Discovery of 4-phenyl-1H-indazole derivatives as novel small-molecule inhibitors targeting the PD-1/PD-L1 interaction.

The inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway with small molecules is a promising approach for cancer immunotherapy. Herein, novel small molecules compounds bearing various scaffolds including thiophene, thiazole, tetrahydroquinoline, benzimidazole and indazole were designed, synthesized and evaluated for their inhibitory activity against the PD-1/PD-L1 interaction. Among them, compound Z13 exhibited the most potent activity with IC50 of 189.6 nM in the homogeneous time-resolved fluorescence (HTRF) binding assay. Surface plasmon resonance (SPR) assay demonstrated that Z13 bound to PD-L1 with high affinity (KD values of 231 nM and 311 nM for hPD-L1 and mPD-L1, respectively). In the HepG2/Jurkat T co-culture cell model, Z13 decreased the viability rate of HepG2 cells in a concentration-dependent manner. In addition, Z13 showed significant in vivo antitumor efficacy (TGI = 52.6 % at 40 mg/kg) without obvious toxicity in the B16-F10 melanoma model. Furthermore, flow cytometry analysis demonstrated that Z13 inhibited tumor growth in vivo by activating the tumor immune microenvironment. These findings indicate that Z13 is a promising PD-1/PD-L1 inhibitor deserving further investigation.

Discovery and Computational Studies of Potent Covalent Kinase Inhibitors with α-Substituent Electrophiles Targeting Cysteine.

Both reversible noncovalent inhibitors and irreversible covalent inhibitors targeting tyrosine kinases have their disadvantages. The reversible covalent inhibitors with electrophilic group cyanoacrylamide as warheads reacting with cysteine residues could solve the dilemmas. However, there are still several unresolved issues regarding the electrophilic groups. In this manuscript, a series of EGFR inhibitors with double electron-withdrawing substituents introduced into the Cα position on the olefin bond were designed and synthesized. The binding structures and characteristics of inhibitors with the kinase in both the first noncovalent binding phase and the second covalent binding step were explored and combined with molecular docking and molecular dynamics simulations. Then, the reverse ß-elimination reactions of the thiol-Michael adducts were investigated by applying density functional theory calculations. In addition, the effects of different electrophilic substituents of Cα on the binding between the inhibitors and kinase were elucidated. The results suggested that the electrophilicity and size of the electron-withdrawing groups play an important role in the specific interactions during the reaction. The compounds with the electron-withdrawing groups that had medium electrostatic and steric complementarity to the kinase active site could cooperatively stabilize the complexes and showed relatively good potent activities in the kinase assay experiment. The mechanical and structural information in this study could enhance our understanding of the functioning of the electron-withdrawing groups in the covalent inhibitors. The results might help to design efficient cysteine targeting inhibitors in the future.

Discovery of conformationally constrained c-Abl inhibitors with potential neuroprotective effects against Parkinson's disease.

Abelson tyrosine kinase (c-Abl) is involved in various biological processes in neurodegenerative diseases and is an attractive target for anti-PD (Parkinson's disease) drug discovery. Based on our previous work, we designed several novel c-Abl inhibitors through a conformational constrained strategy and evaluated their pharmacological activities. Among them, compound A6 exhibited superior inhibitory activity against c-Abl than nilotinib in the homogenous time-resolved fluorescence (HTRF) assay. Furthermore, A6 displayed higher neuroprotective effects against SH-SY5Y cell death induced by MPP+ and lower cytotoxicity than that of nilotinib. Molecular modeling revealed that the 1H-pyrrolo[2,3-B]pyridine ring may contribute to the high affinity of A6 for binding to c-Abl. Collectively, these results suggest that A6 deserves further investigation as a c-Abl inhibitor for neurodegenerative disorders.

Discovery and Characterization of Moracin C as an Anti-Gouty Arthritis/Hyperuricemia Candidate by Docking-Based Virtual Screening and Pharmacological Evaluation.

