Discovery of Novel Small-Molecule-Based Potential PD-L1/EGFR Dual Inhibitors with High Druggability for Glioblastoma Immunotherapy.

Based on the close relationship between programmed death protein ligand 1 (PD-L1) and epidermal growth factor receptor (EGFR) in glioblastoma (GBM), we designed and synthesized a series of small molecules as potential dual inhibitors of EGFR and PD-L1. Among them, compound EP26 exhibited the highest inhibitory activity against EGFR (IC50 = 37.5 nM) and PD-1/PD-L1 interaction (IC50 = 1.77 µM). In addition, EP26 displayed superior in vitro antiproliferative activities and in vitro immunomodulatory effects by promoting U87MG cell death in a U87MG/Jurkat cell coculture model. Furthermore, EP26 possessed favorable pharmacokinetic properties (F = 22%) and inhibited tumor growth (TGI = 92.0%) in a GBM mouse model more effectively than Gefitinib (77.2%) and NP19 (82.8%). Moreover, EP26 increased CD4+ cells and CD8+ cells in tumor microenvironment. Collectively, these results suggest that EP26 represents the first small-molecule-based PD-L1/EGFR dual inhibitor deserving further investigation as an immunomodulating agent for cancer treatment.

Discovery and Crystallography Study of Novel Biphenyl Ether and Oxadiazole Thioether (Non-Arylmethylamine)-Based Small-Molecule PD-1/PD-L1 Inhibitors as Immunotherapeutic Agents.

Current small-molecule PD-1/PD-L1 inhibitors are mainly based on the arylmethylamine/biphenyl core scaffold. Herein, we designed for the first time a series of non-arylmethylamine analogues (oxadiazole thioether derivatives) as small-molecule PD-1/PD-L1 inhibitors. Among them, compound LP23 exhibited the most potent PD-L1 inhibitory activity with an IC50 of 16.7 nM, 3.2-fold better than the lead BMS-202 (IC50 = 53.6 nM). The X-ray crystal structure of LP23 in complex with PD-L1 was solved at a resolution of 2.6 Å, which further confirmed the high binding affinity of LP23 to PD-L1. In the HepG2/Jurkat T cell co-culture model, LP23 effectively promoted HepG2 cell death by restoring the immune function of T cells. In addition, LP23 showed excellent in vivo antitumor efficacy (TGI = 88.6% at 30 mg/kg) and benign toxicity profiles in a B16-F10 tumor model by modulating PD-L1. In summary, LP23 represents the first non-arylmethylamine-based small-molecule PD-1/PD-L1 inhibitor worthy of further investigation.

Discovery of novel resorcinol biphenyl ether-based macrocyclic small molecules as PD-1/PD-L1 inhibitors with favorable pharmacokinetics for cancer immunotherapy.

Programmed death protein 1 (PD-1)/programmed death protein ligand 1 (PD-L1) is one of the most promising immune checkpoints (ICs) in tumor immunology and has been actively pursued as a target for anticancer drug discovery. Based on our previous research in small molecule PD-1/PD-L1 modulators, we designed and synthesized a series of resorcinol biphenyl ether-bearing macrocyclic compounds and evaluated their anti-PD-1/PD-L1 activities. Among them, compound 8d exhibited the highest inhibitory activity against PD-1/PD-L1 interaction with IC50 of 259.7 nM in the homogenous time-resolved fluorescence (HTRF) assay. In addition, 8d displayed in vitro immunomodulatory effects by promoting HepG2 cell death in a HepG2/Jurkat cell co-culture model. Furthermore, 8d effectively inhibited tumor growth (TGI = 74.6% at 40 mg/kg) in a melanoma tumor model in mice without causing obvious toxicity. Moreover, 8d exhibited favorable pharmacokinetics [e.g. high stability, reasonable half-life, and good oral bioavailability (F = 21.5%)]. Finally, molecular modeling studies showed that 8d bound to PD-L1 with high affinity. These results suggest that 8d may serve as a starting point for further development of macrocyclic small molecule-based PD-1/PD-L1 inhibitors for cancer treatment.

