A DXd/TLR7-agonist dual-conjugate anti-HER2 ADC exerts robust anti-tumor activity through tumor cell killing and immune activation.

The emergence of trastuzumab deruxtecan (T-DXd), a new-generation antibody-drug conjugate (ADC), has profoundly altered the therapeutic paradigm for HER2-positive solid tumors, demonstrating remarkable clinical benefits. However, the combined outcomes of T-DXd with immunotherapy agents remain ambiguous. In this study, we introduce Tras-DXd-MTL1, an innovative HER2 targeting ADC that integrates the topoisomerase inhibitor DXd and a toll-like receptor 7 (TLR7) agonist MTT-5, linked to trastuzumab via a GGFG tetrapeptide linker. Mechanistically, Tras-DXd-MTL1 retains the DNA-damaging and cell-killing properties of topoisomerase inhibitors while simultaneously enhancing the immune response within the tumor microenvironment (TME). This is achieved by promoting immune cell infiltration and activating dendritic cells and CD8+T cells via MTT-5. In vivo evaluation of Tras-DXd-MTL1's anti-tumor potency revealed a notably superior performance compared to the T-DXd (Tras-DXd) or Tras-MTL1 in immunocompetent mice with trastuzumab-resistant EMT6-HER2 tumor and immunodeficient mice with JIMT-1 tumor. This improved efficacy is primarily attributed to its dual functions of immune stimulation and cytotoxicity. Our findings highlight the potential of incorporating immunostimulatory agents into ADC design to potentiate antitumor effects and establish durable immune memory, thereby reducing tumor recurrence risks. Therefore, our study offers a novel strategy for the design of immune-activating ADCs and provides a potential approach for targeting solid tumors with different levels of HER2 expression.

Discovery and optimization of 3-(indolin-5-yloxy)pyridin-2-amine derivatives as potent necroptosis inhibitors.

Necroptosis is a form of regulated necrotic cell death and has been confirmed to play pivotal roles in the pathogenesis of multiple autoimmune diseases such as rheumatoid arthritis (RA) and psoriasis. The development of necroptosis inhibitors may offer a promising therapeutic strategy for the treatment of these autoimmune diseases. Herein, starting from the in-house hit compound 1, we systematically performed structural optimization to discover potent necroptosis inhibitors with good pharmacokinetic profiles. The resulting compound 33 was a potent necroptosis inhibitor for both human I2.1 cells (IC50 < 0.2 nM) and murine Hepa1-6 cells (IC50 < 5 nM). Further target identification revealed that compound 33 was an inhibitor of receptor interacting protein kinase 1 (RIPK1) with favorable selectivity. In addition, compound 33 also exhibited favorable pharmacokinetic profiles (T1/2 = 1.32 h, AUC = 1157 ng·h/mL) in Sprague-Dawley rats. Molecular docking and molecular dynamics simulations confirmed that compound 33 could bind to RIPK1 with high affinity. In silico ADMET analysis demonstrated that compound 33 possesses good drug-likeness profiles. Collectively, compound 33 is a promising candidate for antinecroptotic drug discovery.

13,14-seco withaphysalins from Physalis minima and their inhibitory effects on NLRP3 inflammasome activation.

Seven new 13,14-seco withaphysalins including two new skeletons (1 and 9) were isolated from the whole plants of Physalis minima, together with three known analogues (6-8). Among them, compound 1 was an extremely rare steroid with a 6, 8-cyclo ring. Their structures were established by extensive analysis of spectroscopic data, experimental electronic circular dichroism measurements, and single-crystal X-ray crystallographic analysis. In Raw264.7 cells, compounds 1-3, 5, 6, and 8 demonstrated potent ability to reduce the NLRP3-dependent caspase-1 activation. Among these compounds, 1 and 2 showed a superior potential, consistently concentration-dependent downregulating NLRP3-dependent proinflammatory cytokine IL-1ß production in macrophage. Mechanistically, compounds 1 and 2 reduced the cleavage of caspase-1 and GSDMD, and exhibited no obvious impact both on the NF-κB activation and the expression of NLRP3 and IL-1ß, suggesting that the compounds target the activation of the NLRP3 pathway mainly by inhibiting the NLRP3 inflammasome activation step rather than the priming step.

