The combination of methionine adenosyltransferase 2A inhibitor and methyltransferase like 3 inhibitor promotes apoptosis of non-small cell lung cancer cells and produces synergistic anti-tumor activity.

Methionine adenosyltransferase 2 A (MAT2A) mediates the synthesis of methyl donor S-Adenosylmethionine (SAM), providing raw materials for methylation reactions in cells. MAT2A inhibitors are currently used for the treatment of tumors with methylthioadenosine phosphorylase (MTAP) deficiency in clinical research. Methyltransferase like 3 (METTL3) catalyzes N6-methyladenosine (m6A) modification of mRNA in mammalian cells using SAM as the substrate which has been shown to affect the tumorigenesis of non-small cell lung cancer (NSCLC) from multiple perspectives. MAT2A-induced SAM depletion may have the potential to inhibit the methyl transfer function of METTL3. Therefore, in order to expand the applicability of inhibitors, improve anti-tumor effects and reduce toxicity, the combinational effect of MAT2A inhibitor AG-270 and METTL3 inhibitor STM2457 was evaluated in NSCLC. The results showed that this combination induced cell apoptosis rather than cell cycle arrest, which was non-tissue-specific and was independent of MTAP expression status, resulting in a significant synergistic anti-tumor effect. We further elucidated that the combination-induced enhanced apoptosis was associated with the decreased m6A level, leading to downregulation of PI3K/AKT protein, ultimately activating the apoptosis-related proteins. Unexpectedly, although combination therapy resulted in metabolic recombination, no significant change in methionine metabolic metabolites was found. More importantly, the combination also exerted synergistic effects in vivo. In summary, the combination of MAT2A inhibitor and METTL3 inhibitor showed synergistic effects both in vivo and in vitro, which laid a theoretical foundation for expanding the clinical application research of the two types of drugs.

Identification of 2-substituted pyrrolo[1,2-b]pyridazine derivatives as new PARP-1 inhibitors.

A library of new 2-substituted pyrrolo[1,2-b]pyridazine derivatives were rapidly assembled and identified as PARP inhibitors. Structure-activity relationship for this class of inhibitor resulted in the discovery of most potent compounds 15a and 15b that exhibited about 29- and 5- fold selective activity against PARP-1 over PARP-2 respectively. The antiproliferative activity of the as-prepared compounds were demonstrated by further celluar assay in BRCA2-deficient V-C8 and BRCA1-deficient MDA-MB-436 cell lines, displaying that compound 15b could robustly reduce the corresponding cell proliferation and growth with CC50s of 340 and 106 nM respectively. The PK property of 15b was also investigated here.

Discovery of a series of 1H-pyrrolo[2,3-b]pyridine compounds as potent TNIK inhibitors.

In an in-house screening, 1H-pyrrolo[2,3-b]pyridine scaffold was found to have high inhibition on TNIK. Several series of compounds were designed and synthesized, among which some compounds had potent TNIK inhibition with IC50 values lower than 1 nM. Some compounds showed concentration-dependent characteristics of IL-2 inhibition. These results provided new applications of TNIK inhibitors and new prospects of TNIK as a drug target.

A new BET inhibitor, 171, inhibits tumor growth through cell proliferation inhibition more than apoptosis induction.

The bromodomain and extra-terminal domain (BET) family of proteins, especially bromodomain-containing protein 4 (BRD4), has emerged as exciting anti-tumor targets due to their important roles in epigenetic regulation. Therefore, the discovery of BET inhibitors with promising anti-tumor efficacy will provide a novel approach to epigenetic anticancer therapy. Recently, we discovered the new BET inhibitor compound 171, which is derived from a polo-like kinase 1 (PLK1)-BRD4 dual inhibitor based on our previous research. Compound 171 was found to maintain BET inhibition ability without PLK1 inhibition, and there was no selectivity among BET family members. The in vitro and in vivo results both indicated that the overall anti-tumor activity of compound 171 was improved compared with the (+)-JQ-1 or OTX-015 BET inhibitors. Furthermore, we found that compound 171 could regulate the expression of cell cycle-regulating proteins including c-Myc and p21 and induce cell cycle arrest in the G0/G1 phase. However, compound 171 only has a quite limited effect on apoptosis, in considering that apoptosis was only observed at doses greater than 50 µM. To determine the mechanisms underlying cell death, proliferation activity assay was conducted. The results showed that compound 171 induced clear anti-proliferative effects at doses that no obvious apoptosis was induced, which indicated that the cell cycle arresting effect contributed mostly to its anti-tumor activity. The result of this study revealed the anti-tumor mechanism of compound 171, and laid a foundation for the combination therapy in clinical practice, if compound 171 or its series compounds become drug candidates in the future.