In the present study, a natural product database of compounds associated with herbs traditionally verified to treat gout/hyperuricemia/arthritis was constructed. 3D-shape and docking-based virtual screening was conducted. To identify potential xanthine oxidase (XOD) inhibitors in the database, eight compounds with commercial availability were identified as high 3D-shape similarity with febuxostat (1), a known XOD inhibitor. Docking was used to further predict the possible interactions between XOD and these compounds. Moracin C (2), moracin D (3), and isoformononetin (8) exhibited higher docking scores and binding energies than other compounds. In vitro, 2 inhibited XOD with an IC50 value of 0.25 ± 0.14 µM, which is similar to that of 1 (0.16 ± 0.08 µM). In a hyperuricemic mouse model, 5-20 mg/kg 2 exhibited satisfying urate-lowering and XOD inhibitory effects. Compound 2 also exhibited antiarthritis activities. In RAW264.7 cells, 2 at 1-10 µM inhibited the expression of IL-1ß and TNF-α induced by MSU. In an acute gouty arthritis model in SD rats, 5-20 mg/kg 2 significantly alleviated the toe swelling, inflammatory response, and dysfunction disorder caused by monosodium urate (MSU). Compound 2 inhibited serum IL-1ß and TNF-α cytokines as well as reduced the expression of the NLRP3/ASC/caspase-1 inflammasome in joints. In summary, 2 was an effective compound for the treatment of hyperuricemia/gouty arthritis.

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.

Discovery of 4-methyl-3-(pyridin-2-ylamino)benzamide derivatives as C-Abl inhibitors with potential neuroprotective effect.

C-Abl is involved in various biological processes and plays an important role in neurodegenerative diseases, especially Parkinson's disease (PD). Previous studies have found that nilotinib shows a neuroprotective effect in cell and animal models of PD by inhibiting the activation of c-Abl. But the low blood-brain barrier permeability and potential toxicity limit the further use of nilotinib in PD. Based on molecular modeling studies, a series of 4-methyl-3-(pyridin-2-ylamino)benzamide derivatives were designed and synthesized. In particular, compound 9a exhibited significant inhibitory activity against c-Abl and a potent neuroprotective effect against MPP+-induced SH-SY5Y cell death. Moreover, 9a not only displayed lower cell toxicity compared with nilotinib, but also showed higher oral bioavailability and proper permeability of the blood-brain barrier. This paper provides 4-methyl-3-(pyridin-2-ylamino)benzamide derivatives as a new scaffold for c-Abl inhibitor with potential neuroprotective effect.

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.

Discovery of KRas G12C-IN-3 and Pomalidomide-based PROTACs as degraders of endogenous KRAS G12C with potent anticancer activity.

A series of KRAS G12C-targeting PROTACs (PROteolysis TArgeting Chimeras) were designed and synthesized based on KRas G12C-IN-3 (a KRAS G12C inhibitor) and pomalidomide as degraders of KRAS G12C with a molecular weight of < 900. Among them, compound KP-14 (m.w. = 852.16; tPSA = 174.53) showed the highest KRAS G12C-degrading capability in NCI-H358 cancer cells (DC50≈1.25 µM). KP-14 bound to KRAS G12C through the acrylamide warhead and recruited the E3 ligase CRBN, causing rapid and sustained KRAS G12C degradation which led to suppression of MAPK signaling pathway in NCI-H358 cells. In addition, KP-14 selectively induced the degradation of KRAS G12C but not other KRAS isoforms such as G13D via PROTAC mechanism. Furthermore, KP-14 exhibited potent antiproliferative activity against NCI-H358 cancer cells and was able to suppress the formation of NCI-H358 tumor colonies. Collectively, this work suggests that KP-14 may serve as a tool compound for exploring the degradation of KRAS G12C by PROTAC strategy and deserve further investigation as a potential anticancer agent.

Identification of imidazo[4,5-c]pyridin-2-one derivatives as novel Src family kinase inhibitors against glioblastoma.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour in the central nervous system (CNS). As the ideal targets for GBM treatment, Src family kinases (SFKs) have attracted much attention. Herein, a new series of imidazo[4,5-c]pyridin-2-one derivatives were designed and synthesised as SFK inhibitors. Compounds 1d, 1e, 1q, 1s exhibited potential Src and Fyn kinase inhibition in the submicromolar range, of which were next tested for their antiproliferative potency on four GBM cell lines. Compound 1s showed effective activity against U87, U251, T98G, and U87-EGFRvIII GBM cell lines, comparable to that of lead compound PP2. Molecular dynamics (MDs) simulation revealed the possible binding patterns of the most active compound 1s in ATP binding site of SFKs. ADME prediction suggested that 1s accord with the criteria of CNS drugs. These results led us to identify a novel SFK inhibitor as candidate for GBM treatment.

The anti-parasitic drug suramin potently inhibits formation of seminal amyloid fibrils and their interaction with HIV-1.