GV-971 attenuates the progression of neuromyelitis optica in murine models and reverses alterations in gut microbiota and associated peripheral abnormalities.

AIMS: Growing evidence suggests that an imbalanced gut microbiota composition plays a crucial role in the development of neuromyelitis optica spectrum disorders (NMOSD), an inflammatory demyelinating disease primarily affecting the optic nerves and central nervous system (CNS). In light of this, we explored the potential therapeutic benefits of GV-971 in NMOSD. GV-971 is a drug used for treating mild-to-moderate Alzheimer's disease, which targets the gut-brain axis and reduces neuroinflammation. METHODS: To evaluate GV-971's effects, we employed the experimental autoimmune encephalomyelitis (EAE) mouse model to establish NMOSD animal models. This was achieved by injecting NMO-IgG into aged mice (11 months old) or using NMO-IgG along with complement injection and microbubble-enhanced low-frequency ultrasound (MELFUS) techniques in young mice (7 weeks old). We assessed the impact of GV-971 on incidence rate, clinical scores, body weight, and survival, with methylprednisolone serving as a positive control. In NMOSD models of young mice, we analyzed spinal cord samples through H&E staining, immunohistochemistry, and Luxol Fast Blue staining. Fecal samples collected at different time points underwent 16S rRNA gene sequencing, while plasma samples were analyzed using cytokine array and untargeted metabolomics analysis. RESULTS: Our findings indicated that GV-971 significantly reduced the incidence of NMOSD, alleviated symptoms, and prolonged survival in NMOSD mouse models. The NMOSD model exhibited substantial neuroinflammation and injury, accompanied by imbalances in gut microbiota, peripheral inflammation, and metabolic disorders, suggesting a potentially vicious cycle that accelerates disease pathogenesis. Notably, GV-971 effectively reduces neuroinflammation and injury, and restores gut microbiota composition, as well as ameliorates peripheral inflammation and metabolic disorders. CONCLUSIONS: GV-971 attenuates the progression of NMOSD in murine models and reduces neuroinflammation and injury, likely through its effects on remodeling gut microbiota and peripheral inflammation and metabolic disorders.

Identification of benzo[b]thiophene-1,1-dioxide derivatives as novel PHGDH covalent inhibitors.

The increased de novo serine biosynthesis confers many advantages for tumorigenesis and metastasis. Phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in serine biogenesis, exhibits hyperactivity across multiple tumors and emerges as a promising target for cancer treatment. Through screening our in-house compound library, we identified compound Stattic as a potent PHGDH inhibitor (IC50 = 1.98 ± 0.66 µM). Subsequent exploration in structural activity relationships led to the discovery of compound B12 that demonstrated the increased enzymatic inhibitory activity (IC50 = 0.29 ± 0.02 µM). Furthermore, B12 exhibited robust inhibitory effects on the proliferation of MDA-MB-468, NCI-H1975, HT1080 and PC9 cells that overexpress PHGDH. Additionally, using a [U-13C6]-glucose tracing assay, B12 was found to reduce the production of glucose-derived serine in MDA-MB-468 cells. Finally, mass spectrometry-based peptide profiling, mutagenesis experiment and molecular docking study collectively suggested that B12 formed a covalent bond with Cys421 of PHGDH.

Design, synthesis, and evaluation of thiazolecarboxamide derivatives as stimulator of interferon gene inhibitors.