X-ray Crystal Structure-Guided Discovery of Novel Indole Analogues as Colchicine-Binding Site Tubulin Inhibitors with Immune-Potentiating and Antitumor Effects against Melanoma.

A series of novel indole analogues were discovered as colchicine-binding site inhibitors of tubulin. Among them, 3a exhibited the highest antiproliferative activity (average IC50 = 4.5 nM), better than colchicine (IC50 = 65.3 nM). The crystal structure of 3a in complex with tubulin was solved by X-ray crystallography, which explained the improved binding affinity of 3a to tubulin and thus its higher anticancer activity (IC50 = 4.5 nM) than the lead compound 12b (IC50 = 32.5 nM). In vivo, 3a (5 mg/kg) displayed significant antitumor efficacy against B16-F10 melanoma with a TGI of 62.96% and enhanced the antitumor efficacy of a small-molecule PD-1/PD-L1 inhibitor NP19 (TGI = 77.85%). Moreover, 3a potentiated the antitumor immunity of NP19 by activating the tumor immune microenvironment, as demonstrated by the increased tumor-infiltrating lymphocytes (TIL). Collectively, this work shows a successful example of crystal structure-guided discovery of a novel tubulin inhibitor 3a as a potential anticancer and immune-potentiating agent.

Discovery of Novel Acridane-Based Tubulin Polymerization Inhibitors with Anticancer and Potential Immunomodulatory Effects.

A series of novel acridane-based tubulin polymerization inhibitors were designed, synthesized, and bioevaluated as anticancer agents. The most potent compound NT-6 exhibited high tubulin polymerization inhibitory activity (IC50 = 1.5 µM) and remarkable antiproliferative potency against four cancer cell lines with an average IC50 of 30 nM, better than colchicine and the hit compound 1f (IC50 of 65 and 126 nM, respectively). In addition, NT-6 (10 mg/kg) exerted excellent antitumor efficacy in a melanoma tumor model with a tumor growth inhibition (TGI) of 65.1% without apparent toxicity. Importantly, the combination of NT-6 with a small-molecule PD-L1 inhibitor NP-19 decreased tumor burden significantly (TGI% = 77.6%). Moreover, the combination of NT-6 with NP-19 enhanced the antitumor immune response, mediated by a decrease of PD-L1 expression levels and increased infiltration of antitumor CD8+ effector T cells in tumor tissues. Collectively, NT-6 represents a novel tubulin polymerization inhibitor with immunopotentiating effects.

Design, synthesis, and bioevaluation of imidazo [1,2-a] pyrazine derivatives as tubulin polymerization inhibitors with potent anticancer activities.

Through structural optimization and ring fusion strategy, we designed a series of novel imidazo[1,2-a]pyrazine derivatives as potential tubulin inhibitors. These compounds displayed potent anti-proliferative activities (micromolar to nanomolar) against a panel of cancer cell lines (including HepG-2, HCT-116, A549 and MDA-MB-231 cells). Among them, compound TB-25 exhibited the strongest inhibitory effects against HCT-116 cells with an IC50 of 23 nM. Mechanism studies revealed that TB-25 could effectively inhibit tubulin polymerization in vitro, and destroy the dynamic equilibrium of microtubules in HCT-116 cells. In addition, TB-25 dose-dependently induced G2/M phase cell cycle arrest and apoptosis in HCT-116 cells. Furthermore, TB-25 suppressed HCT-116 cell migration in a concentration-dependent manner. Finally, molecular docking showed that TB-25 fitted well in the colchicine binding site of tubulin and overlapped nicely with CA-4. Collectively, these results suggest that TB-25 represents a promising tubulin inhibitor deserving further investigation.

Novel CRBN-Recruiting Proteolysis-Targeting Chimeras as Degraders of Stimulator of Interferon Genes with In Vivo Anti-Inflammatory Efficacy.

