity relationships (SARs) demonstrated that conformational restriction with (S)-indane is superior in potency to inhibit the interaction of PD-1 and PD-L1. Compound D3 was found to be the most potent inhibitor with an IC50 value of 2.2 nM against PD-1/PD-L1 interaction. Cell-based assay showed that D3 significantly induced immune activity of peripheral blood mononuclear cells (PBMCs) against MDA-MB-231 cells and could restore the immune function of T cells by promoting secretion of the IFN-γ. The above results indicate that compound D3 is a promising PD-1/PD-L1 inhibitor that deserves further development.

Discovery of quinazolin-4-one-based non-covalent inhibitors targeting the severe acute respiratory syndrome coronavirus 2 main protease (SARS-CoV-2 Mpro).

The COVID-19 pandemic caused by SARS-CoV-2 continues to pose a great threat to public health while various vaccines are available worldwide. Main protease (Mpro) has been validated as an effective anti-COVID-19 drug target. Using medicinal chemistry and rational drug design strategies, we identified a quinazolin-4-one series of nonpeptidic, noncovalent SARS-CoV-2 Mpro inhibitors based on baicalein, 5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one. In particular, compound C7 exhibits superior inhibitory activity against SARS-CoV-2 Mpro relative to baicalein (IC50 = 0.085 ± 0.006 and 0.966 ± 0.065 µM, respectively), as well as improved physicochemical and drug metabolism and pharmacokinetics (DMPK) properties. In addition, C7 inhibits viral replication in SARS-CoV-2-infected Vero E6 cells more effectively than baicalein (EC50 = 1.10 ± 0.12 and 5.15 ± 1.64 µM, respectively) with low cytotoxicity (CC50 ＞ 50 µM). An X-ray co-crystal structure reveals a non-covalent mechanism of action, and a noncanonical binding mode not observed by baicalein. These results suggest that C7 represents a promising lead for development of more effective SARS-CoV-2 Mpro inhibitors and anti-COVID-19 drugs.

Development and Therapeutic Implications of Tyrosine Kinase 2 Inhibitors.

Janus kinases (JAKs) are central components in cytokine signaling pathways. A number of small molecule JAK inhibitors have been approved to treat a wide range of inflammatory and autoimmune diseases. Due to safety concerns of pan-JAK inhibition, the thrust of current research is toward the discovery of isoform-selective JAK inhibitors. Selective inhibition of tyrosine kinase 2 (TYK2) has the potential to balance efficacy and safety. Substantial efforts have been made to develop selective TYK2 inhibitors: Deucravacitinib (BMS-986165) is a representative allosteric inhibitor that has been approved by the FDA, and ropsacitinib (PF-06826647) is an active site-directed inhibitor currently being evaluated in clinical trials. Herein, we outline the key roles of TYK2 in diseases, review the advances of selective TYK2 inhibitors, and finally discuss future perspectives and challenges in the development of TYK2 inhibitors.

Design, synthesis, biological evaluation of urea substituted 1,2,5-oxadiazole-3-carboximidamides as novel indoleamine 2,3-dioxygenase-1 (IDO1) inhibitors.

Indoleamine 2,3-dioxygenase-1 (IDO1) has been considered as an attractive target for oncology immunotherapy due to its immunosuppressive effects on the tumor microenvironment. The most advanced IDO1 inhibitor epacadostat in combination with anti-PD-1 antibody failed to show desirable objective response. Epacadostat is now reevaluated in phase III clinical trials, but its pharmacokinetic (PK) properties are unsatisfactory. To further unravel the antitumor efficacy of IDO1 inhibitors, we designed a series of epacadostat analogues by introducing various urea-containing side chains. In particular, the most active compound 3 showed superior inhibitory potency against recombinant hIDO1 and hIDO1 in HeLa cells induced by interferon γ (IFNγ) relative to epacadostat (3, biochemical hIDO1 IC50 = 67.4 nM, HeLa hIDO1 IC50 = 17.6 nM; epacadostat, biochemical hIDO1 IC50 = 75.9 nM, HeLa hIDO1 IC50 = 20.6 nM). Moreover, compound 3 exhibited improved physicochemical properties and rat PK profile with better oral exposure and bioavailability compared with epacadostat. Importantly, this compound exhibited comparable antitumor efficacy with epacadostat in LLC syngeneic xenograft models. Hence, compound 3 represents a promising lead compound for discovery of more effective IDO1 inhibitors.