Stimulator of interferon gene (STING) plays critical roles in the cytoplasmic DNA-sensing pathway and in the induction of inflammatory response. Aberrant cytoplasmic DNA accumulation and STING activation are implicated in numerous inflammatory and autoimmune diseases. Here, we reported the discovery of a series of thiazolecarboxamide-based STING inhibitors through a molecular planarity/symmetry disruption strategy. The privileged compound 15b significantly inhibited STING signaling and suppressed immune-inflammatory cytokine levels in both human and murine cells. In vivo experiments demonstrated 15b effectively ameliorated immune-inflammatory cytokines upregulation in MSA-2-stimulated and Trex1-D18N mice. Furthermore, compound 15b exhibited enhanced efficacy in suppressing interferon-stimulated gene 15 (ISG15), a critical positive feedback regulator of STING. Overall, compound 15b deserves further development for the treatment of STING-associated inflammatory and autoimmune diseases.

Oncogenic KRAS Induces Arginine Auxotrophy and Confers a Therapeutic Vulnerability to SLC7A1 Inhibition in Non-Small Cell Lung Cancer.

The urea cycle is frequently rewired in cancer cells to meet the metabolic demands of cancer. Elucidation of the underlying mechanism by which oncogenic signaling mediates urea cycle reprogramming could help identify targetable metabolic vulnerabilities. In this study, we discovered that oncogenic activation of KRAS in non-small cell lung cancer (NSCLC) silenced the expression of argininosuccinate synthase 1 (ASS1), a urea cycle enzyme that catalyzes the production of arginine from aspartate and citrulline, and thereby diverted the utilization of aspartate to pyrimidine synthesis to meet the high demand for DNA replication. Specifically, KRAS signaling facilitated a hypoacetylated state in the promoter region of the ASS1 gene in a histone deacetylase 3-dependent manner, which in turn impeded the recruitment of c-MYC for ASS1 transcription. ASS1 suppression in KRAS-mutant NSCLC cells impaired the biosynthesis of arginine and rendered a dependency on the arginine transmembrane transporter SLC7A1 to import extracellular arginine. Depletion of SLC7A1 in both patient-derived organoid and xenograft models inhibited KRAS-driven NSCLC growth. Together, these findings uncover the role of oncogenic KRAS in rewiring urea cycle metabolism and identify SLC7A1-mediated arginine uptake as a therapeutic vulnerability for treating KRAS-mutant NSCLC. SIGNIFICANCE: ASS1 deficiency is induced by mutant KRAS in NSCLC to facilitate DNA synthesis and creates a dependency on SLC7A1, revealing dietary arginine restriction and SLC7A1 inhibition as potential therapeutic strategies.

Structure-based discovery of a new series of nucleoside-derived ring-opening PRMT5 inhibitors.

The ubiquitous methyltransferases employing SAM as the methyl donor have emerged as potential targets in many disease treatments, especially in anticancer. Therefore, developing SAM-competitive inhibitors of methyltransferases is of great interest to the drug research. To explore this direction, herein, we rationally designed a series of nucleoside derivatives as potent PRMT5 inhibitors with novel scaffold. The representative compounds A2 and A8 exhibited highly potent PRMT5 inhibition activity as well as good selectivity over other PRMTs and PKMTs. Further cellular experiments revealed that compounds A2 and A8 potently reduced the level of sDMA and inhibited the proliferation of Z-138 and MOLM-13 cell lines by inducing apoptosis. Moreover, compounds A8 which had favorable pharmacokinetic properties exhibited potent antitumor efficacy without the loss of body weight in a subcutaneous MOLM-13 xenograft model. In summary, our efforts provided a series of novel nucleoside analogs as potent PRMT5 inhibitors and may also offer a new strategy to develop SAM analogs as other methyltransferases' inhibitors.

Integrated multi-omics analysis reveals gut microbiota dysbiosis and systemic disturbance in major depressive disorder.