The activation of the cyclic GMP-AMP synthase-stimulator of interferon gene (STING) pathway has been associated with the pathogenesis of many autoimmune and inflammatory disorders, and small molecules targeting STING have emerged as a new therapeutic strategy for the treatment of these diseases. While several STING inhibitors have been identified with potent anti-inflammatory effects, we would like to explore STING degraders based on the proteolysis-targeting chimera (PROTAC) technology as an alternative strategy to target the STING pathway. Thus, we designed and synthesized a series of STING protein degraders based on a small-molecule STING inhibitor (C-170) and pomalidomide (a CRBN ligand). These compounds demonstrated moderate STING-degrading activities. Among them, SP23 achieved the highest degradation potency with a DC50 of 3.2 µM. Importantly, SP23 exerted high anti-inflammatory efficacy in a cisplatin-induced acute kidney injury mouse model by modulating the STING signaling pathway. Taken together, SP23 represents the first PROTAC degrader of STING deserving further investigation as a new anti-inflammatory agent.

Discovery of Thieno[2,3-d]pyrimidine-based KRAS G12D inhibitors as potential anticancer agents via combinatorial virtual screening.

A series of novel thieno[2,3-d]pyrimidine analogs were designed and synthesized as KRAS G12D inhibitors via combinatorial virtual screening approach. Most compounds exhibited potent antiproliferative activity on KRAS G12D mutated cancer cell lines (Panc1, SW1990 and CT26) with IC50 values in the low micromolar range. Among them, compound KD-8 showed the highest antiproliferative activity with an average IC50 of 2.1 µM against three KRAS G12D-mutated cells (Panc1, SW1990 and CT26). KD-8 decreased the active form of KRAS (KRAS-GTP) in KRAS G12D mutated cancer cell lines (CT26 and SW1990) but not in KRAS G13D mutated cancer cells (HCT116). Moreover, KD-8 down-regulated the phosphorylated Raf and Erk in CT26 and SW1990 cancer cell lines but not in HeLa cells (KRAS WT). The binding affinity of KD-8 was further confirmed by the isothermal titration calorimetry (ITC) assay in which KD-8 exhibited a KD of 33 nM for binding to KRAS G12D protein. In addition, KD-8 (40 mg/kg or 60 mg/kg) exhibited significant antitumor efficacy in a CT26 tumor model with a tumor growth inhibition (TGI) of 42% or 53% without causing apparent toxicity. Taken together the above results suggest that KD-8 is a promising KRAS G12D inhibitor deserving further investigation.

Discovery of Novel Histone Deacetylase 6 (HDAC6) Inhibitors with Enhanced Antitumor Immunity of Anti-PD-L1 Immunotherapy in Melanoma.

A series of 2-phenylthiazole analogues were designed and synthesized as potential histone deacetylase 6 (HDAC6) inhibitors based on compound 12c (an HDAC6/tubulin dual inhibitor discovered by us recently) and CAY10603 (a known HDAC6 inhibitor). Among them, compound XP5 was the most potent HDAC6 inhibitor with an IC50 of 31 nM and excellent HDAC6 selectivity (SI = 338 for HDAC6 over HDAC3). XP5 also displayed high antiproliferative activity against various cancer cell lines including the HDACi-resistant YCC3/7 gastric cancer cells (IC50 = 0.16-2.31 µM), better than CAY10603. Further, XP5 (50 mg/kg) exhibited significant antitumor efficacy in a melanoma tumor model with a tumor growth inhibition (TGI) of 63% without apparent toxicity. Moreover, XP5 efficiently enhanced the in vivo antitumor immune response when combined with a small-molecule PD-L1 inhibitor, as demonstrated by the increased tumor-infiltrating lymphocytes and reduced PD-L1 expression levels. Taken together, the above results suggest that XP5 is a promising HDAC6 inhibitor deserving further investigation.

Design, synthesis and biological evaluation of novel acridine and quinoline derivatives as tubulin polymerization inhibitors with anticancer activities.