Rare new bicyclic cembranoid ethers and a novel trihydroxy prenylated guaiane from the Xisha soft coral Lobophytum sp.

Seven new cembrane-type diterpenes, lobophytolins C-I (3-9), and one new prenylated-guiane-type diterpene, lobophytolin J (10), along with six known related ones (1, 2, 11-14), have been isolated from the soft coral Lobophytum sp. collected off the Xisha Island in the South China Sea. Their structures were elucidated by extensive spectroscopic analysis and quantum mechanical (QM)-NMR methods. The absolute configuration of lobophytolin H (8) was determined by the application of the modified Mosher's method and chemical transformation. Lobophytolin D (4) exhibited promising cytotoxicities in in vitro bioassays against HT-29, Capan-1, A549, and SNU-398 human cancer cell lines with IC50 values of 4.52, 6.62, 5.17, and 6.15 µM, respectively.

Increased PARP1-DNA binding due to autoPARylation inhibition of PARP1 on DNA rather than PARP1-DNA trapping is correlated with PARP1 inhibitor's cytotoxicity.

PARP1 inhibitors (PARPis) are used clinically during cancer therapy and are thought to exert their cytotoxicity through PARP1 polymerase inhibition and PARP1-DNA trapping. Here, we showed no significant correlation between PARP1-DNA trapping and cytotoxicity induced by PARPis. We complemented PARP1-knockout sublines with wild-type PARP1 and 11 mutants with different point mutations that affect the polymerase activity. When examining the PARPi talazoparib, the induced cytotoxicity was highly significantly correlated with cellular PARP1 polymerase activity, but not with its PARP1-DNA trapping or polymerase inhibition. Similarly, talazoparib's PARP1-DNA trapping revealed significant correlation with the polymerase activity rather than its inhibition. Differently, however, when evaluating purified wild-type and mutated PARP1, we identified an almost linear relationship between PARPis' inhibiting PARP1 dissociation from DNA and their cytotoxicity in 17 cancer cell lines. In contrast, no significant correlation existed between PARP1 polymerase inhibition in the histone-based systems and the cytotoxicity. After careful comparisons on different methods and detection targets, we conclude that the PARPi-mediated increase in PARP1-DNA binding by inhibiting autoPARylation of PARP1 on DNA rather than in PARP1-DNA trapping is correlated with PARPi's cytotoxicity. Accordingly, we established a new PARPi screening model that more closely predicts cytotoxicity.

Cytotoxic Nitrogenous Terpenoids from Two South China Sea Nudibranchs Phyllidiella pustulosa, Phyllidia coelestis, and Their Sponge-Prey Acanthella cavernosa.

A detailed chemical investigation of two South China Sea nudibranchs Phyllidiella pustulosa and Phyllidia coelestis, as well as their possible sponge-prey Acanthella cavernosa, led to the isolation of one new nitrogenous cadinane-type sesquiterpenoid xidaoisocyanate A (1), one new naturally occurring nitrogen-containing kalihinane-type diterpenoid bisformamidokalihinol A (16), along with 17 known nitrogenous terpenoids (2⁻15, 17⁻19). The structures of all the isolates were elucidated by detailed spectroscopic analysis and by the comparison of their spectroscopic data with those reported in the literature. In addition, the absolute stereochemistry of the previously reported axiriabiline A (5) was determined by X-ray diffraction (XRD) analysis. In a bioassay, the bisabolane-type sesquiterpenoids 8, 10, and 11 exhibited cytotoxicity against several human cancer cell lines.

