Design, Synthesis, and Antitumor Activity Evaluation of Novel VISTA Small Molecule Inhibitors.

VISTA (V-domain Ig suppressor of T cell activation) is a novel immune checkpoint protein and represents a promising target for cancer immunotherapy. Here, we report the design, synthesis, and evaluation of a series of methoxy-pyrimidine-based VISTA small molecule inhibitors with potent antitumor activity. By employing molecular docking and microscale thermophoresis (MST) assay, we identified a lead compound A1 that binds to VISTA protein with high affinity and optimized its structure. A4 was then obtained, which exhibited the strongest binding ability to VISTA protein, with a KD value of 0.49 ± 0.20 µM. In vitro, A4 significantly activated peripheral blood mononuclear cells (PBMCs) induced the release of cytokines such as IFN-γ and enhanced the cytotoxicity of PBMCs against tumor cells. In vivo, A4 displayed potent antitumor activity and synergized with PD-L1 antibody to enhance the therapeutic effect against cancer. These results suggest that compound A4 is an effective VISTA small molecule inhibitor, providing a basis for the future development of VISTA-targeted drugs.

Discovery of potent and selective c-Met inhibitors for MET-amplified hepatocellular carcinoma treatment.

Hepatocellular carcinoma (HCC) is a prevalent and lethal malignancy worldwide. The MET gene, which encodes receptor tyrosine kinase c-Met, is aberrantly activated in various solid tumors, including non-small cell lung cancer and HCC. In this study, we identified a novel c-Met inhibitor 54 by virtual screening and structural optimization. Compound 54 showed potent c-Met inhibition with an IC50 value of 0.45 ± 0.06 nM. It also exhibited high selectivity among 370 kinases and potent anti-proliferative activity against MET-amplified HCC cells. Moreover, compound 54 displayed significant anti-tumor efficacy in vivo, making it a potential candidate for HCC treatment in future studies.

Discovery of Novel Fourth-Generation EGFR Inhibitors to Overcome C797S-Mediated Resistance.

Epidermal growth factor receptor (EGFR)-activating mutation is an important oncogenic driver of nonsmall cell lung cancer (NSCLC) patients. Osimertinib has been the first-line treatment for EGFR-mutated NSCLC. However, the tertiary C797S mutation leads to Osimertinib resistance by blocking the covalent binding of Cys797 to Osimertinib. To date, there are no approved inhibitors for the treatment of Osimertinib resistance. Herein, we identified a novel lead compound S8 targeting EGFRL858R/T790M/C797S by structure-based virtual screening and synthesized a series of novel compounds. Representative compound C34 showed potent inhibitory activity against EGFRL858R/T790M/C797S with an IC50 of 5.1 nM and significantly inhibited the proliferation of the H1975-TM cell line harboring EGFRL858R/T790M/C797S with an IC50 of 0.05 µM. Additionally, compound C34 demonstrated good pharmacokinetic properties with an oral bioavailability of 30.72% and significantly inhibited tumor growth in the H1975-TM xenograft tumor model. This study provides a novel thiazole derivative as an EGFR inhibitor to overcome C797S-mediated resistance.

Targeting polyketide synthase 13 for the treatment of tuberculosis.

Tuberculosis (TB) is one of the most threatening diseases for humans, however, the drug treatment strategy for TB has been stagnant and inadequate, which could not meet current treatment needs. TB is caused by Mycobacterial tuberculosis, which has a unique cell wall that plays a crucial role in its growth, virulence, and drug resistance. Polyketide synthase 13 (Pks13) is an essential enzyme that catalyzes the biosynthesis of the cell wall and its critical role is only found in Mycobacteria. Therefore, Pks13 is a promising target for developing novel anti-TB drugs. In this review, we first introduced the mechanism of targeting Pks13 for TB treatment. Subsequently, we focused on summarizing the recent advance of Pks13 inhibitors, including the challenges encountered during their discovery and the rational design strategies employed to overcome these obstacles, which could be helpful for the development of novel Pks13 inhibitors in the future.

Limonin, a natural ERK2 agonist, protects against ischemic acute kidney injury.