Major depressive disorder (MDD) involves systemic changes in peripheral blood and gut microbiota, but the current understanding is incomplete. Herein, we conducted a multi-omics analysis of fecal and blood samples obtained from an observational cohort including MDD patients (n = 99) and healthy control (HC, n = 50). 16S rRNA sequencing of gut microbiota showed structural alterations in MDD, as characterized by increased Enterococcus. Metagenomics sequencing of gut microbiota showed substantial functional alterations including upregulation in the superpathway of the glyoxylate cycle and fatty acid degradation and downregulation in various metabolic pathways in MDD. Plasma metabolomics revealed decreased amino acids and bile acids, together with increased sphingolipids and cholesterol esters in MDD. Notably, metabolites involved in arginine and proline metabolism were decreased while sphingolipid metabolic pathway were increased. Mass cytometry analysis of blood immune cell subtypes showed rises in proinflammatory immune subsets and declines in anti-inflammatory immune subsets in MDD. Furthermore, our findings revealed disease severity-related factors of MDD. Interestingly, we classified MDD into two immune subtypes that were highly correlated with disease relapse. Moreover, we established discriminative signatures that differentiate MDD from HC. These findings contribute to a comprehensive understanding of the MDD pathogenesis and provide valuable resources for the discovery of biomarkers.

Discovery of hematopoietic progenitor kinase 1 inhibitors using machine learning-based screening and free energy perturbation.

Hematopoietic progenitor kinase 1 (HPK1) is a key negative regulator of T-cell receptor (TCR) signaling and a promising target for cancer immunotherapy. The development of novel HPK1 inhibitors is challenging yet promising. In this study, we used a combination of machine learning (ML)-based virtual screening and free energy perturbation (FEP) calculations to identify novel HPK1 inhibitors. ML-based screening yielded 10 potent HPK1 inhibitors (IC50 < 1 µM). The FEP-guided modification of the in-house false-positive hit, DW21302, revealed that a single key atom change could trigger activity cliffs. The resulting DW21302-A was a potent HPK1 inhibitor (IC50 = 2.1 nM) and potently inhibited cellular HPK1 signaling and enhanced T-cell function. Molecular dynamics (MD) simulations and ADME predictions confirmed DW21302-A as candidate compound. This study provides new strategies and chemical scaffolds for HPK1 inhibitor development.Communicated by Ramaswamy H. Sarma.

Low-Dose Chemotherapy Preferentially Shapes the Ileal Microbiome and Augments the Response to Immune Checkpoint Blockade by Activating AIM2 Inflammasome in Ileal Epithelial Cells.

Intervention of the gut microbiome is a promising adjuvant strategy in cancer immunotherapy. Chemotherapeutic agents are recognized for their substantial impacts on the gut microbiome, yet their therapeutic potential as microbiome modulators remains uncertain, due to the complexity of microbiome-host-drug interactions. Here, it is showed that low-dose chemotherapy preferentially shapes the ileal microbiome to augment the extraintestinal immune response to anti-programmed death-1 (anti-PD-1) therapy without causing intestinal toxicity. Mechanistically, low-dose chemotherapy causes DNA damage restricted to highly-proliferative ileal epithelial cells, resulting in the accumulation of cytosolic dsDNA and the activation of the absent in melanoma 2 (AIM2) inflammasome. AIM2-dependent IL-18 secretion triggers the interplay between proximal Th1 cells and Paneth cells in ileal crypts, impairing the local antimicrobial host defense and resulting in ileal microbiome change. Intestinal epithelium-specific knockout of AIM2 in mice significantly attenuates CPT-11-caused IL-18 secretion, Paneth cell dysfunction, and ileal microbiome alteration. Moreover, AIM2 deficiency in mice or antibiotic microbial depletion attenuates chemotherapy-augmented antitumor responses to anti-PD1 therapy. Collectively, these findings provide mechanistic insights into how chemotherapy-induced genomic stress is transduced to gut microbiome change and support the rationale of applying low-dose chemotherapy as a promising adjuvant strategy in cancer immunotherapy with minimal toxicity.