Three New Cytotoxic Monoterpenoid Bisindole Alkaloids from Tabernaemontana bufalina.

Three new bisindole alkaloids, 3'-(2-oxopropyl)-19,20-dihydrotabernamine (1: ), 3'-(2-oxopropyl)-ervahanine B (2: ), 19,20-dihydrovobparicine (3: ), and 20 known compounds were isolated from the aerial parts of Tabernaemontana bufalina. The structures of these alkaloids were elucidated using spectroscopic methods. The absolute configurations of 1: -3: were determined by the circular dichroic exciton chirality method. Compounds 1: -23: were screened for their cytotoxicity against two human cancer cell lines, A-549 and MCF-7. Ten compounds (1: -3, 10, 14, 16, 17, 19, 22: , and 23: ) exhibited inhibitory effects against the two human cancer cells with IC50 values of 1.19 ~ 6.13 µM.

Discovery of potent 2,4-difluoro-linker poly(ADP-ribose) polymerase 1 inhibitors with enhanced water solubility and in vivo anticancer efficacy.

Poly (ADP-ribose) polymerase 1 (PARP1) is overexpressed in a variety of cancers, especially in breast and ovarian cancers; tumor cells that are deficient in breast cancer gene 1/2 (BRCA1/2) are highly sensitive to PARP1 inhibition. In this study, we identified a series of 2,4-difluorophenyl-linker analogs (15-55) derived from olaparib as novel PARP1 inhibitors. Four potent analogs 17, 43, 47, and 50 (IC50=2.2-4.4 nmol/L) effectively inhibited the proliferation of Chinese hamster lung fibroblast V-C8 cells (IC50=3.2-37.6 nmol/L) in vitro, and showed specificity toward BRCA-deficient cells (SI=40-510). The corresponding hydrochloride salts 56 and 57 (based on 43 and 47) were highly water soluble in pH=1.0 buffered salt solutions (1628.2 µg/mL, 2652.5 µg/mL). In a BRCA1-mutated xenograft model, oral administration of compound 56 (30 mg·kg-1·d-1, for 21 d) exhibited more prominent tumor growth inhibition (96.6%) compared with the same dose of olaparib (56.3%); in a BRCA2-mutated xenograft model, oral administration of analog 43 (10 mg·kg-1·d-1, for 28 d) significantly inhibited tumor growth (69.0%) and had no negative effects on the body weights. Additionally, compound 56 exhibited good oral bioavailability (F=32.2%), similar to that of olaparib (F=45.4%). Furthermore, the free base 43 of the hydrochloride salt 56 exhibited minimal hERG inhibition activity (IC50=6.64 µmol/L). Collectively, these data demonstrate that compound 56 may be an excellent drug candidate for the treatment of cancer, particularly BRCA-deficient tumors.

Synthesis of isoquinolinone-based tricycles as novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors.

The isoquinolinone-based tricyclic compounds were designed and synthesized. Preliminary biological study of these compounds provided potent compounds 17a, 33b, 33c, 33d, and 33g with low nanomolar IC50s against PARP-1 enzyme.

Co-inhibition of BET and NAE enhances BIM-dependent apoptosis with augmented cancer therapeutic efficacy.