Dual Nicotinamide Phosphoribosyltransferase (NAMPT) and Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors for the Treatment of Drug-Resistant Nonsmall-Cell Lung Cancer.

Depleting NAD+ by blocking its biosynthesis has emerged as an attractive anticancer strategy. Simultaneous blockade of NAD+ production from the salvage and de novo synthesis pathways by targeting NAMPT and IDO1 could achieve more effective NAD+ reduction and, subsequently, more robust antitumor efficacy. Herein, we report the discovery of the first series of dual NAMPT and IDO1 inhibitors according to multitarget drug rationales. Compound 10e has good and balanced inhibitory potencies against NAMPT and IDO1, and significantly inhibits both proliferation and migration of a NSCLC cell line resistant to taxol and FK866 (A549/R cells). Compound 10e also displays potent antitumor efficacy in A549/R xenograft mouse models with no significant toxicity. Moreover, this compound enhances the susceptibility of A549/R cells to taxol in vitro and in vivo. This work provides an efficient approach to targeting NAD+ metabolism in the area of cancer therapy, especially in the context of drug resistance.

Recent Insights of Metformin on Hepatocellular Carcinoma (HCC).

Metformin is an oral hypoglycemic drug, the first option used to treat type 2 diabetes mellitus due to its high efficacy and low cost. Recently, it has drawn attention among researchers due to its new-found antitumor effect. Growing evidence showed that metformin could inhibit cancer progression, especially in hepatocellular carcinoma, and several clinical trials are underway. However, the underlying mechanisms of the inhibition of hepatocellular carcinoma remain to be further explored and clarified. Herein, we reviewed the latest findings of how metformin acts against hepatocellular carcinoma and the proposed mechanisms. In addition, we included related preclinical trials, along with the limitations and perspectives of its treatment in hepatocellular carcinoma, providing novel ideas for research to conquer hepatocellular carcinoma.

The development of small-molecule inhibitors targeting HPK1.

Hematopoietic progenitor kinase 1 (HPK1), a 97-kDa serine/threonine Ste20-related protein kinase, is a negative regulator of T cells, B cells, and dendritic cells-mediated immune responses and is primarily expressed in hematopoietic lineage cells. HPK1 regulates different cellular processes by interacting with a variety of substrates and adaptors, including immune cell activation, cellular differentiation, proliferation, adhesion, and apoptosis. In HPK1KO mice, T cells over-proliferate in response to stimulation by anti-CD3 and anti-CD28 antibodies, and these cells can secrete more proinflammatory cytokines to enhance T-cell activation and tumor growth inhibition when immunized with T cell-dependent antigens. Therefore, HPK1 may be associated with occurrence and development of human malignant tumors and is an effective antitumor immunotherapy target. In this perspective review, the biological rationale and potential of HPK1 as a promising candidate target for cancer immunotherapies and the latest research progress of HPK1 are summarized, with special emphasis on the current small-molecule inhibitors of HPK1 in preclinical and clinical studies.

Development of Inhibitors Targeting the V-Domain Ig Suppressor of T Cell Activation Signal Pathway.

Blockade of cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) has produced considerable therapeutic effect, but only in a fraction of patients, so more targets are being investigated. VISTA (V-domain Ig suppressor of T cell activation) is a novel immune checkpoint that is broadly expressed within hematopoietic cells and multiple cancers (low expressing frequency on solid tumors), particularly those with a poor immunotherapy response rate. As a result, VISTA has been identified as an appealing target for immunotherapy, and several VISTA inhibitors are currently in clinical and preclinical trials. In this review, the structural features and binding partners of VISTA are summarized, and we describe the latest developments of monoclonal antibodies and small molecules targeting VISTA as well as possible future directions for development of therapies targeting VISTA.

Discovery of Small Molecules Simultaneously Targeting NAD(P)H:Quinone Oxidoreductase 1 and Nicotinamide Phosphoribosyltransferase: Treatment of Drug-Resistant Non-small-Cell Lung Cancer.

