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.

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 hypo-acetylated state in the promoter region of the ASS1 gene in a histone deacetylase 3 (HDAC3)-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.

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.

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.

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.

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.

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.

Fine-tuning Bacterial Cyclic di-AMP Production for Durable Antitumor Effects Through the Activation of the STING Pathway.

The stimulator of interferon genes (STING) protein is an important and promising innate immune target for tumor therapy. However, the instability of the agonists of STING and their tendency to cause systemic immune activation is a hurdle. The STING activator, cyclic di-adenosine monophosphate (CDA), produced by the modified Escherichia coli Nissle 1917, shows high antitumor activity and effectively reduces the systemic effects of the "off-target" caused by the activation of the STING pathway. In this study, we used synthetic biological approaches to optimize the translation levels of the diadenylate cyclase that catalyzes CDA synthesis in vitro. We developed 2 engineered strains, CIBT4523 and CIBT4712, for producing high levels of CDA while keeping their concentrations within a range that did not compromise the growth. Although CIBT4712 exhibited stronger induction of the STING pathway corresponding to in vitro CDA levels, it had lower antitumor activity than CIBT4523 in an allograft tumor model, which might be related to the stability of the surviving bacteria in the tumor tissue. CIBT4523 exhibited complete tumor regression, prolonged survival of mice, and rejection of rechallenged tumors, thus, offering new possibilities for more effective tumor therapy. We showed that the appropriate production of CDA in engineered bacterial strains is essential for balancing antitumor efficacy and self-toxicity.

Development and validation of LC-MS/MS method for quantitation of sodium oligomannate in human plasma, urine and feces.

Background: Sodium oligomannate was approved for marketing by the National Medical Products Administration of China in 2019 for improving cognitive functions in mild-to-moderate Alzheimer's disease patients. Method: LC-MS/MS methods were established and validated for the quantitation of sodium oligomannate in human plasma, urine and feces to support clinical development studies. Samples were prepared using liquid-liquid extraction and analyzed by ion-pair reversed-phase LC-MS/MS with calibration standard curve ranges of 25.0-5000 ng/ml, 0.500-100 µg/ml and 100-10,000 µg/g in plasma, urine and feces, respectively. Results & conclusion: All validation parameters met the respective acceptance criteria established by US FDA and International Council for Harmonisation of Technical Requirements for Human Use guidelines. The validated methods were applied to a pharmacokinetics and excretion study in healthy Chinese subjects.

Structural Optimization of Fibroblast Growth Factor Receptor Inhibitors for Treating Solid Tumors.

Small-molecule fibroblast growth factor receptor (FGFR) inhibitors have emerged as a promising antitumor therapy. Herein, by further optimizing the lead compound 1 under the guidance of molecular docking, we obtained a series of novel covalent FGFR inhibitors. After careful structure-activity relationship analysis, several compounds were identified to exhibit strong FGFR inhibitory activity and relatively better physicochemical and pharmacokinetic properties compared with those of 1. Among them, 2e potently and selectively inhibited the kinase activity of FGFR1-3 wildtype and high-incidence FGFR2-N549H/K-resistant mutant kinase. Furthermore, it suppressed cellular FGFR signaling, exhibiting considerable antiproliferative activity in FGFR-aberrant cancer cell lines. In addition, the oral administration of 2e in the FGFR1-amplified H1581, FGFR2-amplified NCI-H716, and SNU-16 tumor xenograft models demonstrated potent antitumor efficacy, inducing tumor stasis or even tumor regression.

Discovery of HCD3514 as a potent EGFR inhibitor against C797S mutation in vitro and in vivo.

Osimertinib (AZD9291), a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), has significantly improved the survival of non-small cell lung cancer (NSCLC) patients with EGFRT790M mutation, the major mechanism of acquired resistance to first-generation EGFR TKI. However, resistance to AZD9291 arises eventually and EGFRC797S mutation was reported to be a major resistance mechanism. Thus, it is highly valuable to develop novel EGFR fourth-generation inhibitors targeting C797S mutation to override the acquired resistance. In this study, we identified HCD3514 as a novel EGFR fourth-generation inhibitors targeting C797S triple mutation. It strongly inhibited EGFRL858R/T790M/C797S and EGFR19del/T790M/C797S mutations with IC50 values of 1.0 and 2.0 nM, respectively. HCD3514 dose-dependently inhibited the activation of EGFR in both engineered BaF3 cells and tumor cells harboring EGFRC797S triple mutant and thus effectively suppressed the proliferation of the cells. Moreover, HCD3514 caused a dose-dependent increase of apoptosis in C797S triple mutant cells accompanied by increased levels of cleaved caspase-3 and cleaved PARP. Furthermore, HCD3514 induced tumor growth inhibition in EGFR19del/T790M/C797S xenograft model as a single oral agent by decreasing the activation of EGFR. In addition to EGFRC797S triple mutations, HCD3514 also potently and selectively inhibited EGFRT790M double mutations (L858R/T790M and 19del/T790M). Collectively, HCD3514 is a highly selective and potent EGFR inhibitor against EGFRC797S triple mutations as well as EGFRT790M double mutations and is confirmed potently anti-tumor activity in preclinical models.

Structural Modification and Pharmacological Evaluation of Substituted Quinoline-5,8-diones as Potent NSD2 Inhibitors.

The histone lysine methyltransferase NSD2 is overexpressed, translocated, or mutated in multiple types of cancers and has emerged as an attractive therapeutic target. However, the development of small-molecule NSD2 inhibitors is still in its infancy, and selective and efficacious NSD2 inhibitors are highly desirable. Here, in view of the structural novelty of the reported NSD2 inhibitor DA3003-1, we conducted a comprehensive structural optimization based on the quinoline-5,8-dione scaffold. Compound 15a was identified possessing both high NSD2 inhibitory activity and potent anti-proliferative effects in the cell. Meanwhile, compound 15a has an excellent pharmacokinetic profile with high oral bioavailability. Further, this compound was found to display significant antitumor efficacy with desirable safety profile in the multiple myeloma xenograft mice models, thus warranting it as a promising candidate for further investigation.