Preclinical and early clinical studies of a novel compound SYHA1813 that efficiently crosses the blood-brain barrier and exhibits potent activity against glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults and is poorly controlled. Previous studies have shown that both macrophages and angiogenesis play significant roles in GBM progression, and co-targeting of CSF1R and VEGFR is likely to be an effective strategy for GBM treatment. Therefore, this study developed a novel and selective inhibitor of CSF1R and VEGFR, SYHA1813, possessing potent antitumor activity against GBM. SYHA1813 inhibited VEGFR and CSF1R kinase activities with high potency and selectivity and thus blocked the cell viability of HUVECs and macrophages and exhibited anti-angiogenetic effects both in vitro and in vivo. SYHA1813 also displayed potent in vivo antitumor activity against GBM in immune-competent and immune-deficient mouse models, including temozolomide (TMZ) insensitive tumors. Notably, SYHA1813 could penetrate the blood-brain barrier (BBB) and prolong the survival time of mice bearing intracranial GBM xenografts. Moreover, SYHA1813 treatment resulted in a synergistic antitumor efficacy in combination with the PD-1 antibody. As a clinical proof of concept, SYHA1813 achieved confirmed responses in patients with recurrent GBM in an ongoing first-in-human phase I trial. The data of this study support the rationale for an ongoing phase I clinical study (ChiCTR2100045380).

Structure-based drug discovery of novel fused-pyrazolone carboxamide derivatives as potent and selective AXL inhibitors.

As a novel and promising antitumor target, AXL plays an important role in tumor growth, metastasis, immunosuppression and drug resistance of various malignancies, which has attracted extensive research interest in recent years. In this study, by employing the structure-based drug design and bioisosterism strategies, we designed and synthesized in total 54 novel AXL inhibitors featuring a fused-pyrazolone carboxamide scaffold, of which up to 20 compounds exhibited excellent AXL kinase and BaF3/TEL-AXL cell viability inhibitions. Notably, compound 59 showed a desirable AXL kinase inhibitory activity (IC50: 3.5 nmol/L) as well as good kinase selectivity, and it effectively blocked the cellular AXL signaling. In turn, compound 59 could potently inhibit BaF3/TEL-AXL cell viability (IC50: 1.5 nmol/L) and significantly suppress GAS6/AXL-mediated cancer cell invasion, migration and wound healing at the nanomolar level. More importantly, compound 59 oral administration showed good pharmacokinetic profile and in vivo antitumor efficiency, in which we observed significant AXL phosphorylation suppression, and its antitumor efficacy at 20 mg/kg (qd) was comparable to that of BGB324 at 50 mg/kg (bid), the most advanced AXL inhibitor. Taken together, this work provided a valuable lead compound as a potential AXL inhibitor for the further antitumor drug development.

Unprecedented sesterterpenoids, orientanoids A-C: discovery, bioinspired total synthesis and antitumor immunity.

Sesterterpenoids are a very rare class of important natural products. Three new skeletal spiro sesterterpenoids, named orientanoids A-C (1-3), were isolated from Hedyosmum orientale. Their structures were determined by a combination of spectroscopic data, X-ray crystallography, and total synthesis. To obtain adequate materials for biological research, the bioinspired total syntheses of 1-3 were effectively achieved in 7-8 steps in overall yields of 2.3-6.4% from the commercially available santonin without using any protecting groups. In addition, this work also revised the stereochemistry of hedyosumins B (6) and C (10) as 11R-configuration. Tumor-associated macrophages (TAMs) have emerged as important therapeutic targets in cancer therapy. The in-depth biological evaluation revealed that these sesterterpenoids antagonized the protumoral and immunosuppressive functional phenotype of macrophages in vitro. Among them, the most potent and major compound 1 inhibited protumoral M2-like macrophages and activated cytotoxic CD8+ T cells, and consequently inhibited tumor growth in vivo.