Agents that inhibit bromodomain and extra-terminal domain (BET) proteins have been actively tested in the clinic as potential anticancer drugs. NEDD8-activating enzyme (NAE) inhibitors, represented by MLN4924, target the only activation enzyme in the neddylation pathway that has been identified as an attractive target for cancer therapy. In this study, we focus on the combination of BET inhibitors (BETis) and NAE inhibitors (NAEis) as a cancer therapeutic strategy and investigate its underlying mechanisms to explore and expand the application scope of both types of drugs. The results showed that this combination synergistically inhibited the proliferative activity of tumor cells from different tissues. Compared to a single drug, combination therapy had a weak effect on cycle arrest but significantly enhanced cell apoptosis. Furthermore, the growth of NCI-H1975 xenografts in nude mice was significantly inhibited by the combination without obvious body weight loss. Research on the synergistic mechanism demonstrated that combination therapy significantly increased the mRNA and protein levels of the proapoptotic gene BIM. The inhibition and knockout of BIM significantly attenuated the apoptosis induced by the combination, whereas the re-expression of BIM restored the synergistic effects, indicating that BIM induction plays a critical role in mediating the enhanced apoptosis induced by the co-inhibition of BET and NAE. Together, the enhanced transcription mediated by miR-17-92 cluster inhibition and reduced degradation promoted the increase in BIM levels, resulting in a synergistic effect. Collectively, these findings highlight the need for further clinical investigation into the combination of BETi and NAEi as a promising strategy for cancer therapy.

Design, synthesis, and biological evaluation of pyrido[2,3-d]pyrimidin-7(8H)-one derivatives as potent USP1 inhibitors.

USP1 has emerged as a novel and potential target for drug discovery in single therapeutic agents or combination with chemotherapy and molecular targeted therapy. In this study, based on the disclosed structure of ML323 and KSQ-4279, we designed and synthesized a series of pyrido[2,3-d]pyrimidin-7(8H)-one derivatives as potent USP1 inhibitors by cyclization strategy and the systematic structure-activity relationship exploration was conducted. The representative compounds 1k, 1m and 2d displayed excellent USP1/UAF inhibition and exhibited strong antiproliferation effect in NCI-H1299 cells. Further flow cytometry analysis revealed that they could arrest breast cancer cells MDA-MB-436 in the S phase. Inhibition mechanism study of compound 1m indicated these derivatives acted as reversible and noncompetitive USP1 inhibitors. Of note, the combination of compound 1m with PARP inhibitor olaparib generated enhanced cell killing in olaparib-resistant MDA-MB-436/OP cells, and compound 1m exhibited excellent oral pharmacokinetic properties in mice. Overall, our efforts may provide a reliable basis for the development of novel USP1 inhibitor as a single therapeutic agent and in combination with PARP inhibitors.

Proteasome inhibitors reduce CD73 expression partly via decreasing p-ERK in NSCLC cells.

Ecto-5'-nucleotidase (CD73), encoded by the NT5E gene, mediates tumor immunosuppression and has been targeted for the development of new anticancer drugs. Proteasome inhibitors impair protein degradation by inhibiting proteasome and have been used in the clinic for cancer therapy. Here we report that proteasome inhibitors reduce the protein and mRNA levels of CD73. Among 127 tested small-molecule drugs, proteasome inhibitors were found to consistently decrease the protein and mRNA levels of CD73 in NSCLC NCI-H1299 cells. This effect was further confirmed in different NSCLC cells exposed to different proteasome inhibitors. In those treated cells, the protein levels of ERK and its active form p-ERK, the vital components in the MAPK pathway, were reduced. Consistently, inhibitors of MEK and ERK, another two members of the MAPK pathway, also lowered the protein and mRNA levels of CD73. Correspondingly, treatments with fibroblast growth factor 2 (FGF2), an activator of the MAPK pathway, enhanced the levels of p-ERK and partly rescued the proteasome inhibitor-driven reduction of CD73 mRNA and protein in NSCLC cells. However, exogenous CD73 overexpression in murine Lewis lung carcinoma (LLC) cells was not lowered either in vitro or in vivo, by the treatments with proteasome inhibitors and basically, did not affect their in vitro proliferative inhibition either. In contrast, CD73 overexpression dramatically reduced the in vivo anticancer activity of Bortezomib in immunocompetent mice, with tumor growth inhibition rates from 52.18 % for LLC/vector down to 8.75 % for LLC/NT5E homografts. These findings give new insights into the anticancer mechanisms of proteasome inhibitors.

Thioparib inhibits homologous recombination repair, activates the type I IFN response, and overcomes olaparib resistance.