Seminal amyloid fibrils are made up of naturally occurring peptide fragments and are key targets for the development of combination microbicides or antiviral drugs. Previously, we reported that the polysulfonic compound ADS-J1 is a potential candidate microbicide that not only inhibits HIV-1 entry, but also seminal fibrils. However, the carcinogenic azo moieties in ADS-J1 preclude its clinical application. Here, we screened several ADS-J1-like analogs and found that the antiparasitic drug suramin most potently inhibited seminal amyloid fibrils. Using various biochemical methods, including Congo red staining, CD analysis, transmission EM, viral infection assays, surface plasmon resonance imaging, and molecular dynamics simulations, we investigated suramin's inhibitory effects and its putative mechanism of action. We found that by forming a multivalent interaction, suramin binds to proteolytic peptides and mature fibrils, thereby inhibiting seminal fibril formation and blocking fibril-mediated enhancement of viral infection. Of note, suramin exhibited potent anti-HIV activities, and combining suramin with several antiretroviral drugs produced synergistic effects against HIV-1 in semen. Suramin also displayed a good safety profile for vaginal application. Moreover, suramin inhibited the semen-derived enhancer of viral infection (SEVI)/semen-mediated enhancement of HIV-1 transcytosis through genital epithelial cells and the subsequent infection of target cells. Collectively, suramin has great potential for further development as a combination microbicide to reduce the spread of the AIDS pandemic by targeting both viral and host factors involved in HIV-1 sexual transmission.

Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors targeting the Hedgehog pathway with improved antitumor activity in vivo.

Triple-negative breast cancer (TNBC), a subset of breast cancers, have poorer survival than other breast cancer types. Recent studies have demonstrated that the abnormal Hedgehog (Hh) pathway is activated in TNBC and that these treatment-resistant cancers are sensitive to inhibition of the Hh pathway. Smoothened (Smo) protein is a vital constituent in Hh signaling and an attractive drug target. Vismodegib (VIS) is one of the most widely studied Smo inhibitors. But the clinical application of Smo inhibitors is limited to adult patients with BCC and AML, with many side effects. Therefore, it's necessary to develop novel Smo inhibitor with better profiles. Twenty [1,2,4]triazolo[4,3-a]pyridines were designed, synthesized and screened as Smo inhibitors. Four of these novel compounds showed directly bound to Smo protein with stronger binding affinity than VIS. The new compounds showed broad anti-proliferative activity against cancer cell lines in vitro, especially triple-negative breast cancer cells. Mechanistic studies demonstrated that TPB15 markedly induced cell cycle arrest and apoptosis in MDA-MB-468 cells. TPB15 blocked Smo translocation into the cilia and reduced Smo protein and mRNA expression. Furthermore, the expression of the downstream regulatory factor glioma-associated oncogene 1 (Gli1) was significantly inhibited. Finally, TPB15 demonstrated greater anti-tumor activity in our animal models than VIS with lower toxicity. Hence, these results support further optimization of this novel scaffold to develop improved Smo antagonists.

ADS-J1 disaggregates semen-derived amyloid fibrils.

Semen-derived amyloid fibrils, comprising SEVI (semen-derived enhancer of viral infection) fibrils and SEM1 fibrils, could remarkably enhance HIV-1 sexual transmission and thus are potential targets for the development of an effective microbicide. Previously, we found that ADS-J1, apart from being an HIV-1 entry inhibitor, could also potently inhibit seminal amyloid fibrillization and block fibril-mediated enhancement of viral infection. However, the remodeling effects of ADS-J1 on mature seminal fibrils were unexplored. Herein, we investigated the capacity of ADS-J1 to disassemble seminal fibrils and the potential mode of action by applying several biophysical and biochemical measurements, combined with molecular dynamic (MD) simulations. We found that ADS-J1 effectively remodeled SEVI, SEM186-107 fibrils and endogenous seminal fibrils. Unlike epigallocatechin gallate (EGCG), a universal amyloid fibril breaker, ADS-J1 disaggregated SEVI fibrils into monomeric peptides, which was independent of oxidation reaction. MD simulations revealed that ADS-J1 displayed strong binding potency to the full-length PAP248-286 via electrostatic interactions, hydrophobic interactions and hydrogen bonds. ADS-J1 might initially bind to the fibrillar surface and then occupy the amyloid core, which eventually lead to fibril disassembly. Furthermore, the binding of ADS-J1 with PAP248-286 might induce conformational changes of PAP248-286 Disassembled PAP248-286 might not be favorable to re-aggregate into fibrils. ADS-J1 also exerts abilities to remodel a panel of amyloid fibrils, including Aß1-42, hIAPP1-37 and EP2 fibrils. ADS-J1 displays promising potential to be a combination microbicide and an effective lead-product to treat amyloidogenic diseases.