Tetrandrine synergizes with MAPK inhibitors in treating KRAS-mutant pancreatic ductal adenocarcinoma via collaboratively modulating the TRAIL-death receptor axis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies lacking effective therapies. KRAS mutations that occur in over 90% of PDAC are major oncogenic drivers of PDAC. The MAPK signaling pathway plays a central role in KRAS-driven oncogenic signaling. However, pharmacological inhibitors of the MAPK pathway are poorly responded in KRAS-mutant PDAC, raising a compelling need to understand the mechanism behind and to seek new therapeutic solutions. Herein, we perform a screen utilizing a library composed of 800 naturally-derived bioactive compounds to identify natural products that are able to sensitize KRAS-mutant PDAC cells to the MAPK inhibition. We discover that tetrandrine, a natural bisbenzylisoquinoline alkaloid, shows a synergistic effect with MAPK inhibitors in PDAC cells and xenograft models. Mechanistically, pharmacological inhibition of the MAPK pathway exhibits a double-edged impact on the TRAIL-death receptor axis, transcriptionally upregulating TRAIL yet downregulating its agonistic receptors DR4 and DR5, which may explain the limited therapeutic outcomes of MAPK inhibitors in KRAS-mutant PDAC. Of great interest, tetrandrine stabilizes DR4/DR5 protein via impairing ubiquitination-mediated protein degradation, thereby allowing a synergy with MAPK inhibition in inducing apoptosis in KRAS-mutant PDAC. Our findings identify a new combinatorial approach for treating KRAS-mutant PDAC and highlight the role of TRAIL-DR4/DR5 axis in dictating the therapeutic outcome in KRAS-mutant PDAC.

Photochemically controlled activation of STING by CAIX-targeting photocaged agonists to suppress tumor cell growth.

Controllable activation of the innate immune adapter protein - stimulator of interferon genes (STING) pathway is a critical challenge for the clinical development of STING agonists due to the potential "on-target off-tumor" toxicity caused by systematic activation of STING. Herein, we designed and synthesized a photo-caged STING agonist 2 with a tumor cell-targeting carbonic anhydrase inhibitor warhead, which could be readily uncaged by blue light to release the active STING agonist leading to remarkable activation of STING signaling. Furthermore, compound 2 was found to preferentially target tumor cells, stimulate the STING signaling in zebrafish embryo upon photo-uncaging and to induce proliferation of macrophages and upregulation of the mRNA expression of STING as well as its downstream NF-kB and cytokines, thus leading to significant suppression of tumor cell growth in a photo-dependent manner with reduced systemic toxicity. This photo-caged agonist not only provides a powerful tool to precisely trigger STING signalling, but also represents a novel controllable STING activation strategy for safer cancer immunotherapy.

SYK-mediated epithelial cell state is associated with response to c-Met inhibitors in c-Met-overexpressing lung cancer.

Genomic MET amplification and exon 14 skipping are currently clinically recognized biomarkers for stratifying subsets of non-small cell lung cancer (NSCLC) patients according to the predicted response to c-Met inhibitors (c-Metis), yet the overall clinical benefit of this strategy is quite limited. Notably, c-Met protein overexpression, which occurs in approximately 20-25% of NSCLC patients, has not yet been clearly defined as a clinically useful biomarker. An optimized strategy for accurately classifying patients with c-Met overexpression for decision-making regarding c-Meti treatment is lacking. Herein, we found that SYK regulates the plasticity of cells in an epithelial state and is associated with their sensitivity to c-Metis both in vitro and in vivo in PDX models with c-Met overexpression regardless of MET gene status. Furthermore, TGF-ß1 treatment resulted in SYK transcriptional downregulation, increased Sp1-mediated transcription of FRA1, and restored the mesenchymal state, which conferred resistance to c-Metis. Clinically, a subpopulation of NSCLC patients with c-Met overexpression coupled with SYK overexpression exhibited a high response rate of 73.3% and longer progression-free survival with c-Meti treatment than other patients. SYK negativity coupled with TGF-ß1 positivity conferred de novo and acquired resistance. In summary, SYK regulates cell plasticity toward a therapy-sensitive epithelial cell state. Furthermore, our findings showed that SYK overexpression can aid in precisely stratifying NSCLC patients with c-Met overexpression regardless of MET alterations and expand the population predicted to benefit from c-Met-targeted therapy.