Acute kidney injury (AKI) is a refractory clinical syndrome with limited effective treatments. Amid AKI, activation of the extracellular signal-regulated kinase (ERK) cascade plays a critical role in promoting kidney repair and regeneration. However, a mature ERK agonist in treating kidney disease remains lacking. This study identified limonin, a member of the class of compounds known as furanolactones, as a natural ERK2 activator. Employing a multidisciplinary approach, we systemically dissected how limonin mitigates AKI. Compared to vehicles, pretreatment of limonin significantly preserved kidney functions after ischemic AKI. We revealed that ERK2 is a significant protein linked to the limonin's active binding sites through structural analysis. The molecular docking study showed a high binding affinity between limonin and ERK2, which was confirmed by the cellular thermal shift assay and microscale thermophoresis. Mechanistically, we further validated that limonin promoted tubular cell proliferation and reduced cell apoptosis after AKI by activating ERK signaling pathway in vivo. In vitro and ex vivo, blockade of ERK abolished limonin's capacity of preventing tubular cell death under hypoxia stress. Our results indicated that limonin is a novel ERK2 activator with strong translational potential in preventing or mitigating AKI.

Discovery of Potent DYRK2 Inhibitors with High Selectivity, Great Solubility, and Excellent Safety Properties for the Treatment of Prostate Cancer.

Prostate cancer (PCa) is a common male cancer with high incidence and mortality, and hormonal therapy as the major treatment for PCa patients is troubled by the inevitable resistance that makes us identify novel targets for PCa. Dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) was found to be an effective target for the treatment of PCa, but the research on its inhibitors is rather little. In this work, a potent DYRK2 inhibitor 43 (IC50 = 0.6 nM) was acquired through virtual screening and structural optimization, which displayed high selectivity among 205 kinases; meanwhile, detailed interactions of 43 with DYRK2 were illustrated by the cocrystal. Furthermore, 43 possessed great water solubility (29.5 mg/mL), favorable safety properties (LD50 > 10,000 mg/kg), and potent anti-PCa activities, which could be used as a potential candidate in further preclinical studies.

Novel Small-Molecule PD-L1 Inhibitor Induces PD-L1 Internalization and Optimizes the Immune Microenvironment.

Blocking the PD-1/PD-L1 interaction has become an important strategy for tumor therapy, which has shown outstanding therapeutic effects in clinical settings. However, unsatisfactory response rates and immune-related adverse effects limit the use of anti-PD1/PD-L1 antibodies. Here, we report the discovery and identification of S4-1, an innovative small-molecule inhibitor of PD-L1. In vitro, S4-1 effectively altered the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, improved its localization to endoplasmic reticulum, and thus enhanced the cytotoxicity of peripheral blood mononuclear cells toward tumor cells. In vivo, S4-1 significantly inhibited tumor growth in both lung and colorectal cancer models, particularly in colorectal cancer, where it led to complete clearance of a portion of the tumor cells. Furthermore, S4-1 induced T-cell activation and inversed the inhibitory tumor microenvironment, consistent with the PD-L1/PD-1 pathway blockade. These data support the continued evaluation of S4-1 as an alternative ICB therapeutic strategy.

Novel phthalimides regulating PD-1/PD-L1 interaction as potential immunotherapy agents.

Programmed cell death 1(PD-1)/programmed cell death ligand 1(PD-L1) have emerged as one of the most promising immune checkpoint targets for cancer immunotherapy. Despite the inherent advantages of small-molecule inhibitors over antibodies, the discovery of small-molecule inhibitors has fallen behind that of antibody drugs. Based on docking studies between small molecule inhibitor and PD-L1 protein, changing the chemical linker of inhibitor from a flexible chain to an aromatic ring may improve its binding capacity to PD-L1 protein, which was not reported before. A series of novel phthalimide derivatives from structure-based rational design was synthesized. P39 was identified as the best inhibitor with promising activity, which not only inhibited PD-1/PD-L1 interaction (IC50 = 8.9 nmol/L), but also enhanced killing efficacy of immune cells on cancer cells. Co-crystal data demonstrated that P39 induced the dimerization of PD-L1 proteins, thereby blocking the binding of PD-1/PD-L1. Moreover, P39 exhibited a favorable safety profile with a LD50 > 5000 mg/kg and showed significant in vivo antitumor activity through promoting CD8+ T cell activation. All these data suggest that P39 acts as a promising small chemical inhibitor against the PD-1/PD-L1 axis and has the potential to improve the immunotherapy efficacy of T-cells.

Discovery, Optimization, and Evaluation of Selective CDK4/6 Inhibitors for the Treatment of Breast Cancer.

Breast cancer is the most common tumor in women, and selective cyclin-dependent kinase (CDK) 4/6 inhibitors played an important role in the treatment of breast cancer. Therefore, discovering selective CDK4/6 inhibitors with great safety and potent efficacy is beneficial for the breast cancer treatment. In our work, the lead compound 8 was identified through virtual screening; then, systematic structural optimization was conducted to afford 42, which exhibited strong inhibition on CDK4/6 and showed high selectivity among 205 kinases. 42 possessed excellent safety profiles (LD50 > 5,000 mg/kg), favorable pharmacokinetic properties (F % = 43%), and potent efficacy in reducing the burden of breast cancer in vivo. In conclusion, we offered a highly selective CDK4/6 inhibitor, which could be used as a great candidate for further preclinical studies of breast cancer.

A new LKB1 activator, piericidin analogue S14, retards renal fibrosis through promoting autophagy and mitochondrial homeostasis in renal tubular epithelial cells.

Background: Liver kinase B1 (LKB1) is the key regulator of energy metabolism and cell homeostasis. LKB1 dysfunction plays a key role in renal fibrosis. However, LKB1 activators are scarce in commercial nowadays. This study aims to discover a new drug molecule, piericidin analogue S14 (PA-S14), preventing renal fibrosis as a novel activator to LKB1. Methods: Our group isolated PA-S14 from the broth culture of a marine-derived Streptomyces strain and identified its binding site. We adopted various CKD models or AKI-CKD model (5/6 nephrectomy, UUO, UIRI and adriamycin nephropathy models). TGF-ß-stimulated renal tubular cell culture was also tested. Results: We identified that PA-S14 binds with residue D176 in the kinase domain of LKB1, and then induces the activation of LKB1 through its phosphorylation and complex formation with MO25 and STRAD. As a result, PA-S14 promotes AMPK activation, triggers autophagosome maturation, and increases autophagic flux. PA-S14 inhibited tubular cell senescence and retarded fibrogenesis through activation of LKB1/AMPK signaling. Transcriptomics sequencing and mutation analysis further demonstrated our results. Conclusion: PA-S14 is a novel leading compound of LKB1 activator. PA-S14 is a therapeutic potential to renal fibrosis through LKB1/AMPK-mediated autophagy and mitochondrial homeostasis pathways.

Discovery of Novel Phosphoinositide-3-Kinase α Inhibitors with High Selectivity, Excellent Bioavailability, and Long-Acting Efficacy for Gastric Cancer.

Phosphoinositide-3-kinase (PI3K) overexpressed in many tumors is a promising target for cancer therapy. However, due to toxicity from the ubiquitous expression of PI3K in many tissues, the development of PI3K inhibitors with high selectivity and low toxicity has become an urgent need for tumor treatment. Herein, based on the HipHop, we designed and synthesized a series of 6-(4,6-dimorpholino-1,3,5-triazin-2-yl)benzo[d]oxazol-2-amine derivatives as potent, selective, and long-acting PI3Kα inhibitors. Compound 27 was determined with potent PI3Kα inhibitory activity (IC50 = 4.4 nM), which exhibited excellent selectivity for homologous PI3K enzymes and a 370 kinome panel. Meanwhile, 27 featured favorable stability (T1/2 > 10 h) and high bioavailability (130%). Importantly, compound 27 exerted great antigastric cancer activity in vivo when combined with taxol. Collectively, these characteristics suggested 27 to be a promising PI3K agent for cancer treatment.

B7-1 mediates podocyte injury and glomerulosclerosis through communication with Hsp90ab1-LRP5-ß-catenin pathway.

Podocyte injury is a hallmark of glomerular diseases; however, the underlying mechanisms remain unclear. B7-1 is increased in injured podocytes, but its intrinsic role is controversial. The clinical data here revealed the intimate correlation of urinary B7-1 with severity of glomerular injury. Through transcriptomic and biological assays in B7-1 transgenic and adriamycin nephropathy models, we identified B7-1 is a key mediator in podocyte injury and glomerulosclerosis through a series of signal transmission to ß-catenin. Using LC-MS/MS, Hsp90ab1, a conserved molecular chaperone, was distinguished to be an anchor for transmitting signals from B7-1 to ß-catenin. Molecular docking and subsequent mutant analysis further identified the residue K69 in the N terminal domain of Hsp90ab1 was the key binding site for B7-1 to activate LRP5/ß-catenin pathway. The interaction and biological functions of B7-1-Hsp90ab1-LRP5 complex were further demonstrated in vitro and in vivo. We also found B7-1 is a novel downstream target of ß-catenin. Our results indicate an intercrossed network of B7-1, which collectively induces podocyte injury and glomerulosclerosis. Our study provides an important clue to improve the therapeutic strategies to target B7-1.

Development and clinical advancement of small molecules for ex vivo expansion of hematopoietic stem cell.

Hematopoietic stem cell (HSC) transplantation is the only curative therapy for many diseases. HSCs from umbilical cord blood (UCB) source have many advantages over from bone marrow. However, limited HSC dose in a single CB unit restrict its widespread use. Over the past two decades, ex vivo HSC expansion with small molecules has been an effective approach for obtaining adequate HSCs. Till now, several small-molecule compounds have entered the phase I/II trials, showing safe and favorable pharmacological profiles. As HSC expansion has become a hot topic over recent years, many newly identified small molecules along with novel biological mechanisms for HSC expansion would help solve this challenging issue. Here, we will give an overview of HSC biology, discovery and medicinal chemistry development of small molecules, natural products targeting for HSC expansion, and their recent clinical progresses, as well as potential protein targets for HSC expansion.

Targeting dual-specificity tyrosine phosphorylation-regulated kinase 2 with a highly selective inhibitor for the treatment of prostate cancer.

Prostate cancer (PCa) is one of the most prevalent cancers in men worldwide, and hormonal therapy plays a key role in the treatment of PCa. However, the drug resistance of hormonal therapy makes it urgent and necessary to identify novel targets for PCa treatment. Herein, dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is found and confirmed to be highly expressed in the PCa tissues and cells, and knock-down of DYRK2 remarkably reduces PCa burden in vitro and in vivo. On the base of DYRK2 acting as a promising target, we further discover a highly selective DYRK2 inhibitor YK-2-69, which specifically interacts with Lys-231 and Lys-234 in the co-crystal structure. Especially, YK-2-69 exhibits more potent anti-PCa efficacy than the first-line drug enzalutamide in vivo. Meanwhile, YK-2-69 displays favorable safety properties with a maximal tolerable dose of more than 10,000 mg/kg and pharmacokinetic profiles with 56% bioavailability. In summary, we identify DYRK2 as a potential drug target and verify its critical roles in PCa. Meanwhile, we discover a highly selective DYRK2 inhibitor with favorable druggability for the treatment of PCa.

Discovery of Dual CDK6/PIM1 Inhibitors with a Novel Structure, High Potency, and Favorable Druggability for the Treatment of Acute Myeloid Leukemia.

Nowadays, the simultaneous inhibition of two or more pathways plays an increasingly important role in cancer treatment due to the complex and diverse pathogenesis of cancer, and the combination of the cyclin-dependent kinase 6 (CDK6) inhibitor and PIM1 inhibitor was found to generate synergistic effects in acute myeloid leukemia (AML) treatment. Therefore, we discovered a novel lead 1 targeting CDK6/PIM1 via pharmacophore-based and structure-based virtual screening, synthesized five different series of new derivates, and obtained a potent and balanced dual CDK6/PIM1 inhibitor 51, which showed high kinase selectivity. Meanwhile, 51 displayed an excellent safety profile and great pharmacokinetic properties. Furthermore, 51 displayed stronger potency in reducing the burden of AML than palbociclib and SMI-4a in vivo. In summary, we offered a new direction for AML treatment and provided a great lead compound for AML preclinical studies.

Discovery of novel and orally bioavailable CDK 4/6 inhibitors with high kinome selectivity, low toxicity and long-acting stability for the treatment of multiple myeloma.

Multiple myeloma (MM) ranks second in malignant hematopoietic cancers, and the most common anti-MM drugs easily generate resistance. CDK4/6 have been validated to play determinant roles in MM, but no remarkable progress has been obtained from clinical trials of CDK4/6 inhibitors for MM. To discover novel CDK6 inhibitors with better potency and high druggability, structure-based virtual screening was conducted to identify compound 10. Further chemical optimization afforded a better derivative, compound 32, which exhibited strong inhibition of CDK4/6 and showed high selectivity over 360+ kinases, including homologous CDKs. The in vivo evaluation demonstrated that compound 32 possessed low toxicity (LD50 > 10,000 mg/kg), favorable bioavailability (F% = 51%), high metabolic stability (t1/2 > 24 h) and strong anti-MM potency. In summary, we discovered a novel CDK4/6 inhibitor bearing favorable drug-like properties and offered a great candidate for MM preclinical studies.

Tubeimoside-1 induces TFEB-dependent lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting mTOR.

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) cascade is an effective therapeutic target for immune checkpoint blockade (ICB) therapy. Targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity. Using flow cytometry-based assay, we identify tubeimoside-1 (TBM-1) as a promising antitumor immune modulator that negatively regulates PD-L1 level. TBM-1 disrupts PD-1/PD-L1 interaction and enhances the cytotoxicity of T cells toward cancer cells through decreasing the abundance of PD-L1. Furthermore, TBM-1 exerts its antitumor effect in mice bearing Lewis lung carcinoma (LLC) and B16 melanoma tumor xenograft via activating tumor-infiltrating T-cell immunity. Mechanistically, TBM-1 triggers PD-L1 lysosomal degradation in a TFEB-dependent, autophagy-independent pathway. TBM-1 selectively binds to the mammalian target of rapamycin (mTOR) kinase and suppresses the activation of mTORC1, leading to the nuclear translocation of TFEB and lysosome biogenesis. Moreover, the combination of TBM-1 and anti-CTLA-4 effectively enhances antitumor T-cell immunity and reduces immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our findings reveal a previously unrecognized antitumor mechanism of TBM-1 and represent an alternative ICB therapeutic strategy to enhance the efficacy of cancer immunotherapy.

Design, Synthesis, and Biological Evaluation of Icaritin Derivatives as Novel Putative DEPTOR Inhibitors for Multiple Myeloma Treatment.

Icaritin is an active ingredient in Epimedium, which has a variety of pharmacological activities. However, the low activity of Icaritin and the unclear target greatly limit its application. Therefore, based on the structure of Icaritin, we adopted the strategy of replacing toxic groups and introducing active groups to design and synthesize a series of new analogues. The top compound C3 exhibited better antimultiple myeloma activity with an IC50 of 1.09 µM for RPMI 8226 cells, induced RPMI 8226 apoptosis, and blocked the cell cycle in the S phase. Importantly, transcriptome analysis, cellular thermal shift assay, and microscale thermophoresis assay confirmed that DEPTOR was the target of C3. Moreover, we explored its binding mode with C3. Especially, C3 displayed satisfactory inhibition of tumor growth in RPMI 8226 xenografts without obvious side effects. In summary, C3 was discovered as a novel putative inhibitor of DEPTOR for the treatment of multiple myeloma.

Computational discovery, structural optimization and biological evaluation of novel inhibitors targeting transient receptor potential vanilloid type 3 (TRPV3).

Transient receptor potential vanilloid type 3 (TRPV3) is a Ca2+ permeable nonselective cation channel and expressed abundantly in skin keratinocytes. TRPV3 emerges as an attractive target for treatment of pruritic, inflammatory, pain and skin-related diseases. However, only a few reports of TRPV3 inhibitors exist at present besides some patents. Therefore, TRPV3 research has always been fraught with challenges. Through a combination of virtual screening and biological evaluation, compound P1 (10 µM) was identified as a top hit with 34.5% inhibitory effect on 2-APB (1 mM)-evoked currents of mTRPV3-WT. Further structural optimization provided the inhibitor PC5 with the best activity (IC50 = 2.63 ± 0.28 µM), and point mutation assays indicated that amino acids V629 and F633 are crucial for the binding of PC5 and TRPV3. In summary, these newly discovered inhibitors could serve as promising lead compounds for the development of TRPV3 inhibitors in the future.