Discovery of JN122, a Spiroindoline-Containing Molecule that Inhibits MDM2/p53 Protein-Protein Interaction and Exerts Robust In Vivo Antitumor Efficacy.

MDM2 and MDM4 cooperatively and negatively regulate p53, while this pathway is often hijacked by cancer cells in favor of their survival. Blocking MDM2/p53 interaction with small-molecule inhibitors liberates p53 from MDM2 mediated degradation, which is an attractive strategy for drug discovery. We reported herein structure-based discovery of highly potent spiroindoline-containing MDM2 inhibitor (-)60 (JN122), which also exhibited moderate activities against MDM4/p53 interactions. In a panel of cancer cell lines harboring wild type p53, (-)60 efficiently promoted activation of p53 and its target genes, inhibited cell cycle progression, and induced cell apoptosis. Interestingly, (-)60 also promoted degradation of MDM4. More importantly, (-)60 exhibited good PK properties and exerted robust antitumor efficacies in a systemic mouse xenograft model of MOLM-13. Taken together, our study showcases a class of potent MDM2 inhibitors featuring a novel spiro-indoline scaffold, which is promising for future development targeting cancer cells with wild-type p53.

Discovery of toxoflavin, a potent IRE1α inhibitor acting through structure-dependent oxidative inhibition.

Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC50 value of 0.226 µM. Its inhibitory potencies on IRE1α kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α's role in ER stress.

Dual inhibition of CHK1/FLT3 enhances cytotoxicity and overcomes adaptive and acquired resistance in FLT3-ITD acute myeloid leukemia.

FLT3 inhibitors (FLT3i) are widely used for the treatment of acute myeloid leukemia (AML), but adaptive and acquired resistance remains a primary challenge. Inhibitors simultaneously blocking adaptive and acquired resistance are highly demanded. Here, we observed the potential of CHK1 inhibitors to synergistically improve the therapeutic effect of FLT3i in FLT3-mutated AML cells. Notably, the combination overcame adaptive resistance. The simultaneous targeting of FLT3 and CHK1 kinases may overcome acquired and adaptive resistance. A dual FLT3/CHK1 inhibitor 30 with a good oral PK profile was identified. Mechanistic studies indicated that 30 inhibited FLT3 and CHK1, downregulated the c-Myc pathway and further activated the p53 pathway. Functional studies showed that 30 was more selective against cells with various FLT3 mutants, overcame adaptive resistance in vitro, and effectively inhibited resistant FLT3-ITD AML in vivo. Moreover, 30 showed favorable druggability without significant blood toxicity or myelosuppression and exhibited a good oral PK profile with a T1/2 over 12 h in beagles. These findings support the targeting of FLT3 and CHK1 as a novel strategy for overcoming adaptive and acquired resistance to FLT3i therapy in AML and suggest 30 as a potential clinical candidate.

A small-molecule self-assembled nanodrug for combination therapy of photothermal-differentiation-chemotherapy of breast cancer stem cells.

The combination therapy with different treatment modalities has been widely applied in the clinical applications of cancer treatment. However, it stills a considerable challenge to achieve co-delivery of different drugs because of distinct drug encapsulation mechanisms, low drug loading, and high excipient-related toxicity. Cancer stem cells (CSCs) are closely related to tumor metastasis and recurrence due to high chemoresistance. Herein, we report a stimuli-responsive and tumor-targeted small-molecule self-assembled nanodrug for the combination therapy against CSCs and normal cancer cells. The hydrophobic differentiation-inducing agent (all-trans retinoic acid, ATRA) and hydrophilic anticancer drug (irinotecan, IRI) constitute this amphiphilic nanodrug, which could self-assemble into stable nanoparticles and encapsulate the photothermal agent IR825. Upon cellular uptake, this nanodrug display good release profiles in response to acid and esterase microenvironments by ester linkage. The released drugs not only increase chemotherapy sensitivity by the differentiation of CSCs into non-CSCs, but also exhibit superior cytotoxicity in cancer cells. In addition, IR825 within this nanodrug enables in vivo fluorescence/photoacoustic (PA) imaging allowing for tracking drug distribution. Moreover, the DSPE-PEG-RGD-functionalized nanodrug displayed high tumor accumulation and good biocompatibility, enabling efficient inhibition of tumor growth and tumor metastasis in tumor-bearing mice.

Downregulation of c-Myc expression confers sensitivity to CHK1 inhibitors in hematologic malignancies.

Checkpoint kinase 1 inhibitors (CHK1i) have shown impressive single-agent efficacy in treatment of certain tumors, as monotherapy or potentiators of chemotherapy in clinical trials, but the sensitive tumor types and downstream effectors to dictate the therapeutic responses to CHK1i remains unclear. In this study we first analyzed GDSC (Genomics of Drug Sensitivity in Cancer) and DepMap database and disclosed that hematologic malignancies (HMs) were relatively sensitive to CHK1i or CHK1 knockdown. This notion was confirmed by examining PY34, a new and potent in-house selective CHK1i, which exhibited potent anti-HM effect in vitro and in vivo, as single agent. We demonstrated that the downregulation of c-Myc and its signaling pathway was the common transcriptomic profiling response of sensitive HM cell lines to PY34, whereas overexpressing c-Myc could partially rescue the anticancer effect of PY34. Strikingly, we revealed the significant correlations between downregulation of c-Myc and cell sensitivity to PY34 in 17 HM cell lines and 39 patient-derived cell (PDC) samples. Thus, our results demonstrate that HMs are more sensitive to CHK1i than solid tumors, and c-Myc downregulation could represent the CHK1i efficacy in HMs.