Targeting NAD+ metabolism has emerged as an effective anticancer strategy. Inspired by the synergistic antitumor effect between NAD(P)H:quinone oxidoreductase 1 (NQO1) substrates increasing the NAD consumption and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors hampering the NAD synthesis, first-in-class small molecules simultaneously targeting NQO1 and NAMPT were identified through structure-based design. In particular, compound 10d is an excellent NQO1 substrate that is processed faster than TSA by NQO1 and exhibited a slightly decreased NAMPT inhibitory potency than that of FK866. It can selectively inhibit the proliferation of NQO1-overexpressing A549 cells and taxol-resistant A549/taxol cells and also induce cell apoptosis and inhibit cell migration in an NQO1- and NAMPT-dependent manner in A549/taxol cells. Significantly, compound 10d demonstrated excellent in vivo antitumor efficacy in the A549/taxol xenograft models with no significant toxicity. This proof-of-concept study affirms the feasibility of discovering small molecules that target NQO1 and NAMPT simultaneously, and it also provides a novel, effective, and selective anticancer strategy.

Design, Synthesis, and Evaluation of PD-1/PD-L1 Antagonists Bearing a Benzamide Scaffold.

Several antibodies targeting programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) have been approved by the U.S. Food and Drug Administration (FDA) for cancer therapy. Although many small-molecule inhibitors of the PD-1/PD-L1 pathway have been reported, no small-molecule inhibitors have been approved for cancer treatment. In this work, a series of novel benzamide derivatives were designed, synthesized, and evaluated to find effective inhibitors of the PD-1/PD-L1 interaction. The most potent compound D2 exhibited better activity than that of BMS202, with an IC50 of 16.17 nM. D2 could activate the antitumor immunity of T cells efficiently in PBMCs. The proposed binding mode of compound D2 was investigated by docking analysis. These results indicate that compound D2 is a promising lead compound that can be used for further development.

Novel Biphenyl Pyridines as Potent Small-Molecule Inhibitors Targeting the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction.

With the successful clinical application of anti-programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) monoclonal antibodies (mAb), targeting the PD-1/PD-L1 interaction has become a promising method for the discovery of cancer therapy. Due to the inherent limitations of antibodies, it is necessary to search for small-molecule inhibitors against the PD-1/PD-L1 axis. We report the design, synthesis, and evaluation in vitro and in vivo of a series of novel biphenyl pyridines as the inhibitors of PD-1/PD-L1. 2-(((2-Methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethan-1-ol (24) was found to inhibit the PD-1/PD-L1 interaction with an IC50 value of 3.8 ± 0.3 nM and enhance the killing activity of tumor cells by immune cells. Compound 24 displays great pharmacokinetics (oral bioavailability of 22%) and significant in vivo antitumor activity in a CT26 mouse model. Flow cytometry and immunohistochemistry data indicated that compound 24 activates the immune activity in tumors. These results suggest that compound 24 is a promising small-molecule inhibitor against the PD-1/PD-L1 axis and merits further development.

Structure-Based Design of a Selective Class I Histone Deacetylase (HDAC) Near-Infrared (NIR) Probe for Epigenetic Regulation Detection in Triple-Negative Breast Cancer (TNBC).

Abnormally high levels of class I histone deacetylases (HDACs) are associated with triple-negative breast cancer (TNBC) proliferation, malignant transformation, and poor prognosis of patients. Herein, we report a near-infrared imaging probe for TNBC detection via visualizing class I HDACs. Conjugating Cy5.5 to a cyclic depsipeptide inhibitor, we obtained the probe (20-Cy5.5) that retained desirable class I HDAC affinity and selectivity. Then, this probe could visualize epigenetic changes by class I HDACs in TNBC MDA-MB-231 cells and in xenograft tumor models in real time. Treatment with suberoylanilide hydroxamic acid (SAHA) significantly reduced the uptake of the probe in tumors, suggesting its potential use in evaluation of therapeutic responses of HDACi-mediated therapy. Moreover, 20-Cy5.5 could detect class I HDAC expression in TNBC lung metastasis. This novel NIR probe that achieves tumor class I HDAC imaging not only leads to a better understanding of epigenetic regulation in tumors but also has great potential for improving the TNBC diagnosis and treatment.