Scandoside Exerts Anti-Inflammatory Effect Via Suppressing NF-κB and MAPK Signaling Pathways in LPS-Induced RAW 264.7 Macrophages.

The iridoids of Hedyotis diffusa Willd play an important role in the anti-inflammatory process, but the specific iridoid with anti-inflammatory effect and its mechanism has not be thoroughly studied. An iridoid compound named scandoside (SCA) was isolated from H. diffusa and its anti-inflammatory effect was investigated in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Its anti-inflammatory mechanism was confirmed by in intro experiments and molecular docking analyses. As results, SCA significantly decreased the productions of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and inhibited the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and IL-6 messenger RNA (mRNA) expression in LPS-induced RAW 264.7 macrophages. SCA treatment suppressed the phosphorylation of inhibitor of nuclear transcription factor kappa-B alpaha (IκB-α), p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The docking data suggested that SCA had great binding abilities to COX-2, iNOS and IκB. Taken together, the results indicated that the anti-inflammatory effect of SCA is due to inhibition of pro-inflammatory cytokines and mediators via suppressing the nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, which provided useful information for its application and development.

Molecular Dynamics Study to Investigate the Dimeric Structure of the Full-Length α-Synuclein in Aqueous Solution.

The mechanisms of dimerization of α-synuclein from full-length monomers and their structural features have been investigated through molecular dynamics simulations in this study. The dimerization of α-syn plays a critical role in the fibrillogenesis mechanism and could initiate and trigger α-syn to aggregate by conformational transforming. According to the alignment between three regions of α-syn monomer, eight diverse starting structures have been constructed. However, only five configurations show the dimeric structures, and the detailed properties of three dimers of them are discussed. During the simulations, both identical α-syn peptides (P1 and P2) of these three dimers reduce the high contents of α-helix from their native folded structures, while the contents of ß-sheet increase. Antiparallel ß-hairpin motifs within the α-syn peptide are formed by intramolecular interactions. The ß-hairpin regions are adjacent to the nonamyloid ß component (NAC) of α-syn, and these structural features are consistent with the experimental observation. Moreover, intermolecular ß-sheets also are generated between P1 and P2 through hydrogen bonding interactions. The dimers produce both intramolecular ß-hairpin and intermolecular ß-sheet characters; the former is presented in monomer and oligomer of α-syn, and the latter occurs in the fibril structure. The simulations also show several other interactions such as hydrophobic interactions and salt-bridges, which would contribute to making the α-syn dimers more stable with the aforementioned effects. The results may pave the way to design small molecules to inhibit the dimerization in order to block the aggregation of α-syn in the future.

The Effect of the Aqueous Extract of Bidens Pilosa L. on Androgen Deficiency Dry Eye in Rats.

BACKGROUND/AIMS: Bidens pilosa L. (Bp) is widely distributed in China and has been widely used as a traditional Chinese medicine. The aim of this study was to examine the effect of the extract of Bp on androgen deficiency dry eye and determine its possible mechanisms. METHODS: Twenty-four rats were randomly divided into four groups: Group Con (control), Group Sal (physiological saline), Group Fin (oral finasteride), and Group Bp (oral finasteride and Bp). The dry eye model was established in group Fin and group Bp. Aqueous tear quantity was measured with phenol red-impregnated cotton threads with anesthesia. Tear film breakup time (BUT) and corneal epithelial damage were evaluated by fluorescein staining. Animals were sacrificed at 28 days, and ocular tissues (lacrimal gland and cornea) were evaluated with light microscopy; gene microarray analysis for inflammatory cytokines and Western blot were also performed. RESULTS: Finasteride administration effectively induced dry eye in rats by 14 days after administration. Group Fin rats had significantly higher fluorescein staining scores and lower aqueous tear quantity and BUT than the group Con rats, and notable inflammatory cell infiltrates were observed in the lacrimal gland of group Fin rats. The fluorescein staining score, aqueous tear quantity and BUT significantly improved with Bp treatment in the group Bp rats, and the structures of the lacrimal gland were well maintained without significant lymphocyte infiltration. Cytokine antibody array data identified the cytokines B7-2/Cd86, IL-1ß, IL-4, IL-6, IL-10, MMP-8, FasL, TNF-α and TIMP-1 as candidates for validation by Western blot. Expression levels of pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, in group Fin were upregulated compared with group Con. Levels of anti-inflammatory cytokines, such as IL-4 and IL-10, in group Fin were also upregulated compared with those in group Con. Compared with group Fin, IL-1ß, FasL, and TNF-α were significantly decreased in group Bp. CONCLUSION: The extract of Bp appears to be effective for the treatment of androgen deficiency dry eye in rats by improving aqueous tear quantity, maintaining tear film stability, and inhibiting the inflammation of the lacrimal gland.