Poly-ADP-ribose polymerase (PARP) inhibitors (PARPi) have shown great promise for treating BRCA-deficient tumors. However, over 40% of BRCA-deficient patients fail to respond to PARPi. Here, we report that thioparib, a next-generation PARPi with high affinity against multiple PARPs, including PARP1, PARP2, and PARP7, displays high antitumor activities against PARPi-sensitive and -resistant cells with homologous recombination (HR) deficiency both in vitro and in vivo. Thioparib treatment elicited PARP1-dependent DNA damage and replication stress, causing S-phase arrest and apoptosis. Conversely, thioparib strongly inhibited HR-mediated DNA repair while increasing RAD51 foci formation. Notably, the on-target inhibition of PARP7 by thioparib-activated STING/TBK1-dependent phosphorylation of STAT1, triggered a strong induction of type I interferons (IFNs), and resulted in tumor growth retardation in an immunocompetent mouse model. However, the inhibitory effect of thioparib on tumor growth was more pronounced in PARP1 knockout mice, suggesting that a specific PARP7 inhibitor, rather than a pan inhibitor such as thioparib, would be more relevant for clinical applications. Finally, genome-scale CRISPR screening identified PARP1 and MCRS1 as genes capable of modulating thioparib sensitivity. Taken together, thioparib, a next-generation PARPi acting on both DNA damage response and antitumor immunity, serves as a therapeutic potential for treating hyperactive HR tumors, including those resistant to earlier-generation PARPi.

Loss of VOPP1 Contributes to BET Inhibitor Acquired Resistance in Non-Small Cell Lung Cancer Cells.

Inhibitors targeting bromodomain and extraterminal (BET) proteins are promising anticancer drugs. The emergence of drug resistance during treatments will impair their therapeutic effectiveness. To investigate the mechanisms of acquired resistance to BET inhibitors (BETi), we generated a series of drug-resistant sublines by exposing non-small cell lung cancer (NSCLC) NCI-H1975 cells to the BETi ABBV-075. These sublines displayed cross-resistance to other tested BETis, increased migration abilities, reduced growth rates accompanied by an increased proportion of cells in G1 phase and decreased apoptotic responses to BETis. Changes in RNA expression and gene mutation profiles in the resistant variants indicate that emergence of BETi resistance is multifactorial. Importantly, all the tested ABBV-075-resistant variants showed loss of vesicular overexpressed in cancer prosurvival protein 1 (VOPP1) and an increase in the antiapoptotic BCL-2 protein. By knockdown, knockout, and reconstitution of VOPP1 in resistant cells, their parental cells, and other NSCLC cells, we confirmed that the loss of VOPP1 contributed to BETi resistance. Moreover, knockout of VOPP1 in the parental cells caused the increased expression of BCL-2, and the latter directly mediated BETi resistance. Through combined treatments with BETis and BCL-2 inhibitors (BCL-2i), we demonstrated that BCL-2is synergistically sensitized resistant cells to BETis. IMPLICATIONS: Based on these results, for the first time, we establish a causal link from VOPP1 loss to BCL-2 gain and then to BETi resistance, which provides new insights into BETi resistance and paves the way for further testing to circumvent BETi resistance.

Design and development of a novel series of oral bivalent BET inhibitors with potent anticancer activities.

Bromodomain and extraterminal domain (BET) subfamily members are intriguing targets for cancer treatment. Most of the reported BET inhibitors were monovalent inhibitors. Recently, some bivalent inhibitors were disclosed, which bound to two bromodomains simultaneously. They had good activities, however, most of them also showed unsatisfactory pharmacokinetic properties, which were caused by long chain linkers. Based on our previous work on monovalent BRD4 inhibitors, we designed and synthesized a series of novel bivalent inhibitors with short and hydrophilic linkers. These compounds exhibited better activities than the corresponding monovalent inhibitors and good pharmacokinetic properties. Compound 21 showed excellent in vitro activities. And it also demonstrated potent in vivo antitumor efficacy under oral administration and was well tolerated in in vivo tests.

Chemical constituents from the South China sea soft coral Sinularia humilis.