A series of acridine and quinoline derivatives were designed and synthesized based on our previous work as novel tubulin inhibitors targeting the colchicine binding site. Among them, compound 3b exhibited the highest antiproliferative activity with an IC50 of 261 nM against HepG-2 cells (the most sensitive cell line). In addition, compound 3b was able to suppress the formation of HepG-2 colonies. Mechanism studies revealed that compound 3b effectively inhibited tubulin polymerization in vitro and disrupted microtubule dynamics in HepG-2 cells. Furthermore, compound 3b inhibited the migration of cancer cells in a dose dependent manner. Moreover, compound 3b induced cell cycle arrest in G2/M phase and led to cell apoptosis. Finally, docking studies demonstrated that compound 3b fitted nicely in the colchicine binding site of tubulin and overlapped well with CA-4. Collectively, these results suggested that compound 3b represents a novel tubulin inhibitor deserving further investigation.

Synthesis and pharmacological evaluation of a novel synthetic peptide CWHTH based on the Styela clava-derived natural peptide LWHTH with improved antioxidant, hepatoprotective and angiotensin converting enzyme inhibitory activities.

LWHTH (Leu-Trp-His-Thr-His) is an antioxidant pentapeptide isolated from Styela clava tissue. Based on LWHTH, we designed and synthesized a series of novel peptides using the alanine scanning technique and determined the pharmacological activities of these derivatives. Among the ten newly synthesized LWHTH analogs, peptide CWHTH was identified as the most potent compound with prominent antioxidant activity. CWHTH not only showed the ability to scavenge several biologically important radicals, protected cells from H2O2 or APAP-induced damage by activating the PI3K/Akt and suppressing the JNK/c-Jun pathways, but also exerted strong in vivo hepatoprotective effects in an APAP-induced liver injury model in mice. Moreover, it was demonstrated that CWHTH possesses potent angiotensin converting enzyme (ACE) inhibitory activity and high stability against gastrointestinal proteases. In summary, CWHTH is a promising antioxidant peptide worthy of further investigation as a potential hepatoprotective and antihypertensive agent.

Efficient Synthesis and Bioevaluation of Novel Dual Tubulin/Histone Deacetylase 3 Inhibitors as Potential Anticancer Agents.

Novel dual HDAC3/tubulin inhibitors were designed and efficiently synthesized by combining the pharmacophores of SMART (tubulin inhibitor) and MS-275 (HDAC inhibitor), among which compound 15c was found to be the most potent and balanced HDAC3/tubulin dual inhibitor with high HDAC3 activity (IC50 = 30 nM) and selectivity (SI > 1000) as well as excellent antiproliferative potency against various cancer cell lines, including an HDAC-resistant gastric cancer cell line (YCC3/7) with IC50 values in the range of 30-144 nM. Compound 15c inhibited B16-F10 cancer cell migration and colony formation. In addition, 15c demonstrated significant in vivo antitumor efficacy in a B16-F10 melanoma tumor model with a better TGI (70.00%, 10 mg/kg) than that of the combination of MS-275 and SMART. Finally, 15c presented a safe cardiotoxicity profile and did not cause nephro-/hepatotoxicity. Collectively, this work shows that compound 15c represents a novel tubulin/HDAC3 dual-targeting agent deserving further investigation as a potential anticancer agent.

Discovery of Novel Benzimidazole and Indazole Analogues as Tubulin Polymerization Inhibitors with Potent Anticancer Activities.

Novel indazole and benzimidazole analogues were designed and synthesized as tubulin inhibitors with potent antiproliferative activities. Among them, compound 12b exhibited the strongest inhibitory effects on the growth of cancer cells with an average IC50 value of 50 nM, slightly better than colchicine. 12b exhibited nearly equal potency against both, a paclitaxel-resistant cancer cell line (A2780/T, IC50 = 9.7 nM) and the corresponding parental cell line (A2780S, IC50 = 6.2 nM), thus effectively overcoming paclitaxel resistance in vitro. The crystal structure of 12b in complex with tubulin was solved to 2.45 Å resolution by X-ray crystallography, and its direct binding was confirmed to the colchicine site. Furthermore, 12b displayed significant in vivo antitumor efficacy in a melanoma tumor model with tumor growth inhibition rates of 78.70% (15 mg/kg) and 84.32% (30 mg/kg). Collectively, this work shows that 12b is a promising lead compound deserving further investigation as a potential anticancer agent.