Design, Synthesis, and Biological Evaluation of Orally Bioavailable CHK1 Inhibitors Active against Acute Myeloid Leukemia.

Checkpoint kinase 1 (CHK1) is a central component in DNA damage response and has emerged as a target for antitumor therapeutics. Herein, we describe the design, synthesis, and biological evaluation of a novel series of potent diaminopyrimidine CHK1 inhibitors. The compounds exhibited moderate to potent CHK1 inhibition and could suppress the proliferation of malignant hematological cell lines. The optimized compound 13 had a CHK1 IC50 value of 7.73±0.74 nM, and MV-4-11 cells were sensitive to it (IC50 =0.035±0.007 µM). Furthermore, compound 13 was metabolically stable in mouse liver microsomes in vitro and displayed moderate oral bioavailability in vivo. Moreover, treatment of MV-4-11 cells with compound 13 for 2 h led to robust inhibition of CHK1 autophosphorylation on serine 296. Based on these biochemical results, we consider compound 13 to be a promising CHK1 inhibitor and potential anticancer therapeutic agent.

Diffuse large B-cell lymphoma with low 18F-fluorodeoxyglucose avidity features silent B-cell receptor signaling.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of aggressive lymphomas exhibiting increased glucose uptake. However, some DLBCLs featuring relatively low 18F-fluorodeoxyglucose (18F-FDG) uptake denoted by the maximum standardized uptake value (SUVmax) on PET/CT have been identified. The biologic correlates of such a heterogeneity have remained largely unknown. Herein, we immunohistochemically detected and found low FDG-avid DLBCL cases featuring lower expression of some key molecules involved in B-cell receptor (BCR) signaling (pSYK) and glucose metabolism (GLUT1 and HK2). Besides, BCR-deficient DLBCL xenografts were found displaying lower SUVmax and expressions of pSYK, GLUT1, and HK2. Further immunoblotting demonstrated expressions of GLUT1 and HK2 in BCR-dependent DLBCLs could be down-regulated by a chemical SYK inhibition, whereas the inhibitory effects were not observed in BCR-deficient tumors. These findings suggest low FDG-avid DLBCLs display a silent BCR signaling and PET/CT might be utilized to tailor the BCR signaling-inhibitory treatment.

Discovery of (R)-5-((5-(1-methyl-1H-pyrazol-4-yl)-4-(methylamino)pyrimidin-2-yl)amino)-3-(piperidin-3-yloxy)picolinonitrile, a novel CHK1 inhibitor for hematologic malignancies.

Through virtual screening, we identified the lead compound MCL1020, which exhibited modest CHK1 inhibitory activity. Then a series of 5-(pyrimidin-2-ylamino)picolinonitrile derivatives as CHK1 inhibitors were discovered by further rational optimization. One promising molecule, (R)-17, whose potency was one of the best, had an IC50 of 0.4 nM with remarkable selectivity (>4300-fold CHK1 vs. CHK2). Compound (R)-17 effectively inhibited the growth of malignant hematopathy cell lines especially Z-138 (IC50: 0.013 µM) and displayed low affinity for hERG (IC50 > 40 µM). Moreover, (R)-17 significantly suppressed the tumor growth in Z-138 cell inoculated xenograft model (20 mg/kg I.V., TGI = 90.29%) as a single agent with body weight unaffected. Taken together, our data demonstrated compound (R)-17 could be a promising drug candidate for the treatment of hematologic malignancies.

Disruption of the unfolded protein response (UPR) by lead compound selectively suppresses cancer cell growth.

Identifying chemotherapy candidates with high selectivity against cancer cells is a major challenge in cancer treatment. Tumor microenvironments cause chronic endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) as an adaptive response. Here, one novel small-molecule compound, 17#, was discovered as a potent pan-UPR inhibitor. It exhibited good selection for growth inhibition when cancer cells were cultured in 2-deoxy-D-glucose (2DG), mimicking an in vitro glucose-deprived status. Additionally, 17# alone could mildly suppress the growth of HeLa tumor xenografts, and a synergistic anti-cancer effect was observed when 17# was combined with 2DG. A mechanistic study showed that 17#-induced selective anti-cancer effects were highly dependent on UPR inhibition, and overexpressing GRP78 or XBP1s reversed the 17#-induced growth inhibition and cell cycle arrest, partially by delaying the downregulation of the cell cycle regulator cyclin B1. Furthermore, 17# improved the sensitivity of anti-cancer drugs such as doxorubicin or etoposide. Our study presents evidence that disrupting the UPR has selective therapeutic potential and may enhance drug sensitivity.