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.

Largely Improved Creep Resistance and Thermal-Aging Stability of Eco-Friendly Polypropylene High-Voltage Insulation by Long-Chain Branch-Induced Interfacial Constraints.

Polypropylene (PP)-based composites have attracted numerous attention as a replacement of prevailing cross-linked polyethylene (XLPE) for high-voltage insulation due to their ease of processing, recyclability, and excellent electrical performance. However, the poor resistances against high-temperature creep and thermal aging are obstacles to practical applications of PP-based thermoplastic high-voltage insulation. To address these problems, in this Letter, we synthesized an impact polypropylene copolymer (IPC) containing multifold long-chain branched (LCB) structures in phases, especially the interfaces between the PP matrix and the rubber phase. The results indicated that the structural stability of LCBIPC was significantly enhanced under extreme conditions. In comparison to IPC (without LCB structures), 24.1% less creep strain and 75.2% less unrecoverable deformation are achieved in LCBIPC at 90 °C. In addition, the thermal aging experiments were performed at 135 °C for 48 and 88 days for IPC and LCBIPC, respectively. The results show that the resistance against thermal aging was also enhanced in LCBIPC, which showed a 133% longer thermal aging life compared to IPC. Further results revealed that the interfacial layer between the PP matrix and the rubber phase was constructed in LCBIPC. The two phases are tightly linked by chemical bonds in LCB structures, leading to enforced constraints of the rubber phase at the micro level and better resistance performance against creep and thermal aging at the macro level. Evidently, the reported eco-friendly LCBIPC thermoplastic insulation shows great potential for applications in high-voltage cable insulation.

Discovery of Novel HDAC3 Inhibitors with PD-L1 Downregulating/Degrading and Antitumor Immune Effects.

Targeting the programmed cell death-1/ligand 1 (PD-1/PD-L1) pathway is one of the most promising cancer treatment strategies. Studies have shown that HDAC inhibitors can enhance the antitumor immune response by modulating the expression of PD-L1. Herein, we designed and synthesized a series of novel hydrazide-based small molecule HDAC inhibitors; among them, compound HQ-30 showed selective HDAC3 inhibition (IC50 = 89 nM) and remarkable PD-L1-degrading activity (DC50 = 5.7 µM, Dmax = 80% at 10 µM). Further studies revealed that HQ-30 induced the degradation of PD-L1 by regulating cathepsin B (CTSB) in the lysosomes. Further, HQ-30 could enhance the infiltration of CD3+ CD4+ helper T and CD3+ CD8+ cytotoxic T cells in tumors, thus activating the tumor immune microenvironment. Moreover, HQ-30 possessed a benign toxicity profile (LD50 > 1000 mg/kg) and favorable pharmacokinetic properties (F = 57%). Taken together, HQ-30 is worthy of further investigation as a small molecule-based epigenetic modulator of tumor immunotherapy.

Discovery of Novel Potent and Fast BTK PROTACs for the Treatment of Osteoclasts-Related Inflammatory Diseases.

Bruton's tyrosine kinase (BTK) is an attractive target in inflammatory and autoimmune diseases. However, the effectiveness of BTK inhibitors is limited by side effects and drug resistance. In this study, we report the development of novel BTK proteolysis targeting chimeras (PROTACs) with different classes of BTK-targeting ligands (e.g., spebrutinib) other than ibrutinib. Compound 23 was identified as a potent and fast BTK PROTAC degrader, exhibiting outstanding degradation potency and efficiency in Mino cells (DC50, 4 h = 1.29 ± 0.3 nM, t1/2, 20 nM = 0.59 ± 0.20 h). Furthermore, compound 23 forms a stable ternary complex, as confirmed by the HTRF assay. Notably, 23 down-regulated the BTK-PLCγ2-Ca2+-NFATc1 signaling pathway activated by RANKL, thus inhibiting osteoclastogenesis and attenuating alveolar bone resorption in a mouse periodontitis model. These findings suggest that compound 23 is a potent and promising candidate for osteoclast-related inflammatory diseases, expanding the potential of BTK PROTACs.