Design, synthesis and biological evaluation of benz-fused five-membered heterocyclic compounds as tubulin polymerization inhibitors with anticancer activities.

A series of benz-fused five-membered heterocyclic compounds were designed and synthesized as novel tubulin inhibitors targeting the colchicine binding site. Among them, compound 4d displayed the highest antiproliferative activity against four cancer cell lines with an IC50 value of 4.9 µM in B16-F10 cells. Compound 4d effectively inhibited tubulin polymerization in vitro (IC50 of 13.1 µM). Further, 4d induced cell cycle arrest in G2/M phase. Finally, 4d inhibited the migration of cancer cells in a dose-dependent manner. In summary, these results suggest that compound 4d represents a new class of tubulin inhibitors deserving further investigation.

Discovery of novel cell-penetrating and tumor-targeting peptide-drug conjugate (PDC) for programmable delivery of paclitaxel and cancer treatment.

To ameliorate the deficiencies (e.g. solubility, membrane permeability and non-selective cytotoxicity) of paclitaxel (PTX), we synthesized a "smart" PDC (peptide-drug conjugate), by linking PTX with a multifunctional peptide consisting of a tumor targeting peptide (TTP) and a cell penetrating peptide (CPP), to construct the TTP-CPP-PTX conjugate, LTP-1. LTP-1 could intelligently deliver PTX into LHRH receptor-overexpressed MCF-7 cells, showing 2 times higher cellular uptake than PTX, and enhanced cytotoxicity with IC50 of 3.8 nM (vs 6.6 nM for PTX). LTP-1 exhibited less cytotoxicity to normal cells and the ability to overcome PTX-resistance. Furthermore, LTP-1 had higher in vivo antitumor efficacy than PTX (TGI of 83.4% and 65.7% for LTP-1 and PTX, respectively, at 12 mmol/kg) without apparent toxicities. In summary, we proposed and testified the concept of constructing a novel PDC molecule, which can potentially conquer the drawbacks of PTX. LTP-1 represents a new class of antitumor PDC deserving further investigation.

Discovery of Novel and Highly Potent Resorcinol Dibenzyl Ether-Based PD-1/PD-L1 Inhibitors with Improved Drug-like and Pharmacokinetic Properties for Cancer Treatment.

A series of programmed cell death-1 (PD-1)/programmed cell death ligand 1 (PD-L1) inhibitors based on the resorcinol diphenyl ether scaffold were discovered by incorporating hydrophilic moieties into the side chain and converting into the corresponding hydrochloride salt. Among these compounds, P18 showed the highest inhibitory activity against PD-1/PD-L1 with an IC50 value of 9.1 nM in a homogeneous time-resolved fluorescence binding assay. Besides, P18 promoted HepG2 cell death dose dependently in a HepG2/PD-L1 and Jurkat/PD-1 coculture cell model. Further, P18 demonstrated significantly higher water solubility (17.61 mg/mL) and improved pharmacokinetics (e.g., t1/2 of ∼20 h and oral bioavailability of 12%) than the previous analogues. Moreover, P18 was highly effective in suppressing tumor growth in an immune checkpoint humanized mouse model without apparent toxicity. Collectively, these results suggest that compound P18 represents a promising PD-1/PD-L1 inhibitor worthy of further investigation as a potential anticancer agent.

Discovery of Novel Resorcinol Dibenzyl Ethers Targeting the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction as Potential Anticancer Agents.