A new diterpenoid with an unusual capnosane skeleton, sinuhumilol A (1), alone with twelve known diverse compounds (2-13), were isolated from the South China Sea soft coral Sinularia humilis. Their structures and stereochemistry were elucidated by extensive spectroscopic analysis, quantum chemical calculations, and/or by the comparison of the spectroscopic data with those reported in the literature. In bioassay, compound 11 exhibited interesting specific cytotoxicity against the human colon adenocarcinoma cell line HT-29 with IC50 value of 12.5 µM.

Absorption, distribution, metabolism, and excretion of [14C]Mefuparib (CVL218), a novel PARP1/2 inhibitor, in rats.

INTRODUCTION: Mefuparib (CVL218) is a novel second-generation poly-ADP-ribose polymerase (PARP) inhibitor for cancer treatment. CVL218 can easily enter the brain. However, the transport mechanism by which CVL218 crosses the blood-brain barrier (BBB) is unknown. METHODS: (1) [14C] CVL218 metabolism in rats was traced by a liquid scintillation counter and oxidative combustion. (2) Metabolic profiles and metabolites were identified by UHPLC-ß-RAM/UHPLC-Fraction Collector and UHPLC-Q Exactive Plus MS. (3) The partition coefficient Kp,uu,brain value was simulated by two strategies. One strategy was using ACD and GastroPlus Software based on the results of intravenous administration pharmacokinetics and plasma protein-binding studies. The reliability was confirmed by comparison with another strategy (brain/plasma distribution study). RESULTS: (1) Rapid drug elimination was observed 24 h after intragastric administration. The total cumulative excretion in urine and feces within 168 h accounted for 97.15% of the dose. The cumulative radioactive dose recovery in bile was 41.87% within 72 h. The drug-related substances were extensively distributed to the tissues within 48 h. (2) M8 was the major metabolite in plasma, urine, feces and bile. (3) CVL218 exhibited high brain protein-binding rate (88.16%). The Kp,uu,brain value (8.42) simulated by the simple software strategy was similar to that of the brain/plasma distribution study (7.01). CONCLUSIONS: CVL218 is a fast-metabolizing drug and is mainly excreted in feces. The B/P ratio prediction and observation data for CVL218 were consistent. Furthermore, the Kp,uu,brain value indicated that penetration through the BBB might be mediated by uptake transporters.

SOMCL-19-133, a novel, selective, and orally available inhibitor of NEDD8-activating enzyme (NAE) for cancer therapy.

Inhibition of the NEDD8-activating enzyme (NAE), the key E1 enzyme in the neddylation cascade, has been considered an attractive anticancer strategy with the discovery of the first-in-class NAE inhibitor, MLN4924. In this study, we identified SOMCL-19-133 as a highly potent, selective, and orally available NAE inhibitor, which is an analog to AMP. It effectively inhibited NAE with an IC50 value of 0.36 nM and exhibited more than 2855-fold selectivity over the closely related Ubiquitin-activating enzyme (UAE). It is worth noting that treatment with SOMCL-19-133 prominently inhibited Cullin neddylation and delayed the turnover of a panel of Cullin-RING ligases (CRLs) substrates (e.g., Cdt1, p21, p27, and Wee1) at lower effective concentrations than that of MLN4924, subsequently caused DNA damage and Chk1/Chk2 activation, and thus triggered cell cycle arrest and apoptosis. Moreover, SOMCL-19-133 exhibited potent antiproliferative activity against a broad range of human tumor cell lines (mean IC50 201.11 nM), which was about 5.31-fold more potent than that of MLN4924. In vivo, oral delivery treatments with SOMCL-19-133, as well as the subcutaneous injection, led to significant tumor regression in mouse xenograft models. All of the treatments were well tolerated on a continuous daily dosing schedule. Compared with MLN4924, SOMCL-19-133 had a 5-fold higher peak plasma concentration, lower plasma clearance, and a 4-fold larger area under the curve (AUClast). In conclusion, SOMCL-19-133 is a promising preclinical candidate for treating cancers owing to its profound in vitro and in vivo efficacy and favorable pharmacokinetic properties.