Novel small molecule compounds based on various scaffolds including chalcone, flavonoid, and resorcinol dibenzyl ether were designed and tested for their inhibitory activity against the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 (PD-1/PD-L1) pathway. Among them, compound NP19 inhibited the human PD-1/PD-L1 interaction with IC50 values of 12.5 nM in homogeneous time-resolved fluorescence (HTRF) binding assays. In addition, NP19 dose-dependently elevated IFN-γ production in a coculture model of Hep3B/OS-8/hPD-L1 and CD3 T cells. Furthermore, NP19 displayed significant in vivo antitumor efficacy in two different mouse models of cancer (a melanoma B16-F10 tumor model and an H22 hepatoma tumor model). Moreover, H&E staining and flow cytometry data suggested that NP19 activated the immune microenvironment in the tumor, which may contribute to its antitumor effects. This work shows NP19 is a promising lead compound for further development as a new generation of small molecule inhibitors targeting the PD-1/PD-L1 pathway.

Design, synthesis, and bioevaluation of pyrazolo[1,5-a]pyrimidine derivatives as tubulin polymerization inhibitors targeting the colchicine binding site with potent anticancer activities.

A series of Pyrazolo[1,5-a]Pyrimidine analogs were designed and synthesized as novel tubulin inhibitors. Among them, compounds 1a and 1b showed the highest antiproliferative activity against a panel of cancer cell lines with average IC50 values of 24.8 nM and 28 nM, respectively. We determined the crystal structures of 1a and 1b in complex with tubulin and confirmed their direct binding to the colchicine site. Compounds 1a and 1b also effectively inhibited tubulin polymerization in vitro, induced cell cycle arrest in G2/M phase, and inhibited cancer cell migration. In addition, compound 1b exhibited high metabolic stability in human liver microsomes. Finally, 1b was highly effective in suppressing tumor growth in a B16-F10 mouse melanoma model without apparent toxicity. In summary, these results suggest that 1b represents a promising tubulin inhibitor worthy of further investigation.

Discovery of novel resorcinol diphenyl ether-based PROTAC-like molecules as dual inhibitors and degraders of PD-L1.

Novel resorcinol diphenyl ether-based PROTACs (PROteolysis TArgeting Chimeras) were designed and evaluated for their inhibitory activity against the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and their ability to degrade PD-L1 protein. Most of the compounds displayed excellent inhibitory activities against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay, with IC50 values ranging from 25 nM to 200 nM. Among them, compound P22 is one of the best with an IC50 value of 39.2 nM. In addition to inhibiting PD-1/PD-L1 interaction, P22 also significantly restored the immunity repressed in a co-culture model of Hep3B/OS-8/hPD-L1 and CD3 T cells. Furthermore, flow cytometry (FCM) and western-blot data demonstrated that P22 could moderately reduce the protein levels of PD-L1 in a lysosome-dependent manner, which may contribute to its immune effects. Preliminary FCM and western-blot data suggest that it is possible to build PD-L1-targeting PROTAC-like molecules based on PD-1/PD-L1 small molecule inhibitors, though these compounds showed only modest degradation efficiencies. Collectively, this work suggests that P22 may serve as a starting point for exploring the degradation of PD-L1 by PROTAC-like strategy.

Harmines inhibit cancer cell growth through coordinated activation of apoptosis and inhibition of autophagy.

Harmine and its analogs have long been considered as anticancer agents. In vitro analyses suggested that intercalating DNA or inhibiting topoisomerase might contribute to the cytotoxic effect of this class of compound. However, this idea has not been rigorously tested in intact cells. By synthesizing novel derivatives, here we demonstrate that harmines did not activate the DNA damage response, a cellular signaling commonly induced by agents that intercalate DNA or inhibit topoisomerase. These findings suggest that mechanisms other than DNA intercalating or topoisomerase inhibiting contribute to the toxicity of harmines in vivo. Using a novel N2-benzyl and N9-arylated alkyl compound 10f that has good solubility and stability as the model, we show that harmines strongly inhibited the growth of cancer cells originated from breast, lung, bone and pancreas, but not that of normal fibroblasts. We further show that 10f induced apoptosis and inhibited autophagy in a dose and time-dependent manner. An apoptosis inhibitor suppressed 10f-induced cell death. Together, our results reveal previously unidentified insights into the anticancer mechanism of harmines, supporting future development of this compound class in the treatment